Protocols and Video Articles Authored by Yuji Ikegaya

Hippocampal Ripples Down-regulate Synapses Science (New York, N.Y.). | Pubmed ID: 29439023 The functions of sleep for synaptic plasticity remain elusive. We report that mouse hippocampal sharp wave/ripple oscillations serve as intrinsic events that trigger long-lasting synaptic depression. Silencing of sharp waves/ripples during slow-wave states prevented the spontaneous downregulation of net synaptic weights and impaired the learning of new memories. The synaptic downregulation was-methyl-d-aspartate receptor-dependent and input pathway-selective. Thus, our findings are consistent with the role of slow-wave states in refining memory engrams by reducing recent memory-irrelevant neuronal activity and suggest a new role for sharp waves/ripples.

Cortical and Subcortical Responses to Biological Motion NeuroImage. | Pubmed ID: 29524623 Using fMRI and multivariate analyses we sought to understand the neural representations of articulated body shape and local kinematics in biological motion. We show that in addition to a cortical network that includes areas identified previously for biological motion perception, including the posterior superior temporal sulcus, inferior frontal gyrus, and ventral body areas, the ventral lateral nucleus, a presumably motoric thalamic area is sensitive to both form and kinematic information in biological motion. Our findings suggest that biological motion perception is not achieved as an end-point of segregated cortical form and motion networks as often suggested, but instead involves earlier parts in the visual system including a subcortical network.

Monitoring Brain Neuronal Activity with Manipulation of Cardiac Events in a Freely Moving Rat Neuroscience Research. | Pubmed ID: 29454657 Behavioral and cognitive studies have demonstrated that brain functions are affected by the activity states of the peripheral organs, such as the cardiac and respiratory systems. However, detailed neurophysiological mechanisms underlying the body-brain interactions remain unknown. In this study, we developed a method for manipulating activity levels of the heart using direct cardiac stimulation and vagus nerve stimulation that can be combined with recording cerebral local field potentials using a microdrive system, electrocardiograms, electromyograms, in a freely moving rat. With this method, the electrical stimulation to the heart increases heart rates up to 14 Hz, whereas the vagus nerve stimulation decreases heart rates to 3 Hz. Transient electrical artifacts arising from the peripheral stimulation are not contaminated in cortical local field potential signals low-pass filtered at 150 Hz and distinguishable from extracellular multiunit signals. The technique will contribute to understanding the neurophysiological correlate of mind-body associations in health and disease.

[Representation of Time by Hippocampal Neurons] Brain and Nerve = Shinkei Kenkyu No Shinpo. | Pubmed ID: 29172189 The hippocampus is involved in episodic memories of events including time and space. Many studies have focused on the neuronal processing mechanisms underlying spatial cognition and representation in the hippocampus; however, the time-related aspects of memories have only recently become the focus of research. In this review, we first introduce recent reports demonstrating the importance of the hippocampus in the perception of time and then present the hippocampal neuronal dynamics for the representation of time, revealed by large-scale neuronal recording techniques.

Temporally Coordinated Spiking Activity of Human Induced Pluripotent Stem Cell-derived Neurons Co-cultured with Astrocytes Biochemical and Biophysical Research Communications. | Pubmed ID: 29170135 In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes.

Caffeine Increases Hippocampal Sharp Waves in Vitro Biological & Pharmaceutical Bulletin. | Pubmed ID: 28674254 Caffeine promotes memory consolidation. Memory consolidation is thought to depend at least in part on hippocampal sharp waves (SWs). In the present study, we investigated the effect of bath-application of caffeine in spontaneously occurring SWs in mouse acute hippocampal slices. Caffeine induced an about 100% increase in the event frequency of SWs at concentrations of 60 and 200 µM. The effect of caffeine was reversible after washout of caffeine and was mimicked by an adenosine Areceptor antagonist, but not by an Areceptor antagonist. Caffeine increased SWs even in dentate-CA3 mini-slices without the CA2 regions, in which adenosine Areceptors are abundantly expressed in the hippocampus. Thus, caffeine facilitates SWs by inhibiting adenosine Areceptors in the hippocampal CA3 region or the dentate gyrus.

Docosahexaenoic Acid Improves Long-term Potentiation Attenuated by Phospholipase A(2) Inhibitor in Rat Hippocampal Slices British Journal of Pharmacology. | Pubmed ID: 11264234 1. We investigated the possible involvement of phospholipase A(2) (PLA(2)) and its products in long-term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices. 2. Inhibitors of Ca(2+)-independent PLA(2) (iPLA(2)) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca(2+)-dependent PLA(2) inhibitors were virtually ineffective, which suggests a pivotal role of iPLA(2) in the initiation of LTP. 3. We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA(2)-inhibitor) -impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL-attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either. 4. These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

Different Ca2+ Dynamics Between Isolated Hippocampal Pyramidal Cells and Dentate Granule Cells Journal of Neurocytology. Jan, 2002 | Pubmed ID: 12652086 The hippocampal formation contains a variety of neuronal types. The principal neurons are granule cells in the dentate gyrus and pyramidal cells in Ammon's horn. These two neuron types show distinct cell morphology and display a different vulnerability to ischemic injury or various neurotoxins. In order to illustrate the difference in the pathophysiological properties of these neurons, we established a method for separately culturing granule cells and pyramidal cells. They were prepared from the dentate gyrus and Ammon's horn of 3-day-old Wistar rat pups and maintained for 7-9 days in culture. After transient exposure to N-methyl-D-aspartate or glutamate, both the cultured neuron populations displayed somatic Ca(2+) transients with similar amplitudes, but the subsequent recovery to baseline was about twice as fast in granule cells than in pyramidal cells. Similar results were obtained for K(+) depolarization-induced Ca(2+) elevation, suggesting that the relatively rapid Ca(2+) clearance in granule cells is independent of Ca(2+) influx pathways. The present study provides the first evidence for a difference in Ca(2+) dynamics and homeostasis between granule and pyramidal cells and may represent a cellular basis for the differential vulnerability of hippocampal neurons.

Hippocampal Long-term Depression As an Index of Spatial Working Memory The European Journal of Neuroscience. Sep, 2002 | Pubmed ID: 12372034 Long-term potentiation (LTP), a form of synaptic plasticity in the hippocampus, is a cellular model for the neural basis of learning and memory, but few studies have investigated the contribution of long-term depression (LTD), a counterpart of LTP. To address the possible relationship between hippocampal LTD and spatial performance, the spatial cognitive ability of a rat was assessed in a spontaneous alternation test and, thereafter, LTD in response to low-frequency burst stimulation (LFBS) was monitored in the dentate gyrus of the same rat under anaesthesia. To enhance a divergence in the ability for spatial performance, some of the animals received fimbria-fornix (FF) transection 14 days before the experiments. LTD was reliably induced by application of LFBS to the medial perforant path of intact rats, while no apparent LTD was elicited in rats with FF lesions. The behavioural parameters of spatial memory showed a significant correlation with the magnitude of LTD. We found no evidence that the cognitive ability correlated with other electrophysiological parameters, e.g. basal synaptic responses, stimulus intensity to produce half-maximal responses, paired-pulse facilitation or paired-pulse depression. These results suggest that the magnitude of LTD in the dentate gyrus serves as a reliable index of spatial cognitive ability, providing insights into the functional significance of hippocampal LTD.

Brain-derived Neurotrophic Factor Promotes the Maturation of GABAergic Mechanisms in Cultured Hippocampal Neurons The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2002 | Pubmed ID: 12196581 Brain-derived neurotrophic factor (BDNF) has been implicated in activity-dependent plasticity of neuronal function and network arrangement. To clarify how BDNF exerts its action, we evaluated the physiological, histological, and biochemical characteristics of cultured hippocampal neurons after long-term treatment with BDNF. Here we show that BDNF facilitates high K(+)-elicited release of GABA but not of glutamate and induces an increase in immunoreactive signals of glutamic acid decarboxylase, a GABA-synthesizing enzyme. The soma size of GABAergic neurons was enlarged in BDNF-treated cultures, whereas the average soma size of all neurons was virtually unchanged. BDNF also upregulated protein levels of GABA(A) receptors but not of glutamate receptors. These data imply that BDNF selectively advances the maturation of GABAergic synapses. However, immunocytochemical analyses revealed that a significant expression of TrkB, a high-affinity receptor for BDNF, was detected in non-GABAergic as well as GABAergic neurons. BDNF also increased to total amount of synaptic vesicle-associated proteins without affecting the number of presynaptic vesicles that can be labeled with FM1-43 after K(+) depolarization. Together, our findings indicate that BDNF principally promotes GABAergic maturation but may also potentially contribute to excitatory synapse development via increasing resting synaptic vesicles.

Vasopressin Induces Emesis in Suncus Murinus Japanese Journal of Pharmacology. Jul, 2002 | Pubmed ID: 12184741 This paper reports that vasopressin is emetogenic in the house musk shrew Suncus murinus. Either intravenous or intracerebroventricular administration of vasopressin caused vomiting within a few minutes. The ED50 of intravenous vasopressin was as high as 4.67 microg/kg, whereas intracerebroventricularly injected vasopressin was effective at a low dose of 20 ng/brain. The emetogenic target of vasopressin may therefore be present in the central nervous system. We propose the Suncus as a useful animal for investigation of vasopressin-mediated emesis, including motion sickness.

BDNF Attenuates Hippocampal LTD Via Activation of Phospholipase C: Implications for a Vertical Shift in the Frequency-response Curve of Synaptic Plasticity The European Journal of Neuroscience. Jul, 2002 | Pubmed ID: 12153539 Recent evidence shows that neurotrophins are not only involved in neuronal survival and differentiation during development but also in modulating synaptic strength in the mature brain. To understand how neurotrophins alter this synaptic modification, we have investigated the effect of brain-derived neurotrophic factor (BDNF) on long-term depression (LTD) at Schaffer collateral-CA1 synapses in rat hippocampal slices. The slices treated with BDNF for 5 min showed significantly less LTD in response to a 1-Hz tetanus compared with controls but displayed normal LTD when the afferents were tetanized at 10 Hz. Because BDNF enhanced long-term potentiation (LTP) induced by a 30-Hz tetanus, the synaptic modification threshold (theta(m)) as defined in the 'BCM' theory of Bienenstock Cooper & Monroe [Bienenstock et al. (1982), J. Neurosci., 2, 32-48] was not shifted. BNDF is likely to alter the capability of the plastic changes in synaptic efficacy, i.e. to produce an upward shift in the BCM curve. The suppressive effect of BDNF on LTD was prevented by either the tyrosine kinase (Trk) receptor inhibitor K252a or the phospholipase C inhibitor U73122. Thus, TrkB activation may attenuate LTD through phospholipase C signalling pathway.

Cytoskeleton Disruption Causes Apoptotic Degeneration of Dentate Granule Cells in Hippocampal Slice Cultures Neuropharmacology. Jun, 2002 | Pubmed ID: 12128012 Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 microM and 10 microM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca(2+)-free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.

Mossy Fiber Zn2+ Spillover Modulates Heterosynaptic N-methyl-D-aspartate Receptor Activity in Hippocampal CA3 Circuits The Journal of Cell Biology. Jul, 2002 | Pubmed ID: 12119362 Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non-N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor-mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.

Rapid Regrowth of Hippocampal Mossy Fibres and Preceding Maturation of NMDA Receptor-mediated Neurotransmission The European Journal of Neuroscience. Jun, 2002 | Pubmed ID: 12081666 Early in postnatal development, glutamatergic synapses contain primarily NMDA receptors and progressively acquire AMPA receptor function. To determine whether this transformation occurs in a process of regenerative synaptogenesis following axotomy, we investigated the recovery of AMPA and NMDA receptor-mediated neurotransmission after the transection of mossy fibres (MF) in organotypic hippocampal cultures. An NMDA component could already be elicited 1 day after the lesion and reached a saturated level after 3 days. Thereafter, an AMPA component appeared and slowly matured after 10 days. The preceding establishment of NMDA receptor function implies that immature MF synapses are functionally silent at least for the first several days of recovery. The appearance of AMPA receptor-mediated neurotransmission was unchanged in the presence of an NMDA-receptor antagonist or tetrodotoxin, which suggests that the AMPA receptor maturation is virtually independent of neuronal activity. Thus, the conversion of silent to functional synapses is not unique to synaptic plasticity or developmental processes but also occurs in recovery after brain damage, but its mechanism is likely to differ from NMDA receptor-dependent recruitment of AMPA receptors in synaptic plasticity.

Beta-amyloid Enhances Glial Glutamate Uptake Activity and Attenuates Synaptic Efficacy The Journal of Biological Chemistry. Aug, 2002 | Pubmed ID: 12070161 Although amyloid beta-protein (A beta) has long been implicated in the pathogenesis of Alzheimer's disease, little is known about the mechanism by which A beta causes dementia. A beta leads to neuronal cell death in vivo and in vitro, but recent evidence suggests that the property of the amnesic characteristic of Alzheimer's disease can be explained by a malfunction of synapses rather than a loss of neurons. Here we show that prolonged treatment with A beta augments the glutamate clearance ability of cultured astrocytes and induces a dramatic decrease in glutamatergic synaptic activity of neurons cocultured with the astrocytes. Biotinylation assay revealed that the enhancement of glutamate uptake activity was associated with an increase in cell-surface expression of GLAST, a subtype of glial glutamate transporters, without apparent changes in the total amount of GLAST. This phenomenon was blocked efficiently by actin-disrupting agents. Thus, A beta-induced actin-dependent GLAST redistribution and relevant synaptic malfunction may be a cellular basis for the amnesia of Alzheimer's disease.

Hyperpolarization-activated Current I(h) in Nucleus of Solitary Tract Neurons: Regional Difference in Serotonergic Modulation Japanese Journal of Pharmacology. Apr, 2002 | Pubmed ID: 12046990 The nucleus of solitary tract (NTS) contains diverse neural circuits responsible for basic vital functions. We examined the effect of serotonin (5-HT) on hyperpolarization-activated current (I(h)) in neurons acutely isolated from caudal, medial and rostral parts of the NTS. Caudal and medial NTS neurons showed a large amplitude of I(h) compared with rostral neurons. In these neurons, perfusion with 5-HT potentiated Ih amplitude in a concentration-dependent manner. The effect of 5-HT was blocked by NAN-190, a 5-HT1A receptor antagonist. Thus, 5-HT1A receptors may regulate I(h) channel activity in caudal and medial NTS neurons.

Group II Metabotropic Glutamate Receptor Activation is Required for Normal Hippocampal Mossy Fibre Development in the Rat The Journal of Physiology. Feb, 2002 | Pubmed ID: 11850509 Glutamate is the main neurotransmitter at hippocampal mossy fibre (MF) terminals. Because neurotransmitters have been proposed as regulating factors of neural network formation and neurite morphogenesis in the developing CNS, we examined the possible contribution of glutamate to MF pathfinding. Entorhino-hippocampal slices prepared from early postnatal rats were cultivated in the presence of glutamate receptor antagonists. Timm histochemical staining revealed that pharmacological blockade of metabotropic glutamate receptors (mGluR), but not of ionotropic glutamate receptors, induced abnormal outgrowth of the MFs. When slices were cultured in the presence of mGluR antagonists, DiI-labelled MF axons displayed a great degree of defasciculation, and MF-mediated EPSPs in the CA3 pyramidal cells were altered. Similar results were obtained for a selective antagonist of group II mGluR, but not of group I or III mGluR. Glutamate is, therefore, likely to regulate MF outgrowth via activation of group II mGluR. The present study may provide a novel role of glutamate in hippocampal development.

Endothelin Downregulates the Glutamate Transporter GLAST in CAMP-differentiated Astrocytes in Vitro Glia. Feb, 2002 | Pubmed ID: 11754215 Endothelin (ET) is a putative pathogenetic mediator associated with brain trauma and ischemia. Because a link between neuronal damage after these injuries and glial Na(+)-dependent L-glutamate transporter activity has been suggested, we investigated the effect of ET on the glutamate clearance ability of astrocytes. Dibutyryl cyclic adenosine monophosphate (dBcAMP), which is widely used to induce differentiation of cultured astrocytes, markedly increased [(3)H]glutamate transport activity in a concentration- and time-dependent manner. In the presence of ET, however, dBcAMP decreased the glutamate uptake. This effect was efficiently prevented by an antagonist of ET(B) receptor, but not of ET(A) receptor. ET per se was virtually ineffective. Eadie-Hofstee analysis demonstrated that dBcAMP increased the V(max) value of glutamate uptake activity by 43.4% in the absence of ET, but decreased it by 41.4% in the presence of ET, without apparent changes in the K(m) value. Accordingly, Western blot analysis indicated that the change in transport activity correlated closely with that in expression level of the glial glutamate transporter GLAST. These results may represent the mechanisms by which ET aggravates trauma- and ischemia-elicited neuronal damage.

Beta-amyloid Prevents Excitotoxicity Via Recruitment of Glial Glutamate Transporters Naunyn-Schmiedeberg's Archives of Pharmacology. Sep, 2003 | Pubmed ID: 14513203 Amyloid beta-protein (Abeta), a putative pathogenic endotoxin involved in Alzheimer's disease, induces redistribution of glutamate transporters in astrocytes and promotes their pump activity. Because the transporters are assumed to protect neurons against excitotoxicity by removing extracellular glutamate, we hypothesized that Abeta alters the vulnerability of neurons to glutamate. Cerebrocortical neuron-astroglial co-cultures were exposed to glutamate, the concentration of which was selected so that only 20% of the neurons exhibited degeneration. When cultures were pre-treated with Abeta, exposure to the same "mild" glutamate concentration failed to damage neurons. The Abeta-induced protection was abolished by a glial glutamate transporter inhibitor. Thus, Abeta can alleviate excitotoxicity through glutamate transporter activity. The present results may challenge prevailing concepts that Abeta-induced neuron loss causes Alzheimer's dementia and also provide practical insights into neuro-glial interactions in glutamate toxicity.

Activity-evoked Capacitative Ca2+ Entry: Implications in Synaptic Plasticity The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2003 | Pubmed ID: 12944501 The Ca2+ influx controlled by intracellular Ca2+ stores, called store-operated Ca2+ entry (SOC), occurs in various eukaryotic cells, but whether CNS neurons are endowed with SOC capability and how they may operate have been contentious issues. Using Ca2+ imaging, we present evidence for the presence of SOC in cultured hippocampal pyramidal neurons. Depletion of internal Ca2+ stores by thapsigargin caused intracellular Ca2+ elevation, which was prevented by SOC channel inhibitors 2-aminoethoxydiphenyl borate (2-APB), SKF96365, and La3+. Interestingly, these inhibitors also accelerated the decay of NMDA-induced Ca2+ transients without affecting their peak amplitude. In addition, SOC channel inhibitors attenuated tetanus-induced dendritic Ca2+ accumulation and long-term potentiation at Schaffer collateral-CA1 synapses in hippocampal slice preparations. These data suggest a novel link between ionotropic receptor-activated SOC and neuroplasticity.

