Hippocampal Spreading Depression Bilaterally Activates the Caudal Trigeminal Nucleus in Rodents

Hippocampus. 2003  |  Pubmed ID: 14620879

Spreading depression (SD) and migraine aura involve transiently altered (i.e., increased followed by decreased) electrophysiological activity that propagates at the distinctive rate of millimeters per minute (mm/min), leading to the suggestion that they (and perhaps pain from migraine) are causally related via changes in the same brain structure. Neocortex is considered the anatomical zone associated with migraine aura and is the sole area known to induce caudal trigeminal nucleus (TNC) activation from SD in rodents. However, classical evidence of SD in human neocortex is reported only with severe brain disease, while migraine is a common and comparatively benign disorder. Because SD occurs in human hippocampus, and memory dysfunction referable to hippocampus is seen in migraineurs, we determined whether recurrent SD confined to hippocampus in rat could induce TNC activation. Our work shows that recurrent hippocampal SD evoked a significant (P < 0.05-0.001) increase in bilateral c-fos immunostaining within TNC superficial laminae compared with sham controls. Furthermore, hippocampal SD occurred with a correlated and transient change in spontaneous activity and blood flow in the ipsilateral neocortex without spread of SD to that area. Thus, hippocampal SD may be a previously unrecognized, potential trigger for nociceptive activation of TNC perhaps associated with migraine.

Homeostatic Plasticity in Hippocampal Slice Cultures Involves Changes in Voltage-gated Na+ Channel Expression

Brain Research. Feb, 2004  |  Pubmed ID: 14751586

Neurons preserve stable electrophysiological properties despite ongoing changes in morphology and connectivity throughout their lifetime. This dynamic compensatory adjustment, termed 'homeostatic plasticity', may be a fundamental means by which the brain normalizes its excitability, and is possibly altered in disease states such as epilepsy. Despite this significance, the cellular mechanisms of homeostatic plasticity are incompletely understood. Using field potential analyses, we observed a compensatory enhancement of neural excitability after 48 h of activity deprivation via tetrodotoxin (TTX) in hippocampal slice cultures. Because activity deprivation can enhance voltage-gated sodium channel (VGSC) currents, we used Western blot analyses to probe for these channels in control and activity-deprived slice cultures. A significant upregulation of VGSCs expression was evident after activity deprivation. Furthermore, immunohistochemistry revealed this upregulation to occur along primarily pyramidal cell dendrites. Western blot analyses of cultures after 1 day of recovery from activity deprivation showed that VGSC levels returned to control levels, indicating that multiple molecular mechanisms contribute to enhanced excitability. Because of their longevity and in vivo-like cytoarchitecture, we conclude that slice cultures may be highly useful for investigating homeostatic plasticity. Furthermore, we demonstrate that enhanced excitability involves changes in channel expression with a targeted localization likely profound transform the integrative capacities of hippocampal pyramidal cells and their dendrites.

P/Q Ca2+ Channel Blockade Stops Spreading Depression and Related Pyramidal Neuronal Ca2+ Rise in Hippocampal Organ Culture

Hippocampus. 2004  |  Pubmed ID: 15132435

Ca2+ channels and pyramidal cell Ca2+ are involved in hippocampal spreading depression (SD), but their roles remain elusive. Accordingly, we characterized Ca2+ changes during SD in CA3 pyramidal neurons and determined whether Ca2+ channel antagonists could prevent SD. SD was induced in hippocampal organotypic cultures (HOTCs), in which experimental conditions can be rigorously controlled. SD was triggered by transient exposure to sodium acetate (NaAc)-based Ringer's coupled to an electrical pulse in the dentate gyrus and its occurrence confirmed with interstitial DC recordings. Pyramidal cell Ca2+ was measured with fura-2 filled cells and was quantified at the soma, proximal and more distal apical dendrites. Regional Ca2+ changes began simultaneously with the triggering pulse of SD and reached three distinct peaks before returning to baseline concomitant with the interstitial DC potential of SD. The first peak occurred within 5 s of the triggering pulse, was smallest, and heralded the onset of SD. The second Ca2+ change was the greatest and reached a peak 6 s later, during the early phase of SD. The third was intermediate in size and occurred 18 s later, as SD reached its maximum interstitial DC change. SD was prevented by nonselective Ca2+ blockade (Ni2+ and Cd2+) but not by either L-Ca2+ channel (nifedipine) or N-Ca2+ channel inhibition (omega-conotoxin GVIA). Importantly, SD was blocked by P/Q Ca2+ channel antagonism (omega-agatoxin-IVA), which also prompted a significant reduction in pyramidal cell Ca2+ change and hyperexcitability. These results show that the spatiotemporal pattern of pyramidal cell Ca2+ change with SD is multiphasic; they provide further evidence that these changes begin before electrophysiologic evidence of SD. Furthermore, they show that P/Q Ca2+ channel antagonism can prevent SD in HOTCs and it appears to do so by preventing the NaAc-induced increased pyramidal cell excitability from NaAc exposure, which may involve altered GABAergic transmission.

