multi-layer-cortical-ca2-imaging-freely-moving-mice-with-prism-probes

Multi-layer Cortical Ca<sup>2+</sup> Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Inscopix Inc.

Researchers and clinicians are increasingly recognizing that psychological and psychiatric disorders are often developmentally progressive, and that diagnosis often represents a point along that progression that is defined largely by our abilities to detect symptoms. As a result, strategies that guide our searches for the root causes and etiologies of these disorders are beginning to change. This review describes interactions between genetics and experience that influence the development of psychopathologies. Following a discussion of normal brain development that highlights how specific cellular processes may be targeted by genetic or environmental factors, we focus on four disorders whose origins range from genetic (fragile X syndrome) to environmental (fetal alcohol syndrome) or a mixture of both factors (depression and schizophrenia). C.H. Waddington's canalization model (slightly modified) is used as a tool to conceptualize the interactive influences of genetics and experience in the development of these psychopathologies. Although this model was originally proposed to describe the 'canalizing' role of genetics in promoting normative development, it serves here to help visualize, for example, the effects of adverse (stressful) experience in the kindling model of depression, and the multiple etiologies that may underlie the development of schizophrenia. Waddington's model is also useful in understanding the canalizing influence of experience-based therapeutic approaches, which also likely bring about 'organic' changes in the brain. Finally, in light of increased evidence for the role of experience in the development and treatment of psychopathologies, we suggest that future strategies for identifying the underlying causes of these disorders be based less on the mechanisms of action of effective pharmacological treatments, and more on increased knowledge of the brain's cellular mechanisms of plastic change.

Experience effects on brain development: possible contributions to psychopathology.

The dopamine D4 receptor has been investigated for its potential role in several CNS disorders, notably schizophrenia and more recently, erectile dysfunction. Whereas studies have investigated dopamine D4 receptor-mediated signaling in vitro, there have been few, if any, attempts to identify dopamine D4 receptor signal transduction pathways in vivo. In the present studies, the selective dopamine D4 agonist PD168077 induces c-Fos expression and extracellular signal regulated kinase (ERK) phosphorylation in the hypothalamic paraventricular nucleus (PVN), a site known to regulate proerectile activity. The selective dopamine D4 receptor antagonist A-381393 blocked both c-Fos expression and ERK1/2 phosphorylation produced by PD168077. In addition, PD168077-induced ERK1/2 phosphorylation was prevented by SL327, an inhibitor of ERK1/2 phosphorylation. Interestingly, treatment with A-381393 alone significantly reduced the amount of Fos immunoreactivity as compared to basal expression observed in vehicle-treated controls. Dopamine D4 receptor and c-Fos coexpression in the PVN was observed using double immunohistochemical labeling, suggesting that PD168077-induced signaling may result from direct dopamine D4 receptor activation. Our results demonstrate functional dopamine D4 receptor expression and natural coupling in the PVN linked to signal transduction pathways that include immediate early gene and MAP kinase activation. Further, the ability of the selective dopamine D4 antagonist A-381393 alone to reduce c-Fos expression below control levels may imply the presence of a tonic dopamine D4 receptor activation under basal conditions in vivo. These findings provide additional evidence that the PVN may be a site of dopamine D4 receptor-mediated proerectile activity.

Dopamine D4 receptor signaling in the rat paraventricular hypothalamic nucleus: Evidence of natural coupling involving immediate early gene induction and mitogen activated protein kinase phosphorylation.

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties.

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and has generated recent interest as a potential drug target for treating neurodegenerative disorders such as Alzheimer's disease (AD). The property of Ca(2+) permeation associated with alpha7 nAChR agonism may lead to Ca(2+)-dependent intracellular signaling that contribute to the procognitive and neuroprotective effects that have been described with this pharmacology. In this study, we investigated whether alpha7 nAChR agonism leads to increased phosphorylation of the inhibitory regulating amino acid residue Ser-9 on GSK3beta, a major kinase responsible for tau hyperphosphorylation in AD neuropathology. Immunohistochemical analysis revealed that the selective alpha7 agonist A-582941 increased S(9)-GSK3beta phosphorylation in mouse cingulate cortex and hippocampus that was not observed in alpha7 nAChR knock-out mice. A-582941 steady state exposure through continuous (2 wk) infusion also increased S(9)-GSK3beta phosphorylation in the hippocampus of Tg2576 (APP), as well as wild-type mice. Moreover, A-582941 continuous infusion decreased phosphorylation of tau in hippocampal CA3 Mossy fibers and spinal motoneurons in a hypothermia-induced tau hyperphosphorylation mouse model and AD double transgenic APP/tau mouse line, respectively. These studies demonstrate that inactivation of GSK3beta may be associated with alpha7 nAChR-induced signaling leading to attenuated tau hyperphosphorylation, raising the intriguing possibility that alpha7 nAChR agonism may have disease modifying benefit in the treatment of tauopathies, in particular AD.