Mossy Fiber Pathfinding in Multilayer Organotypic Cultures of Rat Hippocampal Slices Cellular and Molecular Neurobiology. Feb, 2003 | Pubmed ID: 12701887 1. Using a novel technique of organotypic cultures, in which two hippocampal slices were cocultured in a bilayer style, we found that the mossy fibers arising from the dentate gyrus grafted onto another dentate tissue grew along the CA3 stratum lucidum of the host hippocampal slice. The same transplantation of a CA1 microslice failed to form a network with the host hippocampus. 2. Thus the type of grafted neurons is important to determine whether they can form an appropriate network after transplantation.

Mossy Fibre Synaptic NMDA Receptors Trigger Non-Hebbian Long-term Potentiation at Entorhino-CA3 Synapses in the Rat The Journal of Physiology. Feb, 2003 | Pubmed ID: 12562995 Hippocampal CA3 pyramidal cells receive two independent afferents from the enthorinal cortex, i.e. a direct input via the temporoammonic pathway (TA, perforant path) and an indirect input via the mossy fibres (MF) of dentate granule cells. In spite of past suggestions that the TA is assigned an important role in exciting the pyramidal cells, little is known about their physiological properties. By surgically making an incision through the sulcus hippocampi and a small part of the dentate molecular layer, we succeeded in isolating TA-mediated monosynaptic responses in CA3 stratum lacunosum-moleculare. The TA-CA3 synaptic transmission was completely blocked by a combination of D,L-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), NMDA and non-NMDA receptor antagonists, respectively, and displayed paired-pulse facilitation and NMDA receptor-dependent long-term potentiation, which are all typical of glutamatergic synapses. We next addressed the heterosynaptic interaction between TA-CA3 and MF-CA3 synapses. The TA-CA3 transmission was partially attenuated by single-pulse MF pre-stimulation at inter-pulse intervals of up to 70 ms. However, surprisingly, burst stimulation of the MF alone induced long-lasting facilitation of TA-CA3 synaptic efficacy. This non-Hebbian form of synaptic plasticity was efficiently prevented by local application of AP5 into the MF synapse-rich area. Therefore, MF-activated NMDA receptors are responsible for the heterosynaptic modification of TA-CA3 transmission, and thereby, the history of MF activity may be etched into TA-CA3 synaptic strength. Our findings predict a novel form of spatiotemporal information processing in the hippocampus, i.e. a use-dependent intersynaptic memory transfer.

Fimbrial Control of Bidirectional Synaptic Plasticity of Medial Perforant Path-dentate Transmission Synapse (New York, N.Y.). Mar, 2003 | Pubmed ID: 12494398 Lesions of the fimbria-fornix (FF) tract cause profound impairments of cognitive ability in animals. Our previous study showed that spatial performance correlates with long-term potentiation (LTP) of the dentate gyrus (DG), but not of the CA1 region, in rats with bilateral FF lesions, suggesting that FF lesions selectively inhibited LTP in the DG. The cortical input to the DG is anatomically and physiologically divided into two types of afferents, i.e., the medial perforant path (MPP) and the lateral perforant path (LPP), which show distinct synaptic properties. To elucidate the difference in the FF modulation of these two inputs, field responses were recorded from MPP- or LPP-DG synapses in anesthetized rats. MPP-DG synapses of rats with FF lesions displayed neither LTP in response to theta-burst stimulation nor long-term depression (LTD) in response to low-frequency burst stimulation. In contrast to the MPP, LPP-DG synapses showed normal LTP in rats with FF lesions. The low-frequency burst stimulation could not induce LTD at LPP-DG synapses in either intact or FF-lesioned rats. These results suggest that the FF pathway selectively supports the mechanisms of bidirectional synaptic plasticity at MPP-DG synapses. This study provides new insights into external control of information processing in the hippocampus.

Interleukin-1beta Abrogates Long-term Depression of Hippocampal CA1 Synaptic Transmission Synapse (New York, N.Y.). Jan, 2003 | Pubmed ID: 12422373 Although interleukin-1beta (IL-1beta) is well known to modulate synaptic transmission and plasticity of the hippocampus, no study has yet evaluated how this cytokine affects long-term depression (LTD), one of the major forms of hippocampal synaptic plasticity. Here we report that at Schaffer collateral-CA1 synapses, bath application of IL-1beta induces a long-lasting decrease in synaptic strength in intact slices, but not in disinhibited slices in the presence of bicuculline, a gamma-aminobutyric acid receptor antagonist. The IL-1beta-induced synaptic depression efficiently foreclosed the subsequent induction of LTD in response to a 1-Hz tetanus and, conversely, it was also prevented by preexisting LTD. These results suggest that IL-1beta-induced, persistent depression of synaptic efficacy is required for GABAergic activation and shares, at least in part, a common cellular mechanism for LTD.

Mossy Fiber Sprouting As a Potential Therapeutic Target for Epilepsy Current Neurovascular Research. Jan, 2004 | Pubmed ID: 16181061 Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.

Amygdala Stimulation Modulates Hippocampal Synaptic Plasticity Proceedings of the National Academy of Sciences of the United States of America. Sep, 2004 | Pubmed ID: 15381775 Experience-dependent synaptic plasticity is a fundamental feature of neural networks involved in the encoding of information, and the capability of synapses to express plasticity is itself activity-dependent. Here, we introduce a "low-frequency burst stimulation" protocol, which can readily induce both long-term potentiation (LTP) and long-term depression (LTD) at in vivo medial perforant path-dentate gyrus synapses. By varying stimulation parameters, we were able to build a stimulus-response map of synaptic plasticity as a LTP-LTD continuum. The response curve displayed a bidirectional shift toward LTP and LTD, depending on the degree and timing of neural activity of the basolateral amygdala. The range of this plastic modulation was also modified by past activity of the basolateral amygdala, suggesting that the amygdala can arrange its ability to regulate the dentate plastic responses. The effects of the BLA activation were replicated by stimulation of the lateral perforant path and, hence, BLA stimulation may recruit the lateral entorhinal cortex. These results represent a high-order dimension of heterosynaptic modulations of hippocampal synaptic plasticity.

Chronometric Readout from a Memory Trace: Gamma-frequency Field Stimulation Recruits Timed Recurrent Activity in the Rat CA3 Network The Journal of Physiology. Nov, 2004 | Pubmed ID: 15375190 Synchronous population activity is prevalent in neurones of the central nervous system and experimentally captured as oscillatory electric fields, the frequency of which can represent the state of the neural circuit, e.g. theta (approximately 5 Hz) and gamma (approximately 40 Hz). Such field oscillations, however, are not merely a result of coherent neuronal activity. They may also play active roles in information processing in the brain. In this study, we observed that, in cultured hippocampal slices, CA3 pyramidal cells responded to single-pulse stimuli with monosynaptic and polysynaptic potentials and firing spikes which occurred after variable latencies. The variability of the spike latencies was greatly reduced in the presence of weak electric field oscillations, especially the oscillation in the gamma-band frequency range, that per se induced only small fluctuations in the subthreshold membrane potential, and this effect was inhibited by blockade of NMDA receptor activity. Furthermore, the latency of the firing spikes changed if the stimulus was applied at a different phase of the imposed gamma oscillations. These results may suggest that the background field oscillations serve as an extracellular time reference and assure accurate and stable decoding of a memory trace present in cortical feedback networks.

Brain-derived Neurotrophic Factor Induces Hyperexcitable Reentrant Circuits in the Dentate Gyrus The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2004 | Pubmed ID: 15317847 Aberrant sprouting and synaptic reorganization of the mossy fiber (MF) axons are commonly found in the hippocampus of temporal lobe epilepsy patients and result in the formation of excitatory feedback loops in the dentate gyrus, a putative cellular basis for recurrent epileptic seizures. Using ex vivo hippocampal cultures, we show that prolonged hyperactivity induces MF sprouting and the resultant network reorganizations and that brain-derived neurotrophic factor (BDNF) is necessary and sufficient to evoke these pathogenic plasticities. Hyperexcitation induced an upregulation of BDNF protein expression in the MF pathway, an effect mediated by L-type Ca2+ channels. The neurotrophin receptor tyrosine kinase (Trk)B inhibitor K252a or function-blocking anti-BDNF antibody prevented hyperactivity-induced MF sprouting. Even under blockade of neural activity, local application of BDNF to the hilus, but not other subregions, was capable of initiating MF axonal remodeling, eventually leading to dentate hyperexcitability. Transfecting granule cells with dominant-negative TrkB prevented axonal branching. Thus, excessive activation of L-type Ca2+ channels causes granule cells to express BDNF, and extracellularly released BDNF stimulates TrkB receptors present on the hilar segment of the MFs to induce axonal branching, which may establish hyperexcitable dentate circuits.

Environmental Control of the Survival and Differentiation of Dentate Granule Neurons Cerebral Cortex (New York, N.Y. : 1991). Dec, 2004 | Pubmed ID: 15192012 Dentate granule cells (DGCs) and their microcircuits have been implicated in hippocampus-dependent memory encoding and epileptogenesis. Little is known about how the proper maturation of DGCs is determined by their intrinsic programs or external factors during development. In order to explore this, we dispersed premature DGCs on living hippocampal slices. Here we report that the survival and network formation of DGCs are supported by local cues present in the dentate gyrus ex vivo. The density of surviving DGCs was almost uniform throughout the host slices 12 h after implantation but gradually became heterogenous across substrata, with the cells engrafted onto the stratum granulosum scoring the highest rate of survival. The mossy fiber axons arising from DGCs growing on this substratum were properly guided towards CA3, whereas other misplaced DGCs exhibited heterotopic axon projection. In particular, about half of the axons originating from the hilus were misguided into the molecular layer, which resembles the supragranular mossy fiber sprouting seen in epileptic disorders. These results suggest that local environmental factors influence the cell adhesion, neurite polarization and axon guidance of DGCs.

Synfire Chains and Cortical Songs: Temporal Modules of Cortical Activity Science (New York, N.Y.). Apr, 2004 | Pubmed ID: 15105494 How can neural activity propagate through cortical networks built with weak, stochastic synapses? We find precise repetitions of spontaneous patterns of synaptic inputs in neocortical neurons in vivo and in vitro. These patterns repeat after minutes, maintaining millisecond accuracy. Calcium imaging of slices reveals reactivation of sequences of cells during the occurrence of repeated intracellular synaptic patterns. The spontaneous activity drifts with time, engaging different cells. Sequences of active neurons have distinct spatial structures and are repeated in the same order over tens of seconds, revealing modular temporal dynamics. Higher order sequences are replayed with compressed timing.

BDNF Boosts Spike Fidelity in Chaotic Neural Oscillations Biophysical Journal. Mar, 2004 | Pubmed ID: 14990508 Oscillatory activity and its nonlinear dynamics are of fundamental importance for information processing in the central nervous system. Here we show that in aperiodic oscillations, brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances the accuracy of action potentials in terms of spike reliability and temporal precision. Cultured hippocampal neurons displayed irregular oscillations of membrane potential in response to sinusoidal 20-Hz somatic current injection, yielding wobbly orbits in the phase space, i.e., a strange attractor. Brief application of BDNF suppressed this unpredictable dynamics and stabilized membrane potential fluctuations, leading to rhythmical firing. Even in complex oscillations induced by external stimuli of 40 Hz (gamma) on a 5-Hz (theta) carrier, BDNF-treated neurons generated more precisely timed spikes, i.e., phase-locked firing, coupled with theta-phase precession. These phenomena were sensitive to K252a, an inhibitor of tyrosine receptor kinases and appeared attributable to BDNF-evoked Na(+) current. The data are the first indication of pharmacological control of endogenous chaos. BDNF diminishes the ambiguity of spike time jitter and thereby might assure neural encoding, such as spike timing-dependent synaptic plasticity.

[Heterosynaptic Plasticity in CA3 Hippocampus] Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Feb, 2004 | Pubmed ID: 14976758

Developmental Switch in Axon Guidance Modes of Hippocampal Mossy Fibers in Vitro Developmental Biology. Mar, 2004 | Pubmed ID: 14975715 Hippocampal mossy fibers (MFs), axons of dentate granule cells, run through a narrow strip, called the stratum lucidum, and make synaptic contacts with CA3 pyramidal cells. This stereotyped pathfinding is assumed to require a tightly controlled guidance system, but the responsible mechanisms have not been proven directly. To clarify the cellular basis for the MF pathfinding, microslices of the dentate gyrus (DG) and Ammon's horn (AH) were topographically arranged in an organotypic explant coculture system. When collagen gels were interposed between DG and AH slices prepared from postnatal day 6 (P6) rats, the MFs passed across this intervening gap and reached CA3 stratum lucidum. Even when the recipient AH was chemically pre-fixed with paraformaldehyde, the axons were still capable of accessing their normal target area only if the DG and AH slices were directly juxtaposed without a collagen bridge. The data imply that diffusible and contact cues are both involved in MF guidance. To determine how these different cues contribute to MF pathfinding during development, a P6 DG slice was apposed simultaneously to two AH slices prepared from P0 and P13 rats. MFs projected normally to both the host slices, whereas they rarely invaded P0 AH when the two hosts were fixed. Early in development, therefore, the MFs are guided mainly by a chemoattractant gradient, and thereafter, they can find their trajectories by a contact factor, probably via fasciculation with pre-established MFs. The present study proposes a dynamic paradigm in CNS axon pathfinding, that is, developmental changes in axon guidance cues.

CAMP Differentially Regulates Axonal and Dendritic Development of Dentate Granule Cells The Journal of Biological Chemistry. Nov, 2005 | Pubmed ID: 16155295 Neurite polarity is a morphological characteristic of dentate gyrus granule cells, which extend axons to the hilar region and dendrites in the opposite direction, i.e. to the molecular layer. This remarkable polarity must require a differential system for axon and dendrite guidance. Here, we report that the axon and dendrites of a granule cell are differentially responsive to cAMP. In developing cultures of dispersed granule cells, dendritic growth cones were increased in number after pharmacological activation of cAMP signaling and decreased after blockade of cAMP signaling. Activation of cAMP signaling antagonized dendritic collapse induced by the potent repellents Sema3F and glutamate. In contrast to dendrites, axons were protected from Sema3F-induced collapse when cAMP signaling was inhibited. Axonal and dendritic growth cones both expressed type 1 adenylyl cyclase, but only axons showed a cAMP increase in response to Sema3F, and the elevated cAMP was sufficient to collapse axonal growth cones. Thus, the axons and dendrites of dentate granule cells differ in the regulation of cAMP levels as well as responsiveness to cAMP. cAMP may be crucial for shaping the information flow polarity in the dentate gyrus circuit.

To BDNF or Not to BDNF: That is the Epileptic Hippocampus The Neuroscientist : a Review Journal Bringing Neurobiology, Neurology and Psychiatry. Aug, 2005 | Pubmed ID: 16061515 Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has drawn much attention as a potential therapeutic target for temporal lobe epilepsy (TLE). TLE seizures are produced by synchronized hyperactivity of neuron populations due to the disruption of a balance between excitatory and inhibitory synaptic transmissions. In epileptogenesis-related brain areas, including the hippocampus, BDNF is up-regulated in the course of the development of epilepsy and induces a collapse of balanced excitation and inhibition, eventually exerting its epileptogenic effects. On the other hand, several reports demonstrate that intrahippocampal infusion of BDNF can attenuate (or retard) the development of epilepsy. This antiepileptogenic effect seems to be mediated mainly by an increase in the expression of neuropeptide Y. These contrasting effects of BDNF have prevented us from concluding whether inhibition or enhancement of BDNF signaling finally achieves the prevention of TLE. To address this question, it is essential to evaluate how BDNF changes its influences depending on conditions, for example, cell specificity, neural networks, and expression timing and loci. In this article, the authors review BDNF-induced acute and long-lasting changes seen in epileptic circuits from the anatomical and functional points of view.

Microglia-specific Expression of Microsomal Prostaglandin E2 Synthase-1 Contributes to Lipopolysaccharide-induced Prostaglandin E2 Production Journal of Neurochemistry. Sep, 2005 | Pubmed ID: 16000148 Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.

Large-scale Imaging of Cortical Network Activity with Calcium Indicators Neuroscience Research. Jun, 2005 | Pubmed ID: 15893573 Bulk loading of calcium indicators has provided a unique opportunity to reconstruct the activity of cortical networks with single-cell resolution. Here we describe the detailed methods of bulk loading of AM dyes we developed and have been improving for imaging with a spinning disk confocal microscope.

Dynamic Synapses As Archives of Synaptic History: State-dependent Redistribution of Synaptic Efficacy in the Rat Hippocampal CA1 The Journal of Physiology. Jul, 2005 | Pubmed ID: 15845579 Plastic modifications of synaptic strength are putative mechanisms underlying information processing in the brain, including memory storage, signal integration and filtering. Here we describe a dynamic interplay between short-term and long-term synaptic plasticity. At rat hippocampal CA1 synapses, induction of both long-term potentiation (LTP) and depression (LTD) was accompanied by changes in the profile of short-term plasticity, termed redistribution of synaptic efficacy (RSE). RSE was presynaptically expressed and associated in part with a persistent alteration in hyperpolarization-activated I(h) channel activity. Already potentiated synapses were still capable of showing RSE in response to additional LTP-triggering stimulation. Strikingly, RSE took place even after reversal of LTP or LTD, that is, the same synapse can display different levels of short-term plasticity without changing synaptic efficacy for the initial spike in burst presynaptic firing, thereby modulating spike transmission in a firing rate-dependent manner. Thus, the history of long-term synaptic plasticity is registered in the form of short-term plasticity, and RSE extends the information storage capacity of a synapse and adds another dimension of functional complexity to neuronal operations.

Calcium Imaging of Cortical Networks Dynamics Cell Calcium. May, 2005 | Pubmed ID: 15820393 Studies relating spontaneous network activities to cognitive processes and/or brain disorders constitute a recently expanding field of investigation. They are mostly based either on cellular recordings--usually performed in pharmacologically induced oscillations in brain slices--or on multi-cellular recordings using tetrodes or multiple electrodes. However, these research strategies cannot link the electrical recordings with morphological characterization of the neurons. The progress made in imaging techniques allows for the first time to have simultaneously a dynamic and global characterization of network activity and to determine the single-cell properties of the unitary microcircuits involved in this activity.