Multiplexed Cytokine Protein Expression Profiles from Spreading Depression in Hippocampal Organotypic Cultures

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Aug, 2004  |  Pubmed ID: 15362713

Cytokines are involved in ischemic tolerance, including that triggered by spreading depression (SD), yet their roles in neuroprotection remain incompletely defined. The latter may stem from the pleiotropic nature of these signaling molecules whose complexities for interaction might be better deciphered through simultaneous measurement of multiple targeted proteins. Accordingly, the authors used microsphere-based flow cytometric immunoassays and hippocampal organotypic cultures (HOTCs) to characterize the magnitude, time course, and diversity of cytokine (interleukin [IL] 1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) response to SD. GM-CSF was not detected in HOTCs or media. However, SD triggered a significant, generalized increase in seven cytokines evident in HOTCs 6 hours later, with the remaining cytokine, IL-1beta, becoming significantly different at 1 and 3 days. Additionally, these changes extended to include surrounding media for IL-6 and TNF-alpha by 1 and 3 days. This increase was localized to microglia via immunostaining for IL-1alpha, IL-1beta, and interferon-y. IL-10, although significantly more abundant in HOTCs 6 hours after SD, was significantly less abundant in surrounding media at that time and at 1 day. Finally, the generalized early increase in tissue cytokines later settled to a pattern at 3 days of recovery centering on changes in IL-1alpha, IL-1beta, and TNF-alpha, cytokines capable of modulating ischemic injury.

Optical Current Source Density Analysis in Hippocampal Organotypic Culture Shows That Spreading Depression Occurs with Uniquely Reversing Currents

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2005  |  Pubmed ID: 15829647

Spreading depression (SD) involves current flow through principal neurons, but the pattern of current flow over the expanse of susceptible tissues or individual principal neurons remains undefined. Accordingly, tissue and single cell maps made from digital imaging of voltage-sensitive dye changes in hippocampal organotypic cultures undergoing SD were processed via optical current source density analysis to reveal the currents associated with pyramidal neurons. Two distinctive current flow patterns were seen. The first was a trilaminar pattern (420 microm2) that developed with the onset of SD in CA3 pyramidal neurons, in which SD most often began. This initial pattern comprised a somatic current sink with current sources to either side in the dendrites that lasted for seconds extending into the first aspect of the classical "inverted saddle" interstitial direct current waveform of SD. Next, the somatic sink backpropagated at a speed of millimeters per minute into the proximal dendrites, resulting in a reversal of the initial current flow pattern to its second orientation, namely dendritic sinks associated with a somatic source. The latter persisted for the remainder of SD in CA3 and was the only pattern seen in CA1, in which SD was rarely initiated. This backpropagating SD current flow resembles that of activity-dependent synaptic activation. Retrograde and associative signaling via principal neuron current flow is a key means to affect tissue function, including synaptic activation and, by extension, perhaps SD. Such current-related postsynaptic signaling might not only help explain SD but also neuroprotection and migraine, two phenomena increasingly recognized as being related to SD.

Enhanced Integrated Stress Response Promotes Myelinating Oligodendrocyte Survival in Response to Interferon-gamma

The American Journal of Pathology. Nov, 2008  |  Pubmed ID: 18818381

The T-cell-derived, pleiotropic cytokine interferon (IFN)-gamma is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2alpha (eIF2alpha). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2alpha, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-gamma in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2alpha (p-eIF2alpha) in myelinating oligodendrocytes when exposure to IFN-gamma, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2alpha and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-gamma. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases.