Selective alpha7 nicotinic acetylcholine receptor activation regulates glycogen synthase kinase3beta and decreases tau phosphorylation in vivo.

Neocortical networks are composed of diverse populations of cells that differ in their chemical content, electrophysiological characteristics, and connectivity. Gamma-frequency oscillatory activity of inhibitory subnetworks has been hypothesized to regulate information processing in the cortex as a whole. Inhibitory neurons in these subnetworks synchronize their firing and selectively innervate the perisomatic compartments of their target neurons, generating both tonic and rapidly fluctuating inhibition. How do different types of cortical neurons respond to changes in the level and structure of perisomatic inhibition? What accounts for response heterogeneity between cell types, and are these response properties fixed or flexible? To answer these questions, we use in vitro whole-cell recording and dynamic-clamp somatic current injection to study six distinct types of cortical neurons. We demonstrate that different types of neurons systematically vary in their receptiveness to fast changes in the structure of inhibition and the range over which changes in inhibitory tone affect their output. Using simple neuron models and model neuron hybrids (dynamic clamp), we determine which intrinsic differences between cell types lead to these variations in receptiveness. These results suggest important differences in the way cell types are affected by gamma-frequency inhibition, which may have important circuit level implications. Although intrinsic differences observed in vitro are useful for the elucidation of basic cellular properties and differences between cell types, we also investigate how the integrative properties of neurons are likely to be rapidly modulated in the context of active networks in vivo.

Cell type-specific control of neuronal responsiveness by gamma-band oscillatory inhibition.

The brain contains an astonishing diversity of neurons, each expressing only one set of ion channels out of the billions of potential channel combinations. Simple organizing principles are required for us to make sense of this abundance of possibilities and wealth of related data. We suggest that energy minimization subject to functional constraints may be one such unifying principle. We compared the energy needed to produce action potentials singly and in trains for a wide range of channel densities and kinetic parameters and examined which combinations of parameters maximized spiking function while minimizing energetic cost. We confirmed these results for sodium channels using a dynamic current clamp in neocortical fast spiking interneurons. We find further evidence supporting this hypothesis in a wide range of other neurons from several species and conclude that the ion channels in these neurons minimize energy expenditure in their normal range of spiking.

Metabolic cost as a unifying principle governing neuronal biophysics.

Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal's state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (∼ 65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders.

Zolpidem reduces hippocampal neuronal activity in freely behaving mice: a large scale calcium imaging study with miniaturized fluorescence microscope.

Optimally orchestrating complex behavioral states, such as the pursuit and consumption of food, is critical for an organism's survival. The lateral hypothalamus (LH) is a neuroanatomical region essential for appetitive and consummatory behaviors, but whether individual neurons within the LH differentially contribute to these interconnected processes is unknown. Here, we show that selective optogenetic stimulation of a molecularly defined subset of LH GABAergic (Vgat-expressing) neurons enhances both appetitive and consummatory behaviors, whereas genetic ablation of these neurons reduced these phenotypes. Furthermore, this targeted LH subpopulation is distinct from cells containing the feeding-related neuropeptides, melanin-concentrating hormone (MCH), and orexin (Orx). Employing in vivo calcium imaging in freely behaving mice to record activity dynamics from hundreds of cells, we identified individual LH GABAergic neurons that preferentially encode aspects of either appetitive or consummatory behaviors, but rarely both. These tightly regulated, yet highly intertwined, behavioral processes are thus dissociable at the cellular level.

Visualizing hypothalamic network dynamics for appetitive and consummatory behaviors.