BDNF Locally Potentiates GABAergic Presynaptic Machineries: Target-selective Circuit Inhibition Cerebral Cortex (New York, N.Y. : 1991). Mar, 2005 | Pubmed ID: 15238431 Inhibitory neurotransmission is critical for neuronal circuit formation. To examine whether inhibitory neurotransmission receives target-selective modulation in the long term, we expressed the cDNA of brain-derived neurotrophic factor (BDNF), which has been shown to induce the augmentation of GABAergic synapses in vivo and in vitro, in a small population of cultured hippocampal neurons. At 48 h after transfection, the expression level of glutamic acid decarboxylase 65 (GAD65), a GABA synthetic enzyme that resides mainly in GABAergic terminals, was selectively enhanced around the BDNF-expressing neurons, in comparison with the neighboring control neurons interposed between the BDNF-expressing neurons and inhibitory neurons. Exogenous BDNF application for 48 h also increased the GAD level and enhanced the GABA release probability. These potentiating effects were attenuated in inhibitory synapses on neurons expressing a dominant negative form of the BDNF receptor (tTrkB). This suggests that postsynaptic BDNF-TrkB signaling contributes to the target-selective potentiation of inhibitory presynaptic machineries. Since BDNF is expressed in an activity-dependent manner in vivo, this selectivity may be one of the key mechanisms by which the independence of functional neuronal circuits is maintained.

[Potential Roles for Mossy Fiber Sprouting in Temporal Lobe Epilepsy] Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. May, 2006 | Pubmed ID: 16819240

Integrative Spike Dynamics of Rat CA1 Neurons: a Multineuronal Imaging Study The Journal of Physiology. Jul, 2006 | Pubmed ID: 16613875 The brain operates through a coordinated interplay of numerous neurons, yet little is known about the collective behaviour of individual neurons embedded in a huge network. We used large-scale optical recordings to address synaptic integration in hundreds of neurons. In hippocampal slice cultures bolus-loaded with Ca2+ fluorophores, we stimulated the Schaffer collaterals and monitored the aggregate presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the CA1 stratum pyramidale. Single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks stably output a linear sum of synaptic inputs. Nonetheless, the network activity, even though given constant stimuli, varied from trial to trial. This variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. We also mapped the input-frequency preference in spiking activity and found that the majority of CA1 neurons fired in response to a limited range of presynaptic firing rates (20-40 Hz), acting like a band-pass filter, although a few neurons had high pass-like or low pass-like characteristics. This frequency selectivity depended on phasic inhibitory transmission. Thus, our imaging approach enables the linking of single-cell behaviours to their communal dynamics, and we discovered that, even in a relatively simple CA1 circuit, neurons could be engaged in concordant information processing.

Nitric Oxide/cyclic Guanosine Monophosphate-mediated Growth Cone Collapse of Dentate Granule Cells Neuroreport. Apr, 2006 | Pubmed ID: 16603931 Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we established an experimental system that can simultaneously monitor the behaviors of axonal and dendritic outgrowth. Our previous study shows that axons and dendrites respond differentially to manipulated cyclic adenosine monophosphate signaling, but we report here that cyclic guanosine monophosphate exerts similar effects on axons and dendrites; that is, both axonal and dendritic growth cones collapsed after activation of cyclic guanosine monophosphate signaling. In addition, nitric oxide donor-induced growth-cone collapse was prevented by the inhibition of cyclic guanosine monophosphate signaling, and this effect again did not differ between axons and dendrites. Thus, unlike cyclic adenosine monophosphate, cyclic guanosine monophosphate modulates extending axons and dendrites in a similar manner.

Soluble Guanylyl Cyclase Inhibitor Prevents Sema3F-induced Collapse of Axonal and Dendritic Growth Cones of Dentate Granule Cells Biological & Pharmaceutical Bulletin. Apr, 2006 | Pubmed ID: 16595920 Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we succeeded in simultaneously monitoring the behaviors of axonal and dendritic outgrowth. Our previous study shows that cAMP contributes differentially to Sema3F-induced responses of axons and dendrites, but we report here that the cGMP modulation does not have such a striking axo-dendritic difference. Treatment with Sema3F induced collapse of about 90% growth cones, and pretreatment with 1 muM LY83583, an inhibitor of soluble guanylyl cyclase, partially alleviated the collapse of both axons and dendrites. Thus, unlike cAMP, cGMP modulates axonal and dendritic extension in a similar manner.

K252a, an Inhibitor of Trk, Disturbs Pathfinding of Hippocampal Mossy Fibers Neuroreport. Apr, 2006 | Pubmed ID: 16543811 Hippocampal mossy fibers, which are the axons of dentate granule cells, are continuously generated owing to adult neurogenesis of granule cells. They extend exclusively into the stratum lucidum, a proximal layer of the CA3 pyramidal cells. We visualized the mossy fiber tracts by Timm histochemical staining and DiI labeling in the cultured hippocampal slices from newborn rats. The fibers were abnormally expanded when the slices were cultured in the presence of K252a, an inhibitor of the neurotrophin receptor Trk. Similar defasciculation was observed with an inhibitor of MEK, which is one of the signaling molecules downstream of Trk. This study suggests for the first time that Trk and the MEK pathway are required for mossy fiber pathfinding.

Single Neurons Can Induce Phase Transitions of Cortical Recurrent Networks with Multiple Internal States Cerebral Cortex (New York, N.Y. : 1991). May, 2006 | Pubmed ID: 16093564 Fluctuations of membrane potential of cortical neurons, referred to here as internal states, are essential for brain function, but little is known about how these internal states emerge and are maintained, or what determines transitions between these states. We performed intracellular recordings from hippocampal CA3 pyramidal cells ex vivo and found that neurons display multiple and hierarchical internal states, which are linked to cholinergic activity and are characterized by several power law structures in membrane potential dynamics. Multiple recordings from adjacent neurons revealed that the internal states were coherent between neurons, indicating that the internal state of any given cell in a local network could represent the network activity state. Repeated stimulation of single neurons led over time to transitions to different internal states in both the stimulated neuron and neighboring neurons. Thus, single-cell activation is sufficient to shift the state of the entire local network. As the states shift to more active levels, theta- and gamma-frequency components developed in the form of subthreshold oscillations. State transitions were associated with changes in membrane conductance but were not accompanied by a change in reversal potential. These data suggest that the recurrent network organizes the internal states of individual neurons into synchronization through network activity with balanced excitation and inhibition, and that this organization is discrete, heterogeneous and dynamic in nature. Thus, neuronal states reflect the 'phase' of an active network, a novel demonstration of the dynamics and flexibility of cortical microcircuitry.

Active Hippocampal Networks Undergo Spontaneous Synaptic Modification PloS One. Nov, 2007 | Pubmed ID: 18043757 The brain is self-writable; as the brain voluntarily adapts itself to a changing environment, the neural circuitry rearranges its functional connectivity by referring to its own activity. How the internal activity modifies synaptic weights is largely unknown, however. Here we report that spontaneous activity causes complex reorganization of synaptic connectivity without any external (or artificial) stimuli. Under physiologically relevant ionic conditions, CA3 pyramidal cells in hippocampal slices displayed spontaneous spikes with bistable slow oscillations of membrane potential, alternating between the so-called UP and DOWN states. The generation of slow oscillations did not require fast synaptic transmission, but their patterns were coordinated by local circuit activity. In the course of generating spontaneous activity, individual neurons acquired bidirectional long-lasting synaptic modification. The spontaneous synaptic plasticity depended on a rise in intracellular calcium concentrations of postsynaptic cells, but not on NMDA receptor activity. The direction and amount of the plasticity varied depending on slow oscillation patterns and synapse locations, and thus, they were diverse in a network. Once this global synaptic refinement occurred, the same neurons now displayed different patterns of spontaneous activity, which in turn exhibited different levels of synaptic plasticity. Thus, active networks continuously update their internal states through ongoing synaptic plasticity. With computational simulations, we suggest that with this slow oscillation-induced plasticity, a recurrent network converges on a more specific state, compared to that with spike timing-dependent plasticity alone.

Cryopreservation of Granule Cells from the Postnatal Rat Hippocampus Journal of Pharmacological Sciences. Aug, 2007 | Pubmed ID: 17675794 Although primary cultures of neurons are essential methods for cell biological and pharmacological researches, many animals must be sacrificed for each experiment. Here we introduce a novel system to cryopreserve hippocampal granule cells (GCs) prepared from postnatal rats. Being thawed after as long as 60 days of cryopreservation, GCs expressed the mature neuronal marker MAP-2 and elongated single tau-1-positive axons and multiple tau-1-negative dendrites. These properties closely resembled intact GCs in primary cultures, providing the advantage of being able to repeatedly prepare stable cultures with a single sacrifice of animals.

A Low-cost Method for Brain Slice Cultures Journal of Pharmacological Sciences. Jun, 2007 | Pubmed ID: 17558179 Low-cost, simple procedures for organotypic tissue cultures are desirable for high-throughput biological experiments such as large-scale medical/drug screening. We present a practical and economical method to cultivate brain slices using hydrophilic filtration membranes. With a cost reduction of more than 90%, this technique allows us to prepare hippocampal slice cultures that are morphologically and functionally indistinguishable from those obtained by the widely used Millicell-CM method.

Watching Neuronal Circuit Dynamics Through Functional Multineuron Calcium Imaging (fMCI) Neuroscience Research. Jul, 2007 | Pubmed ID: 17418439 Functional multineuron calcium imaging (fMCI) is a large-scale optical recording technique that monitors the spatiotemporal pattern of action potentials, all at once, from large neuron populations. fMCI has unique advantages, including: (i) simultaneous recording from >1000 neurons in a wide area, (ii) single-cell resolution, (iii) identifiable location of neurons and (iv) detection of non-active neurons during the observation period. We review herein the principle, history, utility and limitations of fMCI.

Metastability of Active CA3 Networks The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2007 | Pubmed ID: 17234584 The brain is spontaneously active even in the absence of external input. This ongoing background activity impacts neural information processing. We used functional multineuron calcium imaging (fMCI) to analyze the net structure of spontaneous CA3 network activity in hippocampal slice cultures loaded with Oregon Green 488 BAPTA-1 using a spinning disk confocal microscope (10-30 frames/s). Principal component analysis revealed that network states, defined by active cell ensembles, were stable but heterogenous and discrete. These states were stabilized through synaptic activity and maintained against external perturbations. A few discrete states emerged during our observation period of up to 30 min. Networks tended to stay in a single state for tens of seconds and then suddenly jump to a new state. After a state transition, the old state was rarely, if ever, revisited by the network during our observation period. This temporal profile of state transitions could not be simulated by a hidden Markov model, indicating that the state dynamics is nonrandomly organized. Within each state, the pattern of network activity tended to stabilize in a specific configuration. Neither maintenance nor transition of the network states required NMDA receptor activity. These findings suggest that the network states are metastable, rather than multistable, and might be governed by local attractor-like dynamics. The fMCI data analyzed here are available at http://hippocampus.jp/data/

Statistical Significance of Precisely Repeated Intracellular Synaptic Patterns PloS One. 2008 | Pubmed ID: 19096523 Can neuronal networks produce patterns of activity with millisecond accuracy? It may seem unlikely, considering the probabilistic nature of synaptic transmission. However, some theories of brain function predict that such precision is feasible and can emerge from the non-linearity of the action potential generation in circuits of connected neurons. Several studies have presented evidence for and against this hypothesis. Our earlier work supported the precision hypothesis, based on results demonstrating that precise patterns of synaptic inputs could be found in intracellular recordings from neurons in brain slices and in vivo. To test this hypothesis, we devised a method for finding precise repeats of activity and compared repeats found in the data to those found in surrogate datasets made by shuffling the original data. Because more repeats were found in the original data than in the surrogate data sets, we argued that repeats were not due to chance occurrence. Mokeichev et al. (2007) challenged these conclusions, arguing that the generation of surrogate data was insufficiently rigorous. We have now reanalyzed our previous data with the methods introduced from Mokeichev et al. (2007). Our reanalysis reveals that repeats are statistically significant, thus supporting our earlier conclusions, while also supporting many conclusions that Mokeichev et al. (2007) drew from their recent in vivo recordings. Moreover, we also show that the conditions under which the membrane potential is recorded contributes significantly to the ability to detect repeats and may explain conflicting results. In conclusion, our reevaluation resolves the methodological contradictions between Ikegaya et al. (2004) and Mokeichev et al. (2007), but demonstrates the validity of our previous conclusion that spontaneous network activity is non-randomly organized.

Large-scale Recordings for Drug Screening in Neural Circuit Systems Yakugaku Zasshi : Journal of the Pharmaceutical Society of Japan. Sep, 2008 | Pubmed ID: 18758138 Functional multineuron calcium imaging (fMCI) is a large-scale optical technique that records the suprathreshold activity from large neuron populations. fMCI has several advantages, including: i) simultaneous recording from hundreds of neurons, ii) single-cell resolution, iii) identifiable location of neurons, and iv) detection of non-active neurons during the observation period. I review the principle and detailed method of fMCI and also describe the effect of oseltamivir on neuronal network as an example for practical application of fMCI.

[Visualization of Multineuronal Spike Activity] Brain and Nerve = Shinkei Kenkyu No Shinpo. Jul, 2008 | Pubmed ID: 18646614 The neuronal network is a computing system that transforms input to output. This computation involves complex nonlinear processes that are carried out with polysynaptic feedforward and feedback microcircuitry. Thus it cannot be assessed by isolating responses of single neurons or by averaging multineuronal responses. To solve this problem, functional multineuron calcium imaging (fMCI) is a promising option. This is a large-scale recording technique that simultaneously monitors the spatiotemporal patterns of spikes emitted by hundreds of neurons with single-cell resolution. Here, we review the availability and actual applications of fMCI. Using fMCI, we attempt to understand the manner in which information is processed in the hippocampal networks. The hippocampus receives information from the cortex; relays it through the dentate gyrus (DG), Cornu Ammonis (CA) 3, and then CA1; and sends it back to the cortex. We placed 2 stimulation electrodes (Stim A and Stim B) in the DG granule cell layer of cultured hippocampal networks and monitored the firing activity of a population of CA1 pyramidal neurons. In this experimental design, the hippocampal polysynaptic network is regarded as a huge arithmetic operator that converts DG inputs to CA1 outputs. We found that the hippocampal polysynaptic network functions as a complex parallel and distributed processing system.

Spontaneous Plasticity of Multineuronal Activity Patterns in Activated Hippocampal Networks Neural Plasticity. 2008 | Pubmed ID: 18645610 Using functional multineuron imaging with single-cell resolution, we examined how hippocampal networks by themselves change the spatiotemporal patterns of spontaneous activity during the course of emitting spontaneous activity. When extracellular ionic concentrations were changed to those that mimicked in vivo conditions, spontaneous activity was increased in active cell number and activity frequency. When ionic compositions were restored to the control conditions, the activity level returned to baseline, but the weighted spatial dispersion of active cells, as assessed by entropy-based metrics, did not. Thus, the networks can modify themselves by altering the internal structure of their correlated activity, even though they as a whole maintained the same level of activity in space and time.

Fast and Accurate Detection of Action Potentials from Somatic Calcium Fluctuations Journal of Neurophysiology. Sep, 2008 | Pubmed ID: 18596182 Large-scale recording from a population of neurons is a promising strategy for approaching the study of complex brain functions. Taking advantage of the fact that action potentials reliably evoke transient calcium fluctuations in the cell body, functional multineuron calcium imaging (fMCI) monitors the suprathreshold activity of hundreds of neurons. However, a limitation of fMCI is its semi-manual procedure of spike extraction from somatic calcium fluctuations, which is not only time consuming but is also associated with human errors. Here we describe a novel automatic method that combines principal-component analysis and support vector machine. This simple algorithm determines the timings of the spikes in calcium fluorescence traces more rapidly and reliably than human operators.

Long-range Axonal Calcium Sweep Induces Axon Retraction The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2008 | Pubmed ID: 18448637 Axon guidance molecules trigger a cascade of local signal in growth cones and evoke various morphologic responses, including axon attraction, repulsion, elongation, and retraction. However, little is known about whether subcellular compartments, other than axonal growth cones, control axon outgrowth. We found that in isolated dentate granule cells, local application of glutamate to the somatodendritic areas, but not the axon itself, induced rapid axon retraction, during which a calcium wave propagated from the somata to the axon terminals. The calcium wave and axon retraction were both inhibited by blockade of voltage-sensitive calcium channels and intracellular calcium dynamics. A combination of perisomatic application of calcium ionophore and depolarizing current injection induced axonal calcium sweep and axon retraction. Thus, perisomatic environments can modulate axon behavior through long-range intracellular communication.

Early-life Status Epilepticus Induces Ectopic Granule Cells in Adult Mice Dentate Gyrus Experimental Neurology. Jun, 2008 | Pubmed ID: 18420198 A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these "ectopic" GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.

Oseltamivir Enhances Hippocampal Network Synchronization Journal of Pharmacological Sciences. Apr, 2008 | Pubmed ID: 18403897 Oseltamivir, a widely used anti-influenza drug, inhibits virus neuraminidase. A mammalian homologue of this enzyme is expressed in the brain, yet the effect of oseltamivir on central neurons is largely unknown. Patch-clamp recordings ex vivo revealed that oseltamivir enhanced spike synchronization between hippocampal CA3 pyramidal cells. Time-lapse multineuron calcium imaging revealed that oseltamivir and its active metabolite evoked synchronized population bursts that recruited virtually all neurons in the network. This unique, so-far-unknown, event was attenuated by muscarinic receptor antagonist. Thus, oseltamivir is a useful tool for investigating a new aspect of neural circuit operation.

Differential Involvement of Cell Cycle Reactivation Between Striatal and Cortical Neurons in Cell Death Induced by 3-nitropropionic Acid The Journal of Biological Chemistry. Mar, 2008 | Pubmed ID: 18182390 Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.