Monomeric IgG is Neuroprotective Via Enhancing Microglial Recycling Endocytosis and TNF-alpha

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19020014

In brain, monomeric immunoglobin G (IgG) is regarded as quiescent and only poised to initiate potentially injurious inflammatory reactions via immune complex formation associated with phagocytosis and tumor necrosis factor alpha (TNF-alpha) production in response to disease. Using rat hippocampal slice and microglial cultures, here we show instead that physiological levels (i.e., 0.2-20 microg/ml) of monomeric IgG unassociated with disease triggered benign low-level proinflammatory signaling that was neuroprotective against CA1 area excitotoxicity and followed a U-shaped or hormetic dose-response. The data indicate that physiological IgG levels activated microglia by enhancing recycling endocytosis plus TNF-alpha release from these cells to produce the neuroprotection. Minocycline, known for its anti-inflammatory and neuroprotective effects when given after disease onset, abrogated IgG-mediated neuroprotection and related microglial effects when given before injury. In contrast, E-prostanoid receptor subtype 2 (EP2) activation, which served as an exemplary paracrine stimulus like the one expected from neuronal activity, amplified IgG-mediated increased microglial recycling endocytosis and TNF-alpha production. Furthermore, like monomeric IgG these EP2 related effects took days to be effective, suggesting both were adaptive anabolic effects consistent with those seen from other long-term preconditioning stimuli requiring de novo protein synthesis. The data provide the first evidence that brain monomeric IgG at physiological levels can have signaling function via enhanced recycling endocytosis/TNF-alpha production from microglia unassociated with disease and that these IgG-mediated changes may be a means by which paracrine signaling from neuronal activity influences microglia to evoke neuroprotection. The data provide further support that low-level proinflammatory neural immune signaling unassociated with disease enhances brain function.

TNF-α and Microglial Hormetic Involvement in Neurological Health & Migraine

Dose-response : a Publication of International Hormesis Society. 2010  |  Pubmed ID: 21191481

Environmental enrichment, i.e., increased intellectual, social, and physical activity makes brain more resilient to subsequent neurological disease. The mechanisms for this effect remain incompletely defined, but evidence shows tumor necrosis factor-alpha (TNF-α) is involved. TNF-α, at acutely high levels, possesses the intrinsic capacity to enhance injury associated with neurological disease. Conversely, the effect of TNF-α at low-levels is nutritive over time, consistent with physiological conditioning hormesis. Evidence shows that neural activity triggers low-level pro-inflammatory signaling involving TNF-α. This low-level TNF-α signaling alters gene expression, resulting in an enhanced resilience to disease. Brain-immune signaling may become maladaptive when increased activity is chronic without sufficient periods of reduced activity necessary for nutritive adaptation. Such tonically increased activity may explain, for example, the transformation of episodic to chronic migraine with related increased susceptibility to spreading depression, the most likely underlying cause of this malady. Thus, TNF-α, whose function is to alter gene expression, and its principal cellular source, microglia, seem powerfully positioned to orchestrate hormetic immune signaling that establishes the phenotype of neurological health and disease from brain activity.

Astrocyte Precursor Response to Embryonic Brain Injury

Brain Research. May, 2011  |  Pubmed ID: 21396923

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular, trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.

Spreading Depression Sends Microglia on Lévy Flights

PloS One. 2011  |  Pubmed ID: 21541289

Spreading depression (SD) is thought to cause migraine aura, and perhaps migraine, and includes a transient loss of synaptic activity preceded and followed by increased neuronal excitability. Activated microglia influence neuronal activity and play an important role in homeostatic synaptic scaling via release of cytokines. Furthermore, enhanced neuronal function activates microglia to not only secrete cytokines but also to increase the motility of their branches, with somata remaining stationary. While SD also increases the release of cytokines from microglia, the effects on microglial movement from its synaptic activity fluctuations are unknown. Accordingly, we used time-lapse imaging of rat hippocampal slice cultures to probe for microglial movement associated with SD. We observed that in uninjured brain whole microglial cells moved. The movements were well described by the type of Lévy flight known to be associated with an optimal search pattern. Hours after SD, when synaptic activity rose, microglial cell movement was significantly increased. To test how synaptic activity influenced microglial movement, we enhanced neuronal activity with chemical long-term potentiation or LPS and abolished it with TTX. We found that microglial movement was significantly decreased by enhanced neuronal activity and significantly increased by activity blockade. Finally, application of glutamate and ATP to mimic restoration of synaptic activity in the presence of TTX stopped microglial movement that was otherwise seen with TTX. Thus, synaptic activity retains microglial cells in place and an absence of synaptic activity sends them off to influence wider expanses of brain. Perhaps increased microglial movements after SD are a long-lasting, and thus maladaptive, response in which these cells increase neuronal activity via contact or paracrine signaling, which results in increased susceptibility of larger brain areas to SD. If true, then targeting mechanisms that retard activity-dependent microglial Lévy flights may be a novel means to reduce susceptibility to migraine.

