Distinct Features of Interictal Activity Predict Seizure Localization and Burden in a Mouse Model of Childhood Epilepsy.

The epileptic brain is distinguished by spontaneous seizures and interictal epileptiform discharges (IEDs). Basic patterns of mesoscale brain activity outside of seizures and IEDs are also frequently disrupted in the epileptic brain and likely influence disease symptoms, but are poorly understood. We aimed to quantify how interictal brain activity differs from that in healthy individuals, and identify what features of interictal activity influence seizure occurrence in a genetic mouse model of childhood epilepsy. Neural activity across the majority of the dorsal cortex was monitored with widefield Ca2+ imaging in mice of both sexes expressing a human Kcnt1 variant (Kcnt1m/m ) and wild-type controls (WT). Ca2+ signals during seizures and interictal periods were classified according to their spatiotemporal features. We identified 52 spontaneous seizures, which emerged and propagated within a consistent set of susceptible cortical areas, and were predicted by a concentration of total cortical activity within the emergence zone. Outside of seizures and IEDs, similar events were detected in Kcnt1m/m and WT mice, suggesting that the spatial structure of interictal activity is similar. However, the rate of events whose spatial profile overlapped with where seizures and IEDs emerged was increased, and the characteristic global intensity