[Visualization of Neuronal Network Activity] Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Dec, 2009 | Pubmed ID: 19999159

Behaviorally Evoked Transient Reorganization of Hippocampal Spines The European Journal of Neuroscience. Aug, 2009 | Pubmed ID: 19674085 Dendritic spines, microstructures that receive the majority of excitatory synaptic inputs, are fundamental units to integrate and store neuronal information. The morphological reorganization of spines accompanies the functional alterations in synaptic strength underlying memory-relevant modifications of network connectivity. Here we report the rapid dynamics of cell population-selective spine reorganizations related to behavioral experiences. In Thy1-GFP transgenic mice, hippocampal CA1 pyramidal neurons that were putatively activated during environmental explorations were detected with their post hoc immunoreactivity for Arc, an activity-dependent immediately-early gene. Immediately after a 60-min exposure to a familiar environment, the spine densities of Arc-positive and Arc-negative neurons were differently distributed. This density imbalance was due exclusively to changes in the number of small, rather than large, spines. The change disappeared within 60 min after mice were returned to the home cages. Thus, spines possess the ability to rapidly and reversibly alter their morphology in response to a brief environmental change. We propose that these transient spine dynamics represent a latent preliminary stage for longer-term plasticity on demand.

[Calcium Imaging of Multineuron Activity in Brain Slice Preparations] Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Jul, 2009 | Pubmed ID: 19602782

High-temperature, but Not High-pressure, Conditions Alter Neuronal Activity Journal of Pharmacological Sciences. May, 2009 | Pubmed ID: 19430196 We describe the effect of high pressure and high temperature on neuronal activity. Increased intracranial pressure is generally a pathological sign observed in intracerebral hemorrhage, brain edema, and brain tumor, yet little is known about how the hyperbaric pressure per se affects neuronal activity. Using a pressure/temperature-changeable perfusion chamber, we carried out functional multineuron calcium imaging to record spontaneous spiking activity simultaneously from about 100 neurons in hippocampal slice cultures. High-pressure conditions (up to 100 mmHg) did not alter the network excitability, whereas high-temperature conditions (up to 40 degrees C) increased synchronized network activity. Thus, neurons are sensitive to feverish conditions, but the acute hyperbaric circumstance itself is unlikely to exert a detrimental effect on neuronal function.

Laterality of Neocortical Slow-wave Oscillations in Anesthetized Mice Neuroscience Research. Jun, 2009 | Pubmed ID: 19428706 In the slow-wave (SW) state, the vast majority of cortical neurons exhibit mostly synchronized oscillatory activity. In this study, we examined the right-left hemispheric difference in slow-wave timings in urethane-anesthetized mice. We found that interhemispheric cross-correlograms of local field potentials (LFPs) peaked asymmetrically. Double in vivo whole-cell patch-clamp recordings also revealed the interhemispheric temporal disparity of slow wave-relevant synaptic barrages. The data suggest the hemispheric laterality in the slow wave origin.

Reverse Optical Trawling for Synaptic Connections in Situ Journal of Neurophysiology. Jul, 2009 | Pubmed ID: 19386760 We introduce a new method to unveil the network connectivity among dozens of neurons in brain slice preparations. While synaptic inputs were whole cell recorded from given postsynaptic neurons, the spatiotemporal firing patterns of presynaptic neuron candidates were monitored en masse with functional multineuron calcium imaging, an optical technique that records action potential-evoked somatic calcium transients with single-cell resolution. By statistically screening the neurons that exhibited calcium transients immediately before the postsynaptic inputs, we identified the presynaptic cells that made synaptic connections onto the patch-clamped neurons. To enhance the detection power, we devised the following points: 1) [K+]e was lowered and [Ca2+]e and [Mg2+]e were elevated, to reduce background synaptic activity and minimize the failure rate of synaptic transmission; and 2) a small fraction of presynaptic neurons was specifically activated by glutamate applied iontophoretically through a glass pipette that was moved to survey the presynaptic network of interest ("trawling"). Then we could theoretically detect 96% of presynaptic neurons activated in the imaged regions with a 1% false-positive error rate. This on-line probing technique would be a promising tool in the study of the wiring topography of neuronal circuits.

Experience-dependent, Rapid Structural Changes in Hippocampal Pyramidal Cell Spines Cerebral Cortex (New York, N.Y. : 1991). Nov, 2009 | Pubmed ID: 19240139 Morphological changes in dendritic spines may contribute to the fine tuning of neural network connectivity. The relationship between spine morphology and experience-dependent neuronal activity, however, is largely unknown. In the present study, we combined 2 histological analyses to examine this relationship: 1) Measurement of spines of neurons whose morphology was visualized in brain sections of mice expressing membrane-targeted green fluorescent protein (Thy1-mGFP mice) and 2) Categorization of CA1 neurons by immunohistochemical monitoring of Arc expression as a putative marker of recent neuronal activity. After mice were exposed to a novel, enriched environment for 60 min, neurons that expressed Arc had fewer small spines and more large spines than Arc-negative cells. These differences were not observed when the exploration time was shortened to 15 min. This net-balanced structural change is consistent with both synapse-specific enhancement and suppression. These results provide the first evidence of rapid morphological changes in spines that were preferential to a subset of neurons in association with an animal's experiences.

Influence of Brain-derived Neurotrophic Factor on Pathfinding of Dentate Granule Cell Axons, the Hippocampal Mossy Fibers Molecular Brain. Jan, 2009 | Pubmed ID: 19183490 Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.

Regional Difference in Stainability with Calcium-sensitive Acetoxymethyl-ester Probes in Mouse Brain Slices The International Journal of Neuroscience. 2009 | Pubmed ID: 19125375 Loading neurons with membrane permeable Ca2+ indicators is a core experimental procedure in functional multineuron Ca2+ imaging (fMCI), an optical technique for monitoring multiple neuronal activities. Although fMCI has been applied to several brain networks, including cerebral cortex, hippocampus, and cerebellum, no studies have systematically addressed the dye-loading efficiency in different brain regions. Here, we describe the stainability of Oregon Green 488 BAPTA-1AM in mouse acute brain slice preparations. The data are suggestive of the potential usability of fMCI in many brain regions, including olfactory bulb, thalamus, dentate gyrus, habenular nucleus, and pons.

Current Application and Technology of Functional Multineuron Calcium Imaging Biological & Pharmaceutical Bulletin. Jan, 2009 | Pubmed ID: 19122272 Here we describe recent applications and technical advancements of functional multineuron calcium imaging (fMCI), which monitors the firing activity of more than a thousand neurons through their somatic Ca(2+) signals. fMCI is used for analysis of various neural circuits under normal and pathological conditions. In vitro fMCI is made more sophisticated by using multipoint illumination and scanning technology with spinning-disk and low-laser-intensity imaging, electron-multiplying charge-coupled device cameras, etc. In vivo fMCI is still developing. We review optical technologies for fast scanning imaging, deep tissue imaging, and recording from moving animals.

Rapid and Local Autoregulation of Cerebrovascular Blood Flow: a Deep-brain Imaging Study in the Mouse The Journal of Physiology. Feb, 2009 | Pubmed ID: 19074968 The brain obtains energy by keeping the cerebral blood flow constant against unexpected changes in systemic blood pressure. Although this homeostatic mechanism is widely known as cerebrovascular autoregulation, it is not understood how widely and how robustly it works in the brain. Using a needle-like objective lens designed for deep-tissue imaging, we quantified the degree of autoregulation in the mouse hippocampus with single-capillary resolution. On average, hippocampal blood flow exhibited autoregulation over a comparatively broad range of arterial blood pressure and did not significantly respond to pressure changes induced by the pharmacological activation of autonomic nervous system receptors, whereas peripheral tissues showed linear blood flow changes. At the level of individual capillaries, however, about 40% of hippocampal capillaries did not undergo rapid autoregulation. This heterogeneity suggests the presence of a local baroreflex system to implement cerebral autoregulation.

Messages in Spike Timing-dependent Plasticity: Pros and Cons The Chinese Journal of Physiology. Dec, 2010 | Pubmed ID: 21793347 Spike-timing dependent plasticity (STDP), a synaptic modification depending on a relative timing of presynaptic and postsynaptic spikes, has fascinated researchers in the fields of neurophysiology and computational neuroscience, because it is not only conceptually simple or biologically reasonable but is also versatile in neural network simulations. The STDP rule may be valid only under specific conditions, however. We propose herein a method that could find more natural and potent rules of synaptic plasticity.

Return to Forever: Finding the Origin of Neural Synchrony Communicative & Integrative Biology. Nov, 2010 | Pubmed ID: 21331241 Synchronized spikes prevail in cortical networks, and their modulations are associated with attention, sensory processing and motor behaviors, yet it remained unclear how spikes can synchronize at the millisecond precision in a noisy network. Our new evidence shows that neurons do not easily synchronize; rather, in order to make them generate synchronous spikes, "parent" presynaptic neurons commonly shared by them are required to synchronize in advance. Therefore, the conventional style of neurophysiological research is unable to reach the true origin of synchronization.

Analysis and Modeling of Massively Parallel Neural Signals--2010 Special Issue Neural Networks : the Official Journal of the International Neural Network Society. Aug, 2010 | Pubmed ID: 20538425

Circuit Topology for Synchronizing Neurons in Spontaneously Active Networks Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010 | Pubmed ID: 20479225 Spike synchronization underlies information processing and storage in the brain. But how can neurons synchronize in a noisy network? By exploiting a high-speed (500-2,000 fps) multineuron imaging technique and a large-scale synapse mapping method, we directly compared spontaneous activity patterns and anatomical connectivity in hippocampal CA3 networks ex vivo. As compared to unconnected pairs, synaptically coupled neurons shared more common presynaptic neurons, received more correlated excitatory synaptic inputs, and emitted synchronized spikes with approximately 10(7) times higher probability. Importantly, common presynaptic parents per se synchronized more than unshared upstream neurons. Consistent with this, dynamic-clamp stimulation revealed that common inputs alone could not account for the realistic degree of synchronization unless presynaptic spikes synchronized among common parents. On a macroscopic scale, network activity was coordinated by a power-law scaling of synchronization, which engaged varying sets of densely interwired (thus highly synchronized) neuron groups. Thus, locally coherent activity converges on specific cell assemblies, thereby yielding complex ensemble dynamics. These segmentally synchronized pulse packets may serve as information modules that flow in associatively parallel network channels.

Scale-free Topology of the CA3 Hippocampal Network: a Novel Method to Analyze Functional Neuronal Assemblies Biophysical Journal. May, 2010 | Pubmed ID: 20441736 Cognitive mapping functions of the hippocampus critically depend on the recurrent network of the CA3 pyramidal cells. However, it is still not known in detail how network activity patterns emerge, or how they encode information. By using functional multineuron calcium imaging, we simultaneously recorded the activity of >100 neurons in the CA3 region of hippocampal slice cultures. We utilized a novel computational method to analyze the multichannel spike trains and to depict functional neuronal assemblies. By means of event synchronization and the correlation matrix analysis method, we found that: 1), the average functional neuronal cluster consists of 23 neurons, and neurons could be part of multiple assemblies; 2), the clustering strength, size, and mean distance among cells in neuronal assemblies follow a power-law-like distribution; 3), the clustering strength and size of neuronal assemblies are not correlated with the total number of neurons and their physical distance; and 4), the clustering distance of neuronal assemblies is weakly correlated with the total number of neurons and their physical distance. These findings suggest that the functional organization of the spontaneously firing CA3 hippocampal network is a scale-free structure in slice culture.

Functional Multineuron Calcium Imaging for Systems Pharmacology Analytical and Bioanalytical Chemistry. Sep, 2010 | Pubmed ID: 20437032 Functional multineuron calcium imaging (fMCI) is a large-scale technique used to access brain function on a single-neuron scale. It detects the activity of individual neurons by imaging action potential-evoked transient calcium influxes into their cell bodies. fMCI has recently been used as a high-throughput research tool to examine how neuronal activity is altered in animal models of brain diseases, for example stroke, Alzheimer's disease, and epilepsy, and to estimate how pharmacological agents act on normal and abnormal states of neuronal networks. It offers unique opportunities to discover the mechanisms underlying neurological disorders and new therapeutic targets.

Novel Etiological and Therapeutic Strategies for Neurodiseases: Preface Journal of Pharmacological Sciences. 2010 | Pubmed ID: 20424391

Fluorescent Pipettes for Optically Targeted Patch-clamp Recordings Neural Networks : the Official Journal of the International Neural Network Society. Aug, 2010 | Pubmed ID: 20223634 Targeted patch-clamp recordings are a promising technique that can directly address the physiological properties of a specific neuron embedded in a neuronal network. Typically, neurons are visualized through fluorescent dyes or fluorescent proteins with fluorescence microscopy. After switching to transmitted light microscopy, neurons of interest are re-identified and visually approached in situ with patch-clamp pipettes. Here we introduce a simpler method for neuron targeting. With fluorophore-coated pipettes, fluorescently labeled neurons and the pipette tips are simultaneously imaged at the same fluorescence wavelength in the same microscope field, so that the neurons and even their neurites are targeted without suffering from chromatic aberration or mechanical complication in optics. We did not find that the coated fluorophores affected the electric properties of pipettes or neurons. The novel technique will be wildly available for pipette micromanipulation under online visual control.

High-speed Multineuron Calcium Imaging Using Nipkow-type Confocal Microscopy Current Protocols in Neuroscience. Oct, 2011 | Pubmed ID: 21971847 Conventional confocal and two-photon microscopy scan the field of view sequentially with single-point laser illumination. This raster-scanning method constrains video speeds to tens of frames per second, which are too slow to capture the temporal patterns of fast electrical events initiated by neurons. Nipkow-type spinning-disk confocal microscopy resolves this problem by the use of multiple laser beams. We describe experimental procedures for functional multineuron calcium imaging (fMCI) based on Nipkow-disk confocal microscopy, which enables us to monitor the activities of hundreds of neurons en masse at a cellular resolution at up to 2000 fps.

Hippocampal Polysynaptic Computation The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2011 | Pubmed ID: 21917800 Neural circuitry is a self-organizing arithmetic device that converts input to output and thereby remodels its computational algorithm to produce more desired output; however, experimental evidence regarding the mechanism by which information is modified and stored while propagating across polysynaptic networks is sparse. We used functional multineuron calcium imaging to monitor the spike outputs from thousands of CA1 neurons in response to the stimulation of two independent sites of the dentate gyrus in rat hippocampal networks ex vivo. Only pyramidal cells were analyzed based on post hoc immunostaining. Some CA1 pyramidal cells were observed to fire action potentials only when both sites were simultaneously stimulated (AND-like neurons), whereas other neurons fired in response to either site of stimulation but not to concurrent stimulation (XOR-like neurons). Both types of neurons were interlaced in the same network and altered their logical operation depending on the timing of paired stimulation. Repetitive paired stimulation for brief periods induced a persistent reorganization of AND and XOR operators, suggesting a flexibility in parallel distributed processing. We simulated these network functions in silico and found that synaptic modification of the CA3 recurrent excitation is pivotal to the shaping of logic plasticity. This work provides new insights into how microscopic synaptic properties are associated with the mesoscopic dynamics of complex microcircuits.

Development of a Far-red to Near-infrared Fluorescence Probe for Calcium Ion and Its Application to Multicolor Neuronal Imaging Journal of the American Chemical Society. Sep, 2011 | Pubmed ID: 21827169 To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.

Thoracotomy Reduces Intrinsic Brain Movement Caused by Heartbeat and Respiration: a Simple Method to Prevent Motion Artifact for in Vivo Experiments Neuroscience Research. Oct, 2011 | Pubmed ID: 21787813 Recent technical advances in electrophysiological recording and functional imaging from the brain of living animals have promoted our understandings of the brain function, but these in vivo experiments are still technically demanding and often suffer from spontaneous pulsation, i.e., brain movements caused by respiration and heartbeat. Here we report that thoracotomy suppresses the motion artifact to a practically negligible level. This simple method will be useful in a wide variety of in vivo experiments, such as patch-clamp physiology, and optical imaging of neurons, glial cell, and blood vessels.

Intact Internal Dynamics of the Neocortex in Acutely Paralyzed Mice The Journal of Physiological Sciences : JPS. Jul, 2011 | Pubmed ID: 21633910 Animals collect sensory information through self-generated movements. Muscle movements drive active feedback of sensory information and determine large parts of the sensory inputs the animal receives; however, little is known about how this active feedback process modulates the ongoing dynamics of the brain. We made electrophysiological recordings from layer 2/3 neurons of the mouse neocortex and compared spontaneous cortical activity in local field potentials and intracellular potential fluctuations between normal and hypomyotonic conditions. We found that pancuronium-induced paralysis did not affect the electrophysiological properties of ongoing cortical activity and its perturbation evoked by visual and tactile stimuli. Thus, internal cortical dynamics are not much affected by active muscle movements, at least, in an acute phase.

Asynchronously Enhanced Spiking Activity of Ischemic Neuronal Networks Biological & Pharmaceutical Bulletin. 2011 | Pubmed ID: 21532170 Cerebral ischemia causes the depletion of oxygen and nutrition from brain tissues, and when persistent, results in irreversible damage to the cell function and survival. The cellular response to ischemic conditions and its mechanisms have been investigated widely in in vivo and in vitro experimental models, yet no study has addressed the response of a whole neuronal network to energy deprivation with the single-cell resolution. Observations at the level of network are necessary, because the activity of individual neurons is nonlinearly integrated through a network and thereby gives rise to unexpectedly complex dynamics. Here we used functional multineuron calcium imaging (fMCI), an optical recording technique with high temporal and spatial resolution, to visualize the activity of neuron populations in hippocampus CA1 region under ischemia-like conditions ex vivo. We found that, although neurons responded to oxygen and glucose deprivation with an increase in the event frequency, they maintained an asynchronous network state. This is in contrast with other well known pathological states, in which the network hyperexcitability is usually accompanied by an increase in synchrony. We suggest that under ischemic conditions, at least to some time point, the neuronal network maintains the excitatory and inhibitory balance as a whole, whether actively or as a consequence of the cellular response to energy deprivation.

Nipkow Confocal Imaging from Deep Brain Tissues Journal of Integrative Neuroscience. Mar, 2011 | Pubmed ID: 21425486 One of the problems in imaging from brain tissues is light-scattering. Thus, multiphoton laser scanning microscopy is widely used to optically access fluorescent signals located deeply in tissues. Here we report that Nipkow-type spinning-disk one-photon confocal microscopy, which embodies high temporal resolution and slow photobleaching, is also capable of imaging tissues to a depth of up to 150 μm. Using a Nipkow-disk microscope, we conducted functional multi-cell calcium imaging of CA3 neurons from in toto intact hippocampal preparations and astrocytes from in vivo neocortical layer 1. This novel application of Nipkow-disk microscopy expands the potential usefulness of this type of microscopy and will contribute to our understanding of natural neuronal microcircuitry.