Differential Effects of Low-dose Endotoxin on the Cerebral Circulation During Pregnancy

Reproductive Sciences (Thousand Oaks, Calif.). Dec, 2011  |  Pubmed ID: 21693776

It is well-known that the pregnant state is associated with increased sensitivity to endotoxin in renal and uterine circulations; however, the effects on the cerebral circulation are not known. Intravenous infusion of low-dose lipopolysaccharide ([LPS]; 1.5 μg/kg) to pregnant Wistar rats on day 15 of pregnancy caused significantly decreased myogenic tone of posterior cerebral arteries on day 20, which was not seen in similarly treated nonpregnant rats. Pregnancy alone was associated with a 2-to 4-fold increase in inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) messenger RNA (mRNA) in cerebral arteries compared to nonpregnant, suggesting that the cerebral circulation is in a state of inflammation during pregnancy. After LPS treatment, cerebral arteries from pregnant animals had increased iNOS and TNF-α compared to LPS-treated nonpregnant animals, but decreased interleukin 10 (IL-10) and IFN-γ. These results demonstrate that pregnancy enhances sensitivity to the effects of LPS in the cerebral circulation, which may be due to an enhanced inflammatory state during pregnancy.

Seizures in Women with Preeclampsia: Mechanisms and Management

Fetal and Maternal Medicine Review. May, 2011  |  Pubmed ID: 21709815

Cold Pre-conditioning Neuroprotection Depends on TNF-α and is Enhanced by Blockade of Interleukin-11

Journal of Neurochemistry. Apr, 2011  |  Pubmed ID: 21070241

Cold pre-conditioning reduces subsequent brain injury in small animals but the underlying mechanisms remain undefined. As hypothermia triggers systemic macrophage tumor necrosis factor alpha (TNF-α) production and other neural pre-conditioning stimuli depend on this cytokine, we reasoned that microglia and TNF-α would be similarly involved with cold pre-conditioning neuroprotection. Also, as slice cultures closely approximate their in vivo counterpart and include quiescent microglia, we used rat hippocampal slice cultures to confirm this hypothesis. Furthermore, inflammatory cytokine gene screening with subsequent PCR and immunostaining confirmation of targeted mRNA and related protein changes showed that cold pre-conditioning triggered a significant rise in TNF-α that localized to microglia and a significant rise in interleukin (IL)-11 that localized mainly to hippocampal pyramidal neurons and, more rarely, astrocytes. Importantly, co-stimulation with cold and IL-11, an anti-inflammatory cytokine that inhibits TNF-α expression, abrogated the otherwise evident protection. Instead, cold pre-conditioning coupled with blockade of IL-11 signaling further enhanced neuroprotection from that seen with cold pre-conditioning alone. Thus, physiological activation of brain pro-inflammatory cytokine signaling, and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning.

Pregnant Serum Induces Neuroinflammation and Seizure Activity Via TNFα

Experimental Neurology. Jan, 2012  |  Pubmed ID: 22281105

Preeclampsia is a hypertensive disorder of pregnancy that affects many organs including the brain. Neurological complications occur during preeclampsia, the most serious of which is seizure known as eclampsia. Although preeclampsia can precede the eclamptic seizure, it often occurs during normal pregnancy, suggesting that processes associated with normal pregnancy can promote neuronal excitability. Here we investigated whether circulating inflammatory mediators that are elevated late in gestation when seizure also occurs are hyperexcitable to neuronal tissue. Evoked field potentials were measured in hippocampal slices in which control horse serum that slices are normally grown in, was replaced with serum from nonpregnant or late-pregnant Wistar rats for 48h. We found that serum from pregnant, but not nonpregnant rats, caused hyperexcitability to hippocampal neurons and seizure activity that was abrogated by inhibition of tumor necrosis factor alpha (TNFα) signaling. Additionally, application of TNFα mimicked this increased excitability. Pregnant serum also caused morphological changes in microglia characteristic of activation, and increased TNFα mRNA expression that was not seen with exposure to nonpregnant serum. However, TNFα protein was not found to be elevated in pregnant serum itself, suggesting that other circulating factors during pregnancy caused activation of hippocampal slice cells to produce a TNFα-mediated increase in neuronal excitability. Lastly, although pregnant serum caused neuroinflammation and hyperexcitability of hippocampal slices, it did not increase blood-brain barrier permeability, nor were pregnant rats from which the serum was taken undergoing seizure. Thus, the BBB has an important role in protecting the brain from circulating neuroinflammatory mediators that are hyperexcitable to the brain during pregnancy. These studies provide novel insight into the underlying cause of eclampsia without elevated blood pressure and the protective role of the BBB that prevents exposure of the brain to hyperexcitable factors.