Many lines of theoretical and experimental investigation have suggested that gamma oscillations provide a temporal framework for cortical information processing, acting to either synchronize neuronal firing, restrict neuron's relative spike times, and/or provide a global reference signal to which neurons encode input strength. Each theory has been disputed and some believe that gamma is an epiphenomenon. We investigated the biophysical plausibility of these theories by performing in vitro whole-cell recordings from 6 cortical neuron subtypes and examining how gamma-band and slow fluctuations in injected input affect precision and phase of spike timing. We find that gamma is at least partially able to restrict the spike timing in all subtypes tested, but to varying degrees. Gamma exerts more precise control of spike timing in pyramidal neurons involved in cortico-cortical versus cortico-subcortical communication and in inhibitory neurons that target somatic versus dendritic compartments. We also find that relatively few subtypes are capable of phase-based information coding. Using simple neuron models and dynamic clamp, we determine which intrinsic differences lead to these variations in responsiveness and discuss both the flexibility and confounds of gamma-based spike-timing systems.

Cell Type-Specific Control of Spike Timing by Gamma-Band Oscillatory Inhibition.

Prolonged exposure to abnormally high calcium concentrations is thought to be a core mechanism underlying hippocampal damage in epileptic patients; however, no prior study has characterized calcium activity during seizures in the live, intact hippocampus. We have directly investigated this possibility by combining whole-brain electroencephalographic (EEG) measurements with microendoscopic calcium imaging of pyramidal cells in the CA1 hippocampal region of freely behaving mice treated with the pro-convulsant kainic acid (KA). We observed that KA administration led to systematic patterns of epileptiform calcium activity: a series of large-scale, intensifying flashes of increased calcium fluorescence concurrent with a cluster of low-amplitude EEG waveforms. This was accompanied by a steady increase in cellular calcium levels (>5 fold increase relative to the baseline), followed by an intense spreading calcium wave characterized by a 218% increase in global mean intensity of calcium fluorescence (n = 8, range [114-349%], p < 10(-4); t-test). The wave had no consistent EEG phenotype and occurred before the onset of motor convulsions. Similar changes in calcium activity were also observed in animals treated with 2 different proconvulsant agents, N-methyl-D-aspartate (NMDA) and pentylenetetrazol (PTZ), suggesting the measured changes in calcium dynamics are a signature of seizure activity rather than a KA-specific pathology. Additionally, despite reducing the behavioral severity of KA-induced seizures, the anticonvulsant drug valproate (VA, 300 mg/kg) did not modify the observed abnormalities in calcium dynamics. These results confirm the presence of pathological calcium activity preceding convulsive motor seizures and support calcium as a candidate signaling molecule in a pathway connecting seizures to subsequent cellular damage. Integrating in vivo calcium imaging with traditional assessment of seizures could potentially increase translatability of pharmacological intervention, leading to novel drug screening paradigms and therapeutics designed to target and abolish abnormal patterns of both electrical and calcium excitation.

Article	Year
Experience effects on brain development: possible contributions to psychopathology. Journal of child psychology and psychiatry, and allied disciplines\| PubMed ID: 12553412	2003
Dopamine D4 receptor signaling in the rat paraventricular hypothalamic nucleus: Evidence of natural coupling involving immediate early gene induction and mitogen activated protein kinase pho... Neuropharmacology\| PubMed ID: 16324724	2006
Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties. CNS neuroscience & therapeutics\| PubMed ID: 18482100	2008
Selective alpha7 nicotinic acetylcholine receptor activation regulates glycogen synthase kinase3beta and decreases tau phosphorylation in vivo. Brain research\| PubMed ID: 19230830	2009
Cell type-specific control of neuronal responsiveness by gamma-band oscillatory inhibition. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20147542	2010
Metabolic cost as a unifying principle governing neuronal biophysics. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20616090	2010
Zolpidem reduces hippocampal neuronal activity in freely behaving mice: a large scale calcium imaging study with miniaturized fluorescence microscope. PloS one\| PubMed ID: 25372144	2014
Visualizing hypothalamic network dynamics for appetitive and consummatory behaviors. Cell\| PubMed ID: 25635459	2015
Cell Type-Specific Control of Spike Timing by Gamma-Band Oscillatory Inhibition. Cerebral cortex (New York, N.Y. : 1991)\| PubMed ID: 25778344	2016
Direct Imaging of Hippocampal Epileptiform Calcium Motifs Following Kainic Acid Administration in Freely Behaving Mice. Frontiers in neuroscience\| PubMed ID: 26973444	2016

Stephani Otte

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