Large-scale Calcium Waves Traveling Through Astrocytic Networks in Vivo The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2011 | Pubmed ID: 21325528 Macroscopic changes in cerebral blood flow, such as those captured by functional imaging of the brain, require highly organized, large-scale dynamics of astrocytes, glial cells that interact with both neuronal and cerebrovascular networks. However, astrocyte activity has been studied mainly at the level of individual cells, and information regarding their collective behavior is lacking. In this work, we monitored calcium activity simultaneously from hundreds of mouse hippocampal astrocytes in vivo and found that almost all astrocytes participated en masse in regenerative waves that propagated from cell to cell (referred to here as "glissandi"). Glissandi emerged depending on the neuronal activity and accompanied a reduction in infraslow fluctuations of local field potentials and a decrease in the flow of red blood cells. This novel phenomenon was heretofore overlooked, probably because of the high vulnerability of astrocytes to light damage; glissandi occurred only when observed at much lower laser intensities than previously used.

Action-potential Modulation During Axonal Conduction Science (New York, N.Y.). Feb, 2011 | Pubmed ID: 21292979 Once initiated near the soma, an action potential (AP) is thought to propagate autoregeneratively and distribute uniformly over axonal arbors. We challenge this classic view by showing that APs are subject to waveform modulation while they travel down axons. Using fluorescent patch-clamp pipettes, we recorded APs from axon branches of hippocampal CA3 pyramidal neurons ex vivo. The waveforms of axonal APs increased in width in response to the local application of glutamate and an adenosine A(1) receptor antagonist to the axon shafts, but not to other unrelated axon branches. Uncaging of calcium in periaxonal astrocytes caused AP broadening through ionotropic glutamate receptor activation. The broadened APs triggered larger calcium elevations in presynaptic boutons and facilitated synaptic transmission to postsynaptic neurons. This local AP modification may enable axonal computation through the geometry of axon wiring.

Locally Synchronized Astrocytes Cerebral Cortex (New York, N.Y. : 1991). Aug, 2011 | Pubmed ID: 21212170 Astrocytes exhibit spontaneous calcium fluctuations. These activities have not been captured by large-scale recordings, and little is known about their collective dynamics. In situ and in vivo calcium imaging from hundreds (up to 2195) of astrocytes in the mouse hippocampus and neocortex revealed that neighboring astrocytes spontaneously exhibited synchronous calcium elevations and formed locally correlated cell groups ("clusters" of 2 to 5 astrocytes within a diameter of 81 ± 45 μm). Cluster activity accounted for approximately 10% of the astrocytic calcium events, and 44% of the clusters appeared repetitively during our observation period of 60 min. Astrocytic clusters emerged through metabotropic glutamate receptor activation, independently of neuronal activity. Neurons were depolarized by 1.5 mV when clusters appeared near their dendrites. This depolarization was mediated by non-N-methyl-D-aspartate (NMDA) glutamate receptor channels and was replicated by calcium uncaging activation of multiple astrocytes. Importantly, the activation of single astrocytes alone could not depolarize neurons but readily elicited NMDA-dependent slow inward currents in depolarized neurons. Thus, these novel ensemble dynamics of astrocytes, which cannot be captured by conventional small-scale imaging techniques, play a different role in neuronal modulation than does the sporadic activity of single astrocytes.

Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals PloS One. 2012 | Pubmed ID: 23240011 Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.

An Improved Genetically Encoded Red Fluorescent Ca2+ Indicator for Detecting Optically Evoked Action Potentials PloS One. 2012 | Pubmed ID: 22808076 Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.

GABAergic Excitation After Febrile Seizures Induces Ectopic Granule Cells and Adult Epilepsy Nature Medicine. Aug, 2012 | Pubmed ID: 22797810 Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

[Real Time Imaging of Synaptic Inputs] Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Jul, 2012 | Pubmed ID: 22790228

Preinspiratory Calcium Rise in Putative Pre-Botzinger Complex Astrocytes The Journal of Physiology. Oct, 2012 | Pubmed ID: 22777672 The neural inspiratory activity originates from a ventrolateral medullary region called the pre-Bötzinger complex (preBötC), yet the mechanism underlying respiratory rhythmogenesis is not completely understood. Recently, the role of not only neurons but astrocytes in the central respiratory control has attracted considerable attention. Here we report our discovery that an intracellular calcium rise in a subset of putative astrocytes precedes inspiratory neuronal firing in rhythmically active slices. Functional calcium imaging from hundreds of preBötC cells revealed that a subset of putative astrocytes exhibited rhythmic calcium elevations preceding inspiratory neuronal activity with a time lag of approximately 2 s. These preinspiratory putative astrocytes maintained their rhythmic activities even during the blockade of neuronal activity with tetrodotoxin, whereas the rhythm frequency was lowered and the intercellular phases of these rhythms were decoupled. In addition, optogenetic stimulation of preBötC putative astrocytes induced firing of inspiratory neurons. These findings raise the possibility that astrocytes in the preBötC are actively involved in respiratory rhythm generation in rhythmically active slices.

Heterogeneity and Independency of Unitary Synaptic Outputs from Hippocampal CA3 Pyramidal Cells The Journal of Physiology. Oct, 2012 | Pubmed ID: 22733657 The variation of individual synaptic transmission impacts the dynamics of complex neural circuits. We performed whole-cell recordings from monosynaptically connected hippocampal neurons in rat organotypic slice cultures using a synapse mapping method. The amplitude of unitary excitatory postsynaptic current (uEPSC) varied from trial to trial and was independent of the physical distance between cell pairs. To investigate the source of the transmission variability, we obtained patch-clamp recordings from intact axons. Axonal action potentials (APs) were reliably transmitted throughout the axonal arbour and showed modest changes in width. In contrast, calcium imaging from presynaptic boutons revealed that the amplitude of AP-evoked calcium transients exhibited large variations both among different boutons at a given trial and among trials in a given bouton. These results suggest that a factor contributing to the uEPSC fluctuations is the variability in calcium dynamics at presynaptic terminals. Finally, we acquired triple whole-cell recordings from divergent circuit motifs with one presynaptic neuron projecting to two postsynaptic neurons. Consistent with the independency of calcium dynamics among axonal boutons, a series of uEPSC fluctuations was not correlated between the two postsynaptic cells, indicating that different synapses even from the same neuron act independently.We conclude that the intra-bouton and inter-bouton variability in AP-induced calcium dynamics determine the heterogeneity and independency of uEPSCs.

Targeted Axon-attached Recording with Fluorescent Patch-clamp Pipettes in Brain Slices Nature Protocols. May, 2012 | Pubmed ID: 22653161 Understanding the physiology of axons in the central nervous system requires experimental access to intact axons. This protocol describes how to perform cell-attached recordings from narrow axon fibers (ϕ

Muscarinic Receptor Activation Disrupts Hippocampal Sharp Wave-ripples Brain Research. Jun, 2012 | Pubmed ID: 22608077 Cholinergic muscarinic innervations to the hippocampus play a role in learning and memory. Here we report that pharmacological activation of muscarinic receptors eliminates sharp wave-ripple events in the mouse hippocampal CA1 region in vivo and in vitro. This effect was associated with a decorrelation of excitatory synaptic inputs and a net increase in inhibitory conductances in pyramidal neurons. Multineuron calcium imaging revealed that muscarinic activation altered the spatiotemporal pattern of network activities. Thus, cholinergic input is likely to contribute to a neuromodulatory switch of hippocampal network states, as proposed in the "two-stage" model of learning processes.

Normal Learning Ability of Mice with a Surgically Exposed Hippocampus Neuroreport. May, 2012 | Pubmed ID: 22495001 In rats and mice, the hippocampus lies beneath higher than 1 mm of the neocortex. This anatomical feature makes it difficult to experimentally access the hippocampus from the surface of the brain in vivo. This problem may be solved by surgical removal of the cortical tissue above the hippocampus; however, it has not been examined whether this 'hippocampal window' surgery preserves the normal hippocampal function. We bilaterally aspirated the posterior parietal cortex above the dorsal hippocampus of adult male mice. These mice still exhibited normal local field potentials of the hippocampus, normal motor activity, and normal cognitive ability in the water-maze test and contextual fear conditioning, compared with intact or sham-operated controls. Thus, exposed hippocampal preparations provide a useful experimental model to study the physiology of the hippocampus.

Effects of Axonal Topology on the Somatic Modulation of Synaptic Outputs The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2012 | Pubmed ID: 22357869 Depolarization of the neuronal soma augments synaptic output onto postsynaptic neurons via long-range, axonal cable properties. Here, we report that the range of this somatic influence is spatially restricted by not only axonal path length but also a branching-dependent decrease in axon diameter. Cell-attached recordings of action potentials (APs) from multiple axon branches of a rat hippocampal CA3 pyramidal cell revealed that an AP was broadened following a 20 mV depolarization of the soma and reverted to a normal width during propagation down the axon. The narrowing of the AP depended on the distance traveled by the AP and on the number of axon branch points through which the AP passed. These findings were confirmed by optical imaging of AP-induced calcium elevations in presynaptic boutons, suggesting that the somatic membrane potential modifies synaptic outputs near the soma but not long-projection outputs. Consistent with this prediction, whole-cell recordings from synaptically connected neurons revealed that depolarization of presynaptic CA3 pyramidal cells facilitated synaptic transmission to nearby CA3 pyramidal cells, but not to distant pyramidal cells in CA3 or CA1. Therefore, axonal geometry enables the differential modulation of synaptic output depending on target location.

Synchronized Spike Waves in Immature Dentate Gyrus Networks The European Journal of Neuroscience. Mar, 2012 | Pubmed ID: 22332872 At early developmental stages, immature neuronal networks of the neocortex and hippocampus spontaneously exhibit synchronously oscillating activities, which are believed to play roles in normal circuit maturation. The tissue development of the dentate gyrus (DG) in the hippocampal formation is exceptionally late compared with other brain regions and persists until postnatal periods. Using patch-clamp recording and functional multineuron calcium imaging, we found that the DG networks of postnatal day (P)3-7 mice spontaneously generated traveling waves of action potentials, which were initiated at the upper blade of the granule cell layer and propagated to the lower blade. The propagation was dependent on glutamatergic and electrical synapses, but not on GABAergic receptor activity. Remarkably, the DG waves were almost completely abolished in offspring born to female rats exposed to restraint stress during pregnancy. In the prenatally stressed offspring, DG granule cell dendrites developed normally until P3 and showed atrophy by P9. Thus, the DG waves may be required for the maturation of DG granule cells.

Cannabinoid Receptor Activation Disrupts the Internal Structure of Hippocampal Sharp Wave-ripple Complexes Journal of Pharmacological Sciences. 2012 | Pubmed ID: 22293299 Cannabinoid agonists impair hippocampus-dependent learning and memory. Using mouse hippocampal slice preparations, we examined the effect of anandamide, an endogenous cannabinoid, on sharp wave-ripple (SW-R) complexes, which are believed to mediate memory consolidation during slow-wave sleep or behavioral immobility. Anandamide reduced the frequency of SW-Rs recorded from the CA3 region, and this effect was abolished by AM251, a cannabinoid CB1-receptor antagonist. We further addressed the action of anandamide using a functional multineuron calcium imaging technique. Anandamide reduced the firing rate of hippocampal neurons as well as disrupted the temporal coordination of their firings during SW-R.

Locally Synchronized Synaptic Inputs Science (New York, N.Y.). Jan, 2012 | Pubmed ID: 22267814 Synaptic inputs on dendrites are nonlinearly converted to action potential outputs, yet the spatiotemporal patterns of dendritic activation remain to be elucidated at single-synapse resolution. In rodents, we optically imaged synaptic activities from hundreds of dendritic spines in hippocampal and neocortical pyramidal neurons ex vivo and in vivo. Adjacent spines were frequently synchronized in spontaneously active networks, thereby forming dendritic foci that received locally convergent inputs from presynaptic cell assemblies. This precise subcellular geometry manifested itself during N-methyl-D-aspartate receptor-dependent circuit remodeling. Thus, clustered synaptic plasticity is innately programmed to compartmentalize correlated inputs along dendrites and may reify nonlinear synaptic integration.

[Toward a New Era of Pharmacological Therapeutics] Yakugaku Zasshi : Journal of the Pharmaceutical Society of Japan. 2013 | Pubmed ID: 24292180

The Accuracy and Precision of Signal Source Localization with Tetrodes Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference. 2013 | Pubmed ID: 24109741 Four-sensor microelectrodes, commonly referred to as tetrodes, have the ability to significantly increase the signal-to-noise ratio of neuronal extracellular recordings. They also provide spatio-temporal information about extracellular action potentials (EAP) which may be used to localize and resolve individual neuronal signal sources. Since the relative position of sensors and neurons whose EAPs are recorded is not known during in vivo experiments, the accuracy and precision of neuronal source localization algorithms remain untested. In this study, electrical signals generated by a stimulator were recorded simultaneously with four recording micropipettes immersed in artificial cerebrospinal fluid. The location of the source was estimated using the multiple signal classification algorithm, with an accuracy and precision of ~4 µm and ~7 µm, respectively. These results suggest that in vivo localization and resolution of individual neuronal sources is feasible.

Subicular Activation Preceding Hippocampal Ripples in Vitro Scientific Reports. 2013 | Pubmed ID: 24045268 Sharp wave-ripple complexes (SW-Rs), a transient form of high-frequency field oscillations observed in the hippocampus, are thought to mediate memory consolidation. They are initiated mainly in hippocampal CA3 area and propagate to the entorhinal cortex through the subiculum; however, little is known about how SW-Rs are initiated and propagate. Here, we used functional multineuronal calcium imaging to monitor SW-R-relevant neuronal activity from the subiculum at single-cell resolution. An unexpected finding was that a subset of subicular neurons was activated immediately before hippocampal SW-Rs. The SW-R-preceding activity was not abolished by surgical lesion of the CA1-to-subiculum projection, and thus, it probably arose from entorhinal inputs. Therefore, SW-Rs are likely to be triggered by entorhinal-to-CA3/CA1 inputs. Moreover, the subiculum is not merely a passive intermediate region that SW-Rs pass through, but rather, it seems to contribute to an active modification of neural information related to SW-Rs.

[Mechanisms of Memory Bridging Past and Present] Brain and Nerve = Shinkei Kenkyu No Shinpo. Aug, 2013 | Pubmed ID: 23917495 The human mind develops through history within the passing of time. Thus, what determines the passing of time in the human mind? For example, when you are asked about yourself 10 years before now, you are able to answer by tracing back through your own experiences. You will be confident of your answer as far as you rely on your memory. Therefore, your personal memory is critical for the passage of time; however, how memory that allows for mental time travel is formed or maintained in the brain is largely unknown. This type of memory may exist only in humans. In this article, we review past studies on memories that emerge from time information in human and experimental animals.

Multineuronal Spike Sequences Repeat with Millisecond Precision Frontiers in Neural Circuits. 2013 | Pubmed ID: 23801942 Cortical microcircuits are nonrandomly wired by neurons. As a natural consequence, spikes emitted by microcircuits are also nonrandomly patterned in time and space. One of the prominent spike organizations is a repetition of fixed patterns of spike series across multiple neurons. However, several questions remain unsolved, including how precisely spike sequences repeat, how the sequences are spatially organized, how many neurons participate in sequences, and how different sequences are functionally linked. To address these questions, we monitored spontaneous spikes of hippocampal CA3 neurons ex vivo using a high-speed functional multineuron calcium imaging (fMCI) technique that allowed us to monitor spikes with millisecond resolution and to record the location of spiking and non-spiking neurons. Multineuronal spike sequences (MSSs) were overrepresented in spontaneous activity compared to the statistical chance level. Approximately 75% of neurons participated in at least one sequence during our observation period. The participants were sparsely dispersed and did not show specific spatial organization. The number of sequences relative to the chance level decreased when larger time frames were used to detect sequences. Thus, sequences were precise at the millisecond level. Sequences often shared common spikes with other sequences; parts of sequences were subsequently relayed by following sequences, generating complex chains of multiple sequences.

Novel Contribution of Cell Surface and Intracellular M1-muscarinic Acetylcholine Receptors to Synaptic Plasticity in Hippocampus Journal of Neurochemistry. Aug, 2013 | Pubmed ID: 23678982 Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1-mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand-binding experiments with cell-permeable and -impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1-mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic-binding sites were abolished in M1-mAChR-gene-knockout mice. Activation of cell surface M1-mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long-term potentiation. On the other hand, activation of intracellular M1-mAChRs phosphorylated extracellular-regulated kinase 1/2 and gradually enhanced long-term potentiation. Our data thus demonstrate that M1-mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.

Red Fluorescent Probe for Monitoring the Dynamics of Cytoplasmic Calcium Ions Angewandte Chemie (International Ed. in English). Apr, 2013 | Pubmed ID: 23440861

Layer III Neurons Control Synchronized Waves in the Immature Cerebral Cortex The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2013 | Pubmed ID: 23325237 Correlated spiking activity prevails in immature cortical networks and is believed to contribute to neuronal circuit maturation; however, its spatiotemporal organization is not fully understood. Using wide-field calcium imaging from acute whole-brain slices of rat pups on postnatal days 1-6, we found that correlated spikes were initiated in the anterior part of the lateral entorhinal cortex and propagated anteriorly to the frontal cortex and posteriorly to the medial entorhinal cortex, forming traveling waves that engaged almost the entire cortex. The waves were blocked by ionotropic glutamatergic receptor antagonists but not by GABAergic receptor antagonists. During wave events, glutamatergic and GABAergic synaptic inputs were balanced and induced UP state-like depolarization. Magnified monitoring with cellular resolution revealed that the layer III neurons were first activated when the waves were initiated. Consistent with this finding, layer III contained a larger number of neurons that were autonomously active, even under a blockade of synaptic transmission. During wave propagation, the layer III neurons constituted a leading front of the wave. The waves did not enter the parasubiculum; however, in some cases, they were reflected at the parasubicular border and propagated back in the opposite direction. During this reflection process, the layer III neurons in the medial entorhinal cortex maintained persistent activity. Thus, our data emphasize the role of layer III in early network behaviors and provide insight into the circuit mechanisms through which cerebral cortical networks maturate.

Interpyramid Spike Transmission Stabilizes the Sparseness of Recurrent Network Activity Cerebral Cortex (New York, N.Y. : 1991). Feb, 2013 | Pubmed ID: 22314044 Cortical synaptic strengths vary substantially from synapse to synapse and exhibit a skewed distribution with a small fraction of synapses generating extremely large depolarizations. Using multiple whole-cell recordings from rat hippocampal CA3 pyramidal cells, we found that the amplitude of unitary excitatory postsynaptic conductances approximates a lognormal distribution and that in the presence of synaptic background noise, the strongest fraction of synapses could trigger action potentials in postsynaptic neurons even with single presynaptic action potentials, a phenomenon termed interpyramid spike transmission (IpST). The IpST probability reached 80%, depending on the network state. To examine how IpST impacts network dynamics, we simulated a recurrent neural network embedded with a few potent synapses. This network, unlike many classical neural networks, exhibited distinctive behaviors resembling cortical network activity in vivo. These behaviors included the following: 1) infrequent ongoing activity, 2) firing rates of individual neurons approximating a lognormal distribution, 3) asynchronous spikes among neurons, 4) net balance between excitation and inhibition, 5) network activity patterns that was robust against external perturbation, 6) responsiveness even to a single spike of a single excitatory neuron, and 7) precise firing sequences. Thus, IpST captures a surprising number of recent experimental findings in vivo. We propose that an unequally biased distribution with a few select strong synapses helps stabilize sparse neuronal activity, thereby reducing the total spiking cost, enhancing the circuit responsiveness, and ensuring reliable information transfer.

A Statistical Method of Identifying Interactions in Neuron-glia Systems Based on Functional Multicell Ca2+ Imaging PLoS Computational Biology. Nov, 2014 | Pubmed ID: 25393874 Crosstalk between neurons and glia may constitute a significant part of information processing in the brain. We present a novel method of statistically identifying interactions in a neuron-glia network. We attempted to identify neuron-glia interactions from neuronal and glial activities via maximum-a-posteriori (MAP)-based parameter estimation by developing a generalized linear model (GLM) of a neuron-glia network. The interactions in our interest included functional connectivity and response functions. We evaluated the cross-validated likelihood of GLMs that resulted from the addition or removal of connections to confirm the existence of specific neuron-to-glia or glia-to-neuron connections. We only accepted addition or removal when the modification improved the cross-validated likelihood. We applied the method to a high-throughput, multicellular in vitro Ca2+ imaging dataset obtained from the CA3 region of a rat hippocampus, and then evaluated the reliability of connectivity estimates using a statistical test based on a surrogate method. Our findings based on the estimated connectivity were in good agreement with currently available physiological knowledge, suggesting our method can elucidate undiscovered functions of neuron-glia systems.

Sound-induced Modulation of Hippocampal θ Oscillations Neuroreport. Dec, 2014 | Pubmed ID: 25304497 The mechanism of response of hippocampal neurons to a specific feature in sensory stimuli is not fully understood, although the hippocampus is well known to contribute to the formation of episodic memory in the multisensory world. Using in-vivo voltage-clamp recordings from awake mice, we found that sound pulses induced a transient increase in inhibitory, but not excitatory, conductance in hippocampal CA1 pyramidal cells. In local field potentials, sound pulses induced a phase resetting of the θ oscillations, one of the major oscillatory states of the hippocampus. Repetitive sound pulses at 7 Hz (θ rhythm) increased the θ oscillation power, an effect that was abolished by a surgical fimbria-fornix lesion. Thus, tone-induced inhibition is likely of subcortical origin. It may segment hippocampal neural processing and render temporal boundaries in continuously ongoing experiences.

Ex Vivo Cultured Neuronal Networks Emit in Vivo-like Spontaneous Activity The Journal of Physiological Sciences : JPS. Nov, 2014 | Pubmed ID: 25208897 Spontaneous neuronal activity is present in virtually all brain regions, but neither its function nor spatiotemporal patterns are fully understood. Ex vivo organotypic slice cultures may offer an opportunity to investigate some aspects of spontaneous activity, because they self-restore their networks that collapsed during slicing procedures. In hippocampal networks, we compared the levels and patterns of in vivo spontaneous activity to those in acute and cultured slices. We found that the firing rates and excitatory synaptic activity in the in vivo hippocampus are more similar to those in slice cultures compared to acute slices. The soft confidence-weighted algorithm, a machine learning technique without human bias, also revealed that hippocampal slice cultures resemble the in vivo hippocampus in terms of the overall tendency of the parameters of spontaneous activity.

Dopamine Receptor Activation Reorganizes Neuronal Ensembles During Hippocampal Sharp Waves in Vitro PloS One. 2014 | Pubmed ID: 25089705 Hippocampal sharp wave (SW)/ripple complexes are thought to contribute to memory consolidation. Previous studies suggest that behavioral rewards facilitate SW occurrence in vivo. However, little is known about the precise mechanism underlying this enhancement. Here, we examined the effect of dopaminergic neuromodulation on spontaneously occurring SWs in acute hippocampal slices. Local field potentials were recorded from the CA1 region. A brief (1 min) treatment with dopamine led to a persistent increase in the event frequency and the magnitude of SWs. This effect lasted at least for our recording period of 45 min and did not occur in the presence of a dopamine D1/D5 receptor antagonist. Functional multineuron calcium imaging revealed that dopamine-induced SW augmentation was associated with an enriched repertoire of the firing patterns in SW events, whereas the overall tendency of individual neurons to participate in SWs and the mean number of cells participating in a single SW were maintained. Therefore, dopaminergic activation is likely to reorganize cell assemblies during SWs.

Sound-induced Hyperpolarization of Hippocampal Neurons Neuroreport. Sep, 2014 | Pubmed ID: 25050474 The hippocampus is involved in episodic memory, which is composed of subjective experiences in the multisensory world; however, little is known about the subthreshold membrane potential responses of individual hippocampal neurons to sensory stimuli. Using in-vivo whole-cell patch-clamp recordings from hippocampal CA1 neurons in awake mice, we found that almost all hippocampal neurons exhibited a hyperpolarization of 1-2 mV immediately after the onset of a sound. This large-scale hyperpolarization was unaffected by the duration or pitch of the tone. The response was abolished by general anesthesia and a surgical fimbria-fornix lesion.

Synaptic Plasticity Associated with a Memory Engram in the Basolateral Amygdala The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2014 | Pubmed ID: 25009263 Synaptic plasticity is a cellular mechanism putatively underlying learning and memory. However, it is unclear whether learning induces synaptic modification globally or only in a subset of neurons in associated brain regions. In this study, we genetically identified neurons activated during contextual fear learning and separately recorded synaptic efficacy from recruited and nonrecruited neurons in the mouse basolateral amygdala (BLA). We found that the fear learning induces presynaptic potentiation, which was reflected by an increase in the miniature EPSC frequency and by a decrease in the paired-pulse ratio. Changes occurred only in the cortical synapses targeting the BLA neurons that were recruited into the fear memory trace. Furthermore, we found that fear learning reorganizes the neuronal ensemble responsive to the conditioning context in conjunction with the synaptic plasticity. In particular, the neuronal activity during learning was associated with the neuronal recruitment into the context-responsive ensemble. These findings suggest that synaptic plasticity in a subset of BLA neurons contributes to fear memory expression through ensemble reorganization.

Fear Extinction Requires Arc/Arg3.1 Expression in the Basolateral Amygdala Molecular Brain. Apr, 2014 | Pubmed ID: 24758170 Prolonged re-exposure to a fear-eliciting cue in the absence of an aversive event extinguishes the fear response to the cue, and has been clinically used as an exposure therapy. Arc (also known as Arg3.1) is implicated in synaptic and experience-dependent plasticity. Arc is regulated by the transcription factor cAMP response element binding protein, which is upregulated with and necessary for fear extinction. Because Arc expression is also activated with fear extinction, we hypothesized that Arc expression is required for fear extinction.

Astrocyte Calcium Signalling Orchestrates Neuronal Synchronization in Organotypic Hippocampal Slices The Journal of Physiology. Jul, 2014 | Pubmed ID: 24710057 Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level.

Operant Conditioning of Synaptic and Spiking Activity Patterns in Single Hippocampal Neurons The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2014 | Pubmed ID: 24695722 Learning is a process of plastic adaptation through which a neural circuit generates a more preferable outcome; however, at a microscopic level, little is known about how synaptic activity is patterned into a desired configuration. Here, we report that animals can generate a specific form of synaptic activity in a given neuron in the hippocampus. In awake, head-restricted mice, we applied electrical stimulation to the lateral hypothalamus, a reward-associated brain region, when whole-cell patch-clamped CA1 neurons exhibited spontaneous synaptic activity that met preset criteria. Within 15 min, the mice learned to generate frequently the excitatory synaptic input pattern that satisfied the criteria. This reinforcement learning of synaptic activity was not observed for inhibitory input patterns. When a burst unit activity pattern was conditioned in paired and nonpaired paradigms, the frequency of burst-spiking events increased and decreased, respectively. The burst reinforcement occurred in the conditioned neuron but not in other adjacent neurons; however, ripple field oscillations were concomitantly reinforced. Neural conditioning depended on activation of NMDA receptors and dopamine D1 receptors. Acutely stressed mice and depression model mice that were subjected to forced swimming failed to exhibit the neural conditioning. This learning deficit was rescued by repetitive treatment with fluoxetine, an antidepressant. Therefore, internally motivated animals are capable of routing an ongoing action potential series into a specific neural pathway of the hippocampal network.

Interneuron Firing Precedes Sequential Activation of Neuronal Ensembles in Hippocampal Slices The European Journal of Neuroscience. Jun, 2014 | Pubmed ID: 24645643 Neuronal firing sequences that occur during behavioral tasks are precisely reactivated in the neocortex and the hippocampus during rest and sleep. These precise firing sequences are likely to reflect latent memory traces, and their reactivation is believed to be essential for memory consolidation and working memory maintenance. However, how the organized repeating patterns emerge through the coordinated interplay of distinct types of neurons remains unclear. In this study, we monitored ongoing spatiotemporal firing patterns using a multi-neuron calcium imaging technique and examined how the activity of individual neurons is associated with repeated ensembles in hippocampal slice cultures. To determine the cell types of the imaged neurons, we applied an optical synapse mapping method that identifies network connectivity among dozens of neurons. We observed that inhibitory interneurons exhibited an increase in their firing rates prior to the onset of repeating sequences, while the overall activity level of excitatory neurons remained unchanged. A specific repeating sequence emerged preferentially after the firing of a specific interneuron that was located close to the neuron first activated in the sequence. The times of repeating sequences could be more precisely predicted based on the activity patterns of inhibitory cells than excitatory cells. In line with these observations, stimulation of a single interneuron could trigger the emergence of repeating sequences. These findings provide a conceptual framework that interneurons serve as a key regulator of initiating sequential spike activity.

Unbalanced Excitability Underlies Offline Reactivation of Behaviorally Activated Neurons Nature Neuroscience. Apr, 2014 | Pubmed ID: 24633127 Hippocampal sharp waves (SWs)/ripples represent the reactivation of neurons involved in recently acquired memory and are crucial for memory consolidation. By labeling active cells with fluorescent protein under the control of an immediate-early gene promoter, we found that neurons that had been activated while mice explored a novel environment were preferentially reactivated during spontaneous SWs in hippocampal slices in vitro. During SWs, the reactivated neurons received strong excitatory synaptic inputs as opposed to a globally tuned network balance between excitation and inhibition.

Large-scale Imaging of Subcellular Calcium Dynamics of Cortical Neurons with G-CaMP6-actin Neuroreport. May, 2014 | Pubmed ID: 24468806 Understanding the information processing performed by a single neuron requires the monitoring of physiological dynamics from a variety of subcellular compartments including dendrites and axons. In this study, we showed that the expression of a fusion protein, consisting of a Ca²⁺ indicator protein (G-CaMP6) and a cytoskeleton protein (actin), enabled large-scale recording of Ca²⁺ dynamics from hundreds of postsynaptic spines and presynaptic boutons in a cortical pyramidal cell. At dendritic spines, G-CaMP6-actin had the potential to detect localized Ca²⁺ activity triggered by subthreshold synaptic inputs. Back-propagating action potentials reliably induced Ca²⁺ fluorescent increases in all spines. At axonal boutons, G-CaMP6-actin reported action potential trains propagating along axonal collaterals. The detectability of G-CaMP6-actin should contribute toward a deeper understanding of neural network architecture and dynamics at the level of individual synapses.

Erratum: Simultaneous Silence Organizes Structured Higher-order Interactions in Neural Populations Scientific Reports. Oct, 2015 | Pubmed ID: 26449163

Subtle Modulation of Ongoing Calcium Dynamics in Astrocytic Microdomains by Sensory Inputs Physiological Reports. Oct, 2015 | Pubmed ID: 26438730 Astrocytes communicate with neurons through their processes. In vitro experiments have demonstrated that astrocytic processes exhibit calcium activity both spontaneously and in response to external stimuli; however, it has not been fully determined whether and how astrocytic subcellular domains respond to sensory input in vivo. We visualized the calcium signals in astrocytes in the primary visual cortex of awake, head-fixed mice. Bias-free analyses of two-photon imaging data revealed that calcium activity prevailed in astrocytic subcellular domains, was coordinated with variable spot-like patterns, and was dominantly spontaneous. Indeed, visual stimuli did not affect the frequency of calcium domain activity, but it increased the domain size, whereas tetrodotoxin reduced the sizes of spontaneous calcium domains and abolished their visual responses. The "evoked" domain activity exhibited no apparent orientation tuning and was distributed unevenly within the cell, constituting multiple active hotspots that were often also recruited in spontaneous activity. The hotspots existed dominantly in the somata and endfeet of astrocytes. Thus, the patterns of astrocytic calcium dynamics are intrinsically constrained and are subject to minor but significant modulation by sensory input.

Neocortical Rebound Depolarization Enhances Visual Perception PLoS Biology. Aug, 2015 | Pubmed ID: 26274866 Animals are constantly exposed to the time-varying visual world. Because visual perception is modulated by immediately prior visual experience, visual cortical neurons may register recent visual history into a specific form of offline activity and link it to later visual input. To examine how preceding visual inputs interact with upcoming information at the single neuron level, we designed a simple stimulation protocol in which a brief, orientated flashing stimulus was subsequently coupled to visual stimuli with identical or different features. Using in vivo whole-cell patch-clamp recording and functional two-photon calcium imaging from the primary visual cortex (V1) of awake mice, we discovered that a flash of sinusoidal grating per se induces an early, transient activation as well as a long-delayed reactivation in V1 neurons. This late response, which started hundreds of milliseconds after the flash and persisted for approximately 2 s, was also observed in human V1 electroencephalogram. When another drifting grating stimulus arrived during the late response, the V1 neurons exhibited a sublinear, but apparently increased response, especially to the same grating orientation. In behavioral tests of mice and humans, the flashing stimulation enhanced the detection power of the identically orientated visual stimulation only when the second stimulation was presented during the time window of the late response. Therefore, V1 late responses likely provide a neural basis for admixing temporally separated stimuli and extracting identical features in time-varying visual environments.

Prefrontal Dopamine Regulates Fear Reinstatement Through the Downregulation of Extinction Circuits ELife. Jul, 2015 | Pubmed ID: 26226637 Prevention of relapses is a major challenge in treating anxiety disorders. Fear reinstatement can cause relapse in spite of successful fear reduction through extinction-based exposure therapy. By utilising a contextual fear-conditioning task in mice, we found that reinstatement was accompanied by decreased c-Fos expression in the infralimbic cortex (IL) with reduction of synaptic input and enhanced c-Fos expression in the medial subdivision of the central nucleus of the amygdala (CeM). Moreover, we found that IL dopamine plays a key role in reinstatement. A reinstatement-inducing reminder shock induced c-Fos expression in the IL-projecting dopaminergic neurons in the ventral tegmental area, and the blocking of IL D1 signalling prevented reduction of synaptic input, CeM c-Fos expression, and fear reinstatement. These findings demonstrate that a dopamine-dependent inactivation of extinction circuits underlies fear reinstatement and may explain the comorbidity of substance use disorders and anxiety disorders.

Memory Formation and Retrieval of Neuronal Silencing in the Auditory Cortex Proceedings of the National Academy of Sciences of the United States of America. Aug, 2015 | Pubmed ID: 26199415 Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.

Microglia in the Pathogenesis of Autism Spectrum Disorders Neuroscience Research. Nov, 2015 | Pubmed ID: 26116891 Proper synaptic pruning is essential for the development of functional neural circuits. Impairments in synaptic pruning disrupt the excitatory versus inhibitory balance (E/I balance) of synapses, which may cause neurodevelopmental disorders such as autism spectrum disorder (ASD). Recent studies have determined molecular mechanisms by which microglia, the brain's resident immune cells, engulf inappropriate and less active synapses. Thus, microglial dysfunction may be involved in the pathogenesis of ASD through attenuated or excess synaptic pruning. In this review, we discuss recent animal and human studies that report an E/I imbalance and the characteristics of microglia in ASD. We will further discuss whether and how synaptic pruning by microglia is involved in the pathogenesis of ASD.

Novelty-Induced Phase-Locked Firing to Slow Gamma Oscillations in the Hippocampus: Requirement of Synaptic Plasticity Neuron. Jun, 2015 | Pubmed ID: 26050043 Temporally precise neuronal firing phase-locked to gamma oscillations is thought to mediate the dynamic interaction of neuronal populations, which is essential for information processing underlying higher-order functions such as learning and memory. However, the cellular mechanisms determining phase locking remain unclear. By devising a virus-mediated approach to perform multi-tetrode recording from genetically manipulated neurons, we demonstrated that synaptic plasticity dependent on the GluR1 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor mediates two dynamic changes in neuronal firing in the hippocampal CA1 area during novel experiences: the establishment of phase-locked firing to slow gamma oscillations and the rapid formation of the spatial firing pattern of place cells. The results suggest a series of events potentially underlying the acquisition of new spatial information: slow gamma oscillations, originating from the CA3 area, induce the two GluR1-dependent changes of CA1 neuronal firing, which in turn determine information flow in the hippocampal-entorhinal system.

Topological Organization of CA3-to-CA1 Excitation The European Journal of Neuroscience. Sep, 2015 | Pubmed ID: 26036915 The CA1-projecting axons of CA3 pyramidal cells, called Schaffer collaterals, constitute one of the major information flow routes in the hippocampal formation. Recent anatomical studies have revealed the non-random structural connectivity between CA3 and CA1, but little is known regarding the functional connectivity (i.e. how CA3 network activity is functionally transmitted downstream to the CA1 network). Using functional multi-neuron calcium imaging of rat hippocampal slices, we monitored the spatiotemporal patterns of spontaneous CA3 and CA1 burst activity under pharmacological GABAergic blockade. We found that spatially clustered CA3 activity patterns were transformed into layered CA1 activity sequences. Specifically, synchronized bursts initiated from multiple hot spots in CA3 ensembles, and CA1 neurons located deeper in the pyramidal cell layer were recruited during earlier phases of the burst events. The order of these sequential activations was maintained across the bursts, but the sequence velocity varied depending on the inter-burst intervals. Thus, CA3 axons innervate CA1 neurons in a highly topographical fashion.

A Neural Network Model of Reliably Optimized Spike Transmission Cognitive Neurodynamics. Jun, 2015 | Pubmed ID: 25972976 We studied the detailed structure of a neuronal network model in which the spontaneous spike activity is correctly optimized to match the experimental data and discuss the reliability of the optimized spike transmission. Two stochastic properties of the spontaneous activity were calculated: the spike-count rate and synchrony size. The synchrony size, expected to be an important factor for optimization of spike transmission in the network, represents a percentage of observed coactive neurons within a time bin, whose probability approximately follows a power-law. We systematically investigated how these stochastic properties could matched to those calculated from the experimental data in terms of the log-normally distributed synaptic weights between excitatory and inhibitory neurons and synaptic background activity induced by the input current noise in the network model. To ensure reliably optimized spike transmission, the synchrony size as well as spike-count rate were simultaneously optimized. This required changeably balanced log-normal distributions of synaptic weights between excitatory and inhibitory neurons and appropriately amplified synaptic background activity. Our results suggested that the inhibitory neurons with a hub-like structure driven by intensive feedback from excitatory neurons were a key factor in the simultaneous optimization of the spike-count rate and synchrony size, regardless of different spiking types between excitatory and inhibitory neurons.

Heterogeneous Effects of Antiepileptic Drugs in an in Vitro Epilepsy Model--a Functional Multineuron Calcium Imaging Study The European Journal of Neuroscience. Jul, 2015 | Pubmed ID: 25967117 Epilepsy is a chronic brain disease characterised by recurrent seizures. Many studies of this disease have focused on local neuronal activity, such as local field potentials in the brain. In addition, several recent studies have elucidated the collective behavior of individual neurons in a neuronal network that emits epileptic activity. However, little is known about the effects of antiepileptic drugs on neuronal networks during seizure-like events (SLEs) at single-cell resolution. Using functional multineuron Ca(2+) imaging (fMCI), we monitored the activities of multiple neurons in the rat hippocampal CA1 region on treatment with the proconvulsant bicuculline under Mg(2+) -free conditions. Bicuculline induced recurrent synchronous Ca(2+) influx, and the events were correlated with SLEs. Other proconvulsants, such as 4-aminopyridine, pentetrazol, and pilocarpine, also induced synchronous Ca(2+) influx. We found that the antiepileptic drugs phenytoin, flupirtine, and ethosuximide, which have different mechanisms of action, exerted heterogeneous effects on bicuculline-induced synchronous Ca(2+) influx. Phenytoin and flupirtine significantly decreased the peak, the amount of Ca(2+) influx and the duration of synchronous events in parallel with the duration of SLEs, whereas they did not abolish the synchronous events themselves. Ethosuximide increased the duration of synchronous Ca(2+) influx and SLEs. Furthermore, the magnitude of the inhibitory effect of phenytoin on the peak synchronous Ca(2+) influx level differed according to the peak amplitude of the synchronous event in each individual cell. Evaluation of the collective behavior of individual neurons by fMCI seems to be a powerful tool for elucidating the profiles of antiepileptic drugs.

[Memory Engram of Brain Circuit] Brain and Nerve = Shinkei Kenkyu No Shinpo. May, 2015 | Pubmed ID: 25957206 How are memories stored in the brain and retrieved on demand? This is a frequently asked question. Indeed, we acquire new memories daily and remember old ones. However, how we can memorize one-time experiences is yet to be investigated. Here, we review possible mechanisms by which memories are maintained in neural networks.

Simultaneous Silence Organizes Structured Higher-order Interactions in Neural Populations Scientific Reports. Apr, 2015 | Pubmed ID: 25919985 Activity patterns of neural population are constrained by underlying biological mechanisms. These patterns are characterized not only by individual activity rates and pairwise correlations but also by statistical dependencies among groups of neurons larger than two, known as higher-order interactions (HOIs). While HOIs are ubiquitous in neural activity, primary characteristics of HOIs remain unknown. Here, we report that simultaneous silence (SS) of neurons concisely summarizes neural HOIs. Spontaneously active neurons in cultured hippocampal slices express SS that is more frequent than predicted by their individual activity rates and pairwise correlations. The SS explains structured HOIs seen in the data, namely, alternating signs at successive interaction orders. Inhibitory neurons are necessary to maintain significant SS. The structured HOIs predicted by SS were observed in a simple neural population model characterized by spiking nonlinearity and correlated input. These results suggest that SS is a ubiquitous feature of HOIs that constrain neural activity patterns and can influence information processing.

Visual Cortical Prosthesis with a Geomagnetic Compass Restores Spatial Navigation in Blind Rats Current Biology : CB. Apr, 2015 | Pubmed ID: 25843028 Allocentric sense is one of the major components that underlie spatial navigation. In blind patients, the difficulty in spatial exploration is attributed, at least partly, to the deficit of absolute direction perception. In support of this notion, we announce that blind adult rats can perform spatial tasks normally when externally provided with real-time feedback of their head directions. Head-mountable microstimulators coupled with a digital geomagnetic compass were bilaterally implanted in the primary visual cortex of adult rats whose eyelids had been sutured. These "blind" rats were trained to seek food pellets in a T-shaped maze or a more complicated maze. Within tens of trials, they learned to manage the geomagnetic information source to solve the mazes. Their performance levels and navigation strategies were similar to those of normal sighted, intact rats. Thus, blind rats can recognize self-location through extrinsically provided stereotactic cues.

Brief Fear Preexposure Facilitates Subsequent Fear Conditioning Neuroscience Research. Jun, 2015 | Pubmed ID: 25683290 Post-traumatic stress disorder (PTSD) is an anxiety disorder that occurs following an unexpected exposure to a severe psychological event. A history of a brief trauma is reported to affect a risk for future PTSD development; however, little is known about the mechanisms by which a previous trauma exposure drives the sensitivity to a late-coming trauma. Using a mouse PTSD model, we found that a prior foot shock enhances contextual fear conditioning. This shock-induced facilitation of fear conditioning (i.e., priming effect) persisted for 7 days and was prevented by MK801, an N-methyl-D-aspartate receptor antagonist. Other types of trauma, such as forced swimming or tail pinch, did not induce a priming effect on fear conditioning. Thus, a trauma is unlikely generalized to modify the sensitivity to other traumatic experiences. The behavioral procedure employed in this study may be a useful tool to elucidate the etiology of PTSD.

Long-delayed Expression of the Immediate Early Gene Arc/Arg3.1 Refines Neuronal Circuits to Perpetuate Fear Memory The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2015 | Pubmed ID: 25589774 Fear memories typically persist for long time periods, and persistent fear memories contribute to post-traumatic stress disorder. However, little is known about the cellular and synaptic mechanisms that perpetuate long-term memories. Here, we find that mouse hippocampal CA1 neurons exhibit biphasic Arc (also known as Arg3.1) elevations after fear experience and that the late Arc expression regulates the perpetuation of fear memoires. An early Arc increase returned to the baseline after 6 h, followed by a second Arc increase after 12 h in the same neuronal subpopulation; these elevations occurred via distinct mechanisms. Antisense-induced blockade of late Arc expression disrupted memory persistence but not formation. Moreover, prolonged fear memories were associated with the delayed, specific elimination of dendritic spines and the reactivation of neuronal ensembles formed during fear experience, both of which required late Arc expression. We propose that late Arc expression refines functional circuits in a delayed fashion to prolong fear memory.

Frontal Association Cortex is Engaged in Stimulus Integration During Associative Learning Current Biology : CB. Jan, 2015 | Pubmed ID: 25496961 The frontal association cortex (FrA) is implicated in higher brain function. Aberrant FrA activity is likely to be involved in dementia pathology. However, the functional circuits both within the FrA and with other regions are unclear. A recent study showed that inactivation of the FrA impairs memory consolidation of an auditory fear conditioning in young mice. In addition, dendritic spine remodeling of FrA neurons is sensitive to paired sensory stimuli that produce associative memory. These findings suggest that the FrA is engaged in neural processes critical to associative learning. Here we characterize stimulus integration in the mouse FrA during associative learning. We experimentally separated contextual fear conditioning into context exposure and shock, and found that memory formation requires protein synthesis associated with both context exposure and shock in the FrA. Both context exposure and shock trigger Arc, an activity-dependent immediate-early gene, expression in the FrA, and a subset of FrA neurons was dually activated by both stimuli. In addition, we found that the FrA receives projections from the perirhinal (PRh) and insular (IC) cortices and basolateral amygdala (BLA), which are implicated in context and shock encoding. PRh and IC neurons projecting to the FrA were activated by context exposure and shock, respectively. Arc expression in the FrA associated with context exposure and shock depended on PRh activity and both IC and BLA activities, respectively. These findings indicate that the FrA is engaged in stimulus integration and contributes to memory formation in associative learning.

Microglia and Neurogenesis in the Epileptic Dentate Gyrus Neurogenesis (Austin, Tex.). 2016 | Pubmed ID: 27928548 Microglia are recognized as major immune cells in the brain. They have been traditionally studied in various contexts of disease, where their activation has been assumed to induce mostly detrimental effects. Recent studies, however, have challenged the current view of microglia, clarifying their essential contribution to the development of neural circuits and brain function. In this review, we particularly discuss the role of microglia as the major orchestrators that regulate adult neurogenesis in the hippocampus. We also review the roles of microglia in seizure-induced adult neurogenesis in the epileptic dentate gyrus. Specifically, we introduce our recent study, in which we identified a novel mechanism by which viable newborn cells in the adult dentate gyrus are phagocytosed and eliminated by microglia after status epilepticus, maintaining homeostasis of the dentate circuitry. This review aims to reconsider the microglial function in adult neurogenesis, especially when they are activated during epileptogenesis, challenging the dogma that microglia are harmful neurotoxic cells.

Functional Organization of Flash-Induced V1 Offline Reactivation The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2016 | Pubmed ID: 27852780 The primary visual cortex exhibits a late, long response with a latency of >300 ms and an immediate early response that occurs ∼100 ms after a visual stimulus. The late response is thought to contribute to visual functions such as sensory perception, iconic memory, working memory, and forming connections between temporally separated stimuli. However, how the visual late response is generated and organized is not completely understood. In the mouse primary visual cortex in vivo, we isolated long-delayed responses by using a brief light-flash stimulus for which the stimulus late response occurred long after the stimulus offset and was not contaminated by the instantaneous response evoked by the stimulus. Using whole-cell patch-clamp recordings, we demonstrated that the late rebound response was shaped by a net-balanced increase in excitatory and inhibitory synaptic conductances, whereas transient imbalances were caused by intermittent inhibitory barrage. In contrast to the common assumption that the neocortical late response reflects a feedback signal from the downstream higher-order cortical areas, our pharmacological and optogenetic analyses demonstrated that the late responses likely have a thalamic origin. Therefore, the late component of a sensory-evoked cortical response should be interpreted with caution.

3-Hz Subthreshold Oscillations of CA2 Neurons In Vivo Hippocampus. Dec, 2016 | Pubmed ID: 27650674 The CA2 region is unique in the hippocampus; it receives direct synaptic innervations from several hypothalamic nuclei and expresses various receptors of neuromodulators, including adenosine, vasopressin, and oxytocin. Furthermore, the CA2 region may have distinct brain functions, such as the control of instinctive and social behaviors; however, little is known about the dynamics of the subthreshold membrane potentials of CA2 neurons in vivo. We conducted whole-cell current-clamp recordings from CA2 pyramidal cells in urethane-anesthetized mice and monitored the intrinsic fluctuations in their membrane potentials. The CA2 pyramidal cells emitted spontaneous action potentials at mean firing rates of ∼0.8 Hz. In approximately half of the neurons, the subthreshold membrane potential oscillated at ∼3 Hz. In two neurons, we obtained simultaneous recordings of local field potentials from the CA1 stratum radiatum and demonstrated that the 3-Hz oscillations of CA2 neurons were not correlated with CA1 field potentials. In tetrodotoxin-perfused acute hippocampal slices, the membrane potentials of CA2 pyramidal cells were not preferentially entrained to 3-Hz sinusoidal current inputs, which suggest that intracellular 3-Hz oscillations reflect the neuronal dynamics of the surrounding networks. © 2016 Wiley Periodicals, Inc.

Selective Attenuation of Electrophysiological Activity of the Dentate Gyrus in a Social Defeat Mouse Model The Journal of Physiological Sciences : JPS. Aug, 2016 | Pubmed ID: 27573168 Current research on stress pathology has revealed a set of molecular and cellular mechanisms through which psychosocial stress impairs brain function. However, there are few studies that have examined how chronic stress exposure alters neuronal activity patterns at a network level. Here, we recorded ensemble neuronal activity patterns of the cortico-hippocampal network from urethane-anesthetized mice that were subjected to repeated social defeat stress. In socially defeated mice, the magnitudes of local field potential signals, including theta, slow gamma, and fast gamma oscillations, were significantly reduced in the dentate gyrus, whereas they remained unchanged in the hippocampus and somatosensory cortex. In accordance with the vast majority of histological and biochemical studies, our evidence from electrophysiological investigations highlights the dentate gyrus as a key brain area that is primarily susceptible to stress-induced dysfunction.

Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death EBioMedicine. Aug, 2016 | Pubmed ID: 27544065 Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca(2+) release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca(2+) release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca(2+) release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca(2+) release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.

Autism Spectrum Disorders and Epilepsy Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Aug, 2016 | Pubmed ID: 27478051

A New Device for the Simultaneous Recording of Cerebral, Cardiac, And muscular Electrical Activity in Freely Moving Rodents Journal of Pharmacological Sciences. Sep, 2016 | Pubmed ID: 27430984 We present a new technique for the simultaneous capture of bioelectrical time signals from the brain and peripheral organs of freely moving rodents. The recording system integrates all systemic signals into an electrical interface board that is mounted on an animal's head for an extended period. The interface board accommodates up to 48 channels, enabling us to analyze neuronal activity patterns in multiple brain regions by comparing a variety of physiological body states over weeks and months. This technique will advance the understanding of the neurophysiological correlate of mind-body associations in health and disease.

Development of Practical Red Fluorescent Probe for Cytoplasmic Calcium Ions with Greatly Improved Cell-membrane Permeability Cell Calcium. Oct, 2016 | Pubmed ID: 27349490 Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.

A Computationally Efficient Filter for Reducing Shot Noise in Low S/N Data PloS One. 2016 | Pubmed ID: 27304217 Functional multineuron calcium imaging (fMCI) provides a useful experimental platform to simultaneously capture the spatiotemporal patterns of neuronal activity from a large cell population in situ. However, fMCI often suffers from low signal-to-noise ratios (S/N). The main factor that causes the low S/N is shot noise that arises from photon detectors. Here, we propose a new denoising procedure, termed the Okada filter, which is designed to reduce shot noise under low S/N conditions, such as fMCI. The core idea of the Okada filter is to replace the fluorescence intensity value of a given frame time with the average of two values at the preceding and following frames unless the focused value is the median among these three values. This process is iterated serially throughout a time-series vector. In fMCI data of hippocampal neurons, the Okada filter rapidly reduces background noise and significantly improves the S/N. The Okada filter is also applicable for reducing shot noise in electrophysiological data and photographs. Finally, the Okada filter can be described using a single continuous differentiable equation based on the logistic function and is thus mathematically tractable.

Microglia Engulf Viable Newborn Cells in the Epileptic Dentate Gyrus Glia. Sep, 2016 | Pubmed ID: 27301702 Microglia, which are the brain's resident immune cells, engulf dead neural progenitor cells during adult neurogenesis in the subgranular zone (SGZ) of the dentate gyrus (DG). The number of newborn cells in the SGZ increases significantly after status epilepticus (SE), but whether and how microglia regulate the number of newborn cells after SE remain unclear. Here, we show that microglia rapidly eliminate newborn cells after SE by primary phagocytosis, a process by which viable cells are engulfed, thereby regulating the number of newborn cells that are incorporated into the DG. The number of newborn cells in the DG was increased at 5 days after SE in the adult mouse brain but rapidly decreased to the control levels within a week. During this period, microglia in the DG were highly active and engulfed newborn cells. We found that the majority of engulfed newborn cells were caspase-negative viable cells. Finally, inactivation of microglia with minocycline maintained the increase in the number of newborn cells after SE. Furthermore, minocycline treatment after SE induced the emergence of hilar ectopic granule cells. Thus, our findings suggest that microglia may contribute to homeostasis of the dentate neurogenic niche by eliminating excess newborn cells after SE via primary phagocytosis. GLIA 2016;64:1508-1517.

Structural Insight into Photoactivation of an Adenylate Cyclase from a Photosynthetic Cyanobacterium Proceedings of the National Academy of Sciences of the United States of America. 06, 2016 | Pubmed ID: 27247413 Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Depth and Time-dependent Heterogeneity of Microglia in Mouse Hippocampal Slice Cultures Neuroscience Research. Oct, 2016 | Pubmed ID: 27211829 Microglia are the brain-resident immune cells with the phagocytic capacity to engulf dead and living neurons in health and disease. However, the mechanisms underlying the neuron-microglia interaction remain elusive partly because proper in vitro systems are lacking. Specifically, the highly activated status of microglia with amoeboid morphology in primary culture is different from the 'resting' microglia with multiple processes in vivo. Here, we performed a detailed investigation of microglial properties in mouse hippocampal slice cultures, focusing on the changes in morphology in the activated state, finding a depth and time-dependent localization of in vivo-like microglia in slice cultures.

Empirical Bayesian Significance Measure of Neuronal Spike Response BMC Neuroscience. May, 2016 | Pubmed ID: 27209433 Functional connectivity analyses of multiple neurons provide a powerful bottom-up approach to reveal functions of local neuronal circuits by using simultaneous recording of neuronal activity. A statistical methodology, generalized linear modeling (GLM) of the spike response function, is one of the most promising methodologies to reduce false link discoveries arising from pseudo-correlation based on common inputs. Although recent advancement of fluorescent imaging techniques has increased the number of simultaneously recoded neurons up to the hundreds or thousands, the amount of information per pair of neurons has not correspondingly increased, partly because of the instruments' limitations, and partly because the number of neuron pairs increase in a quadratic manner. Consequently, the estimation of GLM suffers from large statistical uncertainty caused by the shortage in effective information.

Differential Expression of Axon-sorting Molecules In mouse Olfactory Sensory Neurons The European Journal of Neuroscience. Aug, 2016 | Pubmed ID: 27207328 In the mouse olfactory system, the axons of olfactory sensory neurons that express the same type of odorant receptor (OR) converge to a specific set of glomeruli in the olfactory bulb (OB). It is widely accepted that expressed OR molecules instruct glomerular segregation by regulating the expression of axon-sorting molecules. Although the relationship between the expression of axon-sorting molecules and OR types has been analyzed in detail, those between the expressions of axon-sorting molecules remain to be elucidated. Here we collected the expression profiles of four axon-sorting molecules from a large number of glomeruli in the OB. These molecules demonstrated position-independent mosaic expressions, but their patterns were not identical in the OB. Comparing their expressions identified positive and negative correlations between several pairs of genes even though they showed various expressions. Furthermore, the principal component analysis revealed that the factor loadings in the principal component 1, which explain the largest amount of variation, were most likely to reflect the degree of the cyclic nucleotide-gated (CNG) channel dependence on the expression of axon-sorting molecules. Thus, neural activity generated through the CNG channel is a major component in the generation of a wide variety of expressions of axon-sorting molecules in glomerular segregation.

Re-analysis on Geometric Energy Anatomical Science International. Sep, 2016 | Pubmed ID: 27147443

Subcellular Calcium Dynamics During Juvenile Development in Mouse Hippocampal Astrocytes The European Journal of Neuroscience. Apr, 2016 | Pubmed ID: 27041234 Astrocytes generate calcium signals throughout their fine processes, which are assumed to locally regulate neighbouring neurotransmission and blood flow. The intercellular morphological relationships mature during juvenile periods when astrocytes elongate highly ramified processes. In this study, we examined developmental changes in calcium activity patterns of single hippocampal astrocytes using a transgenic mouse line in which astrocytes selectively express a genetically encoded calcium indicator, Yellow Cameleon-Nano50. Compared with postnatal day 7, astrocytes at postnatal day 30 showed larger subcellular calcium events and a greater proportion of somatic events. At both ages, the calcium activity was abolished by removal of extracellular calcium ions. Calcium events in late juvenile astrocytes were not affected by spontaneously occurring sharp waves that trigger synchronized neuronal spikes, implying the independence of astrocyte calcium signals from neuronal synchronization. These results demonstrate that astrocytes undergo dynamic changes in their activity patterns during juvenile development.

Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA Chembiochem : a European Journal of Chemical Biology. Jul, 2016 | Pubmed ID: 27038199 Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

Homeostatic Changes in Neuronal Network Oscillations in Response to Continuous Hypoperfusion in the Mouse Forebrain Neuroscience Research. Aug, 2016 | Pubmed ID: 26945618 Neuronal activity is highly sensitive to changes in oxygen tension. In this study, we examined the impact of hypoxic/ischemic conditions on neuronal ensemble activity patterns in the mouse brain using in vivo extracellular electrophysiological recordings from up to 8 sites in the thalamus, dorsal hippocampus, and neocortex, while cerebral hypoperfusion was induced by unilateral carotid artery occlusion. After a few minutes, the occlusion triggered a rapid change in the power of the local field oscillations. In the hippocampus, but not in the neocortex, the absolute power changes at all frequency ranges (relative to the baseline) became less pronounced with time, and no significant changes were observed 30min after the occlusion-induced hypoperfusion. We also tested whether continuous hypoperfusion induced by the occlusion for up to 1 week alters neuronal activity. In the hippocampus and the thalamus, the chronic occlusion did not lead to a reduction in the power of the local field oscillations. These results indicate that certain neuronal populations have the ability to maintain internal neurophysiological homeostasis against continuous hypoperfusion.

Early Failures Benefit Subsequent Task Performance Scientific Reports. Feb, 2016 | Pubmed ID: 26883387 Animals navigate using cognitive maps. However, how they adaptively exploit these maps in changing environments is not fully understood. In this study, we investigated the problem-solving behaviors of mice in a complicated maze in which multiple routes with different intersections were available (Test 1). Although all mice eventually settled on the shortest route, mice that initially exhibited more trial-and-error exploration solved the maze more rapidly. We then introduced one or two barriers that obstructed learned routes such that mice had to establish novel roundabout detours (Tests 2/3). Solutions varied among mice but were predictable based on individual early trial-and-error patterns observed in Test 1: mice that had initially explored more extensively found better solutions. Finally, when the barriers were removed (Test 4), all mice reverted to the best solution after active exploration. Thus, early active exploration helps mice to develop optimal strategies.

Late Arc/Arg3.1 Expression in the Basolateral Amygdala is Essential for Persistence of Newly-acquired and Reactivated Contextual Fear Memories Scientific Reports. Feb, 2016 | Pubmed ID: 26880136 A feature of fear memory is its persistence, which could be a factor for affective disorders. Memory retrieval destabilizes consolidated memories, and then rapid molecular cascades contribute to early stabilization of reactivated memories. However, persistence of reactivated memories has been poorly understood. Here, we discover that late Arc (also known as Arg3.1) expression in the mouse basolateral amygdala (BLA) is involved in persistence of newly-acquired and reactivated fear memories. After both fear learning and retrieval, Arc levels increased at 2 h, returned to basal levels at 6 h but increased again at 12 h. Inhibiting late Arc expression impaired memory retention 7 d, but not 2 d, after fear learning and retrieval. Moreover, blockade of NR2B-containing N-methyl-D-aspartate receptors (NMDARs) prevented memory destabilization and inhibited late Arc expression. These findings indicate that NR2B-NMDAR and late Arc expression plays a critical role in the destabilization and persistence of reactivated memories.

Subcellular Imbalances in Synaptic Activity Cell Reports. Feb, 2016 | Pubmed ID: 26854220 The dynamic interactions between synaptic excitation and inhibition (E/I) shape membrane potential fluctuations and determine patterns of neuronal outputs; however, the spatiotemporal organization of these interactions within a single cell is poorly understood. Here, we investigated the relationship between local synaptic excitation and global inhibition in hippocampal pyramidal neurons using functional dendrite imaging in combination with whole-cell recordings of inhibitory postsynaptic currents. We found that the sums of spine inputs over dendritic trees were counterbalanced by a proportional amount of somatic inhibitory inputs. This online E/I correlation was maintained in dendritic segments that were longer than 50 μm. However, at the single spine level, only 22% of the active spines were activated with inhibitory inputs. This inhibition-coupled activity occurred mainly in the spines with large heads. These results shed light on a microscopic E/I-balancing mechanism that operates at selected synapses and that may increase the accuracy of neural information.

Photoactivated Adenylyl Cyclase (PAC) Reveals Novel Mechanisms Underlying CAMP-dependent Axonal Morphogenesis Scientific Reports. Jan, 2016 | Pubmed ID: 26795422 Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

Neuraminidase-Dependent Degradation of Polysialic Acid Is Required for the Lamination of Newly Generated Neurons PloS One. 2016 | Pubmed ID: 26731280 Hippocampal granule cells (GCs) are generated throughout the lifetime and are properly incorporated into the innermost region of the granule cell layer (GCL). Hypotheses for the well-regulated lamination of newly generated GCs suggest that polysialic acid (PSA) is present on the GC surface to modulate GC-to-GC interactions, regulating the process of GC migration; however, direct evidence of this involvement is lacking. We show that PSA facilitates the migration of newly generated GCs and that the activity of N-acetyl-α-neuraminidase 1 (NEU1, sialidase 1) cleaves PSA from immature GCs, terminating their migration in the innermost GCL. Developing a migration assay of immature GCs in vitro, we found that the pharmacological depletion of PSA prevents the migration of GCs, whereas the inhibition of PSA degradation with a neuraminidase inhibitor accelerates this migration. We found that NEU1 is highly expressed in immature GCs. The knockdown of NEU1 in newly generated GCs in vivo increased PSA presence on these cells, and attenuated the proper termination of GC migration in the innermost GCL. In conclusion, this study identifies a novel mechanism that underlies the proper lamination of newly generated GCs through the modulation of PSA presence by neuronal NEU1.

Accurate Detection of Low Signal-to-noise Ratio Neuronal Calcium Transient Waves Using a Matched Filter Journal of Neuroscience Methods. Feb, 2016 | Pubmed ID: 26561771 Calcium imaging has become a fundamental modality for studying neuronal circuit dynamics both in vitro and in vivo. However, identifying calcium events (CEs) from spectral data remains laborious and difficult, especially since the signal-to-noise ratio (SNR) often falls below 2. Existing automated signal detection methods are generally applied at high SNRs, leaving a large need for an automated algorithm that can accurately extract CEs from fluorescence intensity data of SNR 2 and below.

Spatial Clusters of Constitutively Active Neurons in Mouse Visual Cortex Anatomical Science International. Mar, 2016 | Pubmed ID: 25940680 The networks of neocortical neurons are coordinated by spontaneous activity, the level of which exhibits high heterogeneity among neurons, ranging from low activity levels to very high activity levels, even in the same network. Highly active neurons represent a small proportion in the cerebral cortex and are mingled in a web of the vast majority of neurons with low firing rates. However, little is known about the spatial arrangement of these highly active cells within the cerebral cortex. Here, we visualized their spatial distribution by labeling them with c-Fos, a neuronal activity marker, in the mouse primary visual cortex. By introducing energy-like and entropy-like parameters that did not require arbitrary thresholds for c-Fos positivity, we found that strongly c-Fos-expressing neurons were clustered in the vicinity. The cluster size measured approximately 100 μm in diameter and was smaller in layer 2/3 than in layers 5 and 6. Layer 1 neurons did not exhibit a clustered pattern of c-Fos-expressing neurons. Our novel statistical approaches are not subject to human bias and are thus widely applicable to evaluate the spatial bias of any particles.

Juvenile Hippocampal CA2 Region Expresses Aggrecan Frontiers in Neuroanatomy. 2017 | Pubmed ID: 28539874 Perineuronal nets (PNNs) are distributed primarily around inhibitory interneurons in the hippocampus, such as parvalbumin-positive interneurons. PNNs are also present around excitatory neurons in some brain regions and prevent plasticity in these neurons. A recent study demonstrated that PNNs also exist around mouse hippocampal pyramidal cells, which are the principle type of excitatory neurons, in the CA2 subregion and modulate the excitability and plasticity of these neurons. However, the development of PNNs in the CA2 region during postnatal maturation was not fully investigated. This study found that a main component of PNNs, aggrecan, existed in the pyramidal cell layer of the putative CA2 subarea prior to the appearance of the CA2 region, which was defined by the CA2 marker protein regulator of G protein signaling 14 (RGS14). We also found that aggrecan immunoreactivity was more evident in the anterior sections of the CA2 area than the posterior sections, which suggests that the function of CA2 PNNs varies along the anterior-posterior axis.

Flashing Lights Induce Prolonged Distortions in Visual Cortical Responses and Visual Perception ENeuro. May-Jun, 2017 | Pubmed ID: 28508035 The primary sensory neocortex generates an internal representation of the environment, and its circuit reorganization is thought to lead to a modification of sensory perception. This reorganization occurs primarily through activity-dependent plasticity and has been well documented in animals during early developmental stages. Here, we describe a new method for the noninvasive induction of long-term plasticity in the mature brain: simple transient visual stimuli (i.e., flashing lights) can be used to induce prolonged modifications in visual cortical processing and visually driven behaviors. Our previous studies have shown that, in the primary visual cortex (V1) of mice, a flashing light stimulus evokes a long-delayed response that persists for seconds. When the mice were repetitively presented with drifting grating stimuli (conditioned stimuli) during the flash stimulus-evoked delayed response period, the V1 neurons exhibited a long-lasting decrease in responsiveness to the conditioned stimuli. The flash stimulus-induced underrepresentation of the grating motion was specific to the direction of the conditioned stimuli and was associated with a decrease in the animal's ability to detect the motion of the drifting gratings. The neurophysiological and behavioral plasticity both persisted for at least several hours and required N-methyl-d-aspartate receptor activation in the visual cortex. We propose that flashing light stimuli can be used as an experimental tool to investigate the visual function and plasticity of neuronal representations and perception after a critical period of neocortical plasticity.

Synthesis of Practical Red Fluorescent Probe for Cytoplasmic Calcium Ions with Greatly Improved Cell-membrane Permeability Data in Brief. Jun, 2017 | Pubmed ID: 28491938 In this data article, we described the detailed synthetic procedure and the experimental data for the synthesis of a red-fluorescent probe for calcium ions (Ca(2+)) with improved water solubility. This Ca(2+) red-fluorescent probe CaTM-3 AM could be applied to fluorescence imaging of physiological Ca(2+) concentration changes in not only live cells, but also brain slices, with high cell-membrane permeability leading to bright fluorescence in biosamples. The data provided herein are in association with the research article "The Development of Practical Red Fluorescent Probe for Cytoplasmic Calcium Ions with Greatly Improved Cell-membrane Permeability" in Cell Calcium (Hirabayashi et al., 2016) [1].

Simultaneous Recordings of Central and Peripheral Bioelectrical Signals in a Freely Moving Rodent Biological & Pharmaceutical Bulletin. 2017 | Pubmed ID: 28458358 Understanding physiological interactions between the central and peripheral nervous systems requires an experimental strategy to simultaneously monitor activity patterns of the brain and peripheral organs. In this study, we developed a novel method to record extracellular field potential signals from a wide range of brain regions together with electrocardiograms, electromyograms, and breathing signals from a freely moving rodent. This method collects all recorded signals into a single device mounted on an animal's head, allowing the reduction of experimental costs and the simplification of data processing. The methodological concept is applicable to a number of biological research issues of how the brain-body association is altered in response to various environmental changes, emotional challenges, and acute and chronic dysfunction of internal organs.

[Recent Advancements in Understanding Hippocampal Functions] Brain and Nerve = Shinkei Kenkyu No Shinpo. Apr, 2017 | Pubmed ID: 28424399 The hippocampal formation located in the medial temporal lobe of the brain has been studied extensively for several decades, both in in vitro slice preparations and in vivo living animals. Recent advancements in large-scale recording techniques, such as multisite silicon probes and tetrodes, enable us to examine the detailed characteristics of temporal spiking patterns of tens or hundreds of neurons in the hippocampal circuit. Here, we introduce how such tools contribute to our understanding of the hippocampal network dynamics and review several hippocampal functions for information processing in the brain, including pattern separation and completion, spatial representation, and encoding of positive and negative valence.

Activation of Perineuronal Net-expressing Excitatory Neurons During Associative Memory Encoding and Retrieval Scientific Reports. Apr, 2017 | Pubmed ID: 28378772 Perineuronal nets (PNNs), proteoglycan-rich extracellular matrix structures, are thought to be expressed around inhibitory neurons and contribute to critical periods of brain function and synaptic plasticity. However, in some specific brain regions such as the amygdala, PNNs were predominantly expressed around excitatory neurons. These neurons were recruited during auditory fear conditioning and memory retrieval. Indeed, the activation of PNN-expressing excitatory neurons predicted cognitive performance.

Machine Learning-based Prediction of Adverse Drug Effects: An Example of Seizure-inducing Compounds Journal of Pharmacological Sciences. Feb, 2017 | Pubmed ID: 28215473 Various biological factors have been implicated in convulsive seizures, involving side effects of drugs. For the preclinical safety assessment of drug development, it is difficult to predict seizure-inducing side effects. Here, we introduced a machine learning-based in vitro system designed to detect seizure-inducing side effects. We recorded local field potentials from the CA1 alveus in acute mouse neocortico-hippocampal slices, while 14 drugs were bath-perfused at 5 different concentrations each. For each experimental condition, we collected seizure-like neuronal activity and merged their waveforms as one graphic image, which was further converted into a feature vector using Caffe, an open framework for deep learning. In the space of the first two principal components, the support vector machine completely separated the vectors (i.e., doses of individual drugs) that induced seizure-like events and identified diphenhydramine, enoxacin, strychnine and theophylline as "seizure-inducing" drugs, which indeed were reported to induce seizures in clinical situations. Thus, this artificial intelligence-based classification may provide a new platform to detect the seizure-inducing side effects of preclinical drugs.

Differential Timing of Neurogenesis Underlies Dorsal-ventral Topographic Projection of Olfactory Sensory Neurons Neural Development. Feb, 2017 | Pubmed ID: 28193234 The mammalian primary olfactory system has a spatially-ordered projection in which olfactory sensory neurons (OSNs) located in the dorsomedial (DM) and ventrolateral (VL) region of the olfactory epithelium (OE) send their axons to the dorsal and ventral region of the olfactory bulb (OB), respectively. We previously found that OSN axonal projections occur sequentially, from the DM to the VL region of the OE. The differential timing of axonal projections is important for olfactory map formation because early-arriving OSN axons secrete guidance cues at the OB to help navigate late-arriving OSN axons. We hypothesized that the differential timing of axonal projections is regulated by the timing of OSN neurogenesis. To test this idea, we investigated spatiotemporal patterns of OSN neurogenesis during olfactory development.

CAMP-Dependent Calcium Oscillations of Astrocytes: An Implication for Pathology Cerebral Cortex (New York, N.Y. : 1991). Feb, 2017 | Pubmed ID: 26803165 Astrocytes in various brain regions exhibit spontaneous intracellular calcium elevations both in vitro and in vivo; however, neither the temporal pattern underlying this activity nor its function has been fully evaluated. Here, we utilized a long-term optical imaging technique to analyze the calcium activity of more than 4000 astrocytes in acute hippocampal slices as well as in the neocortex and hippocampus of head-restrained mice. Although astrocytic calcium activity was largely sparse and irregular, we observed a subset of cells in which the fluctuating calcium oscillations repeated at a regular interval of ∼30 s. These intermittent oscillations i) depended on type 2 inositol 1,4,5-trisphosphate receptors; ii) consisted of a complex reverberatory interaction between the soma and processes of individual astrocytes; iii) did not synchronize with those of other astrocytes; iv) did not require neuronal firing; v) were modulated through cAMP-protein kinase A signaling; vi) were facilitated under pathological conditions, such as energy deprivation and epileptiform hyperexcitation; and vii) were associated with enhanced hypertrophy in astrocytic processes, an early hallmark of reactive gliosis, which is observed in ischemia and epilepsy. Therefore, calcium oscillations appear to be associated with a pathological state in astrocytes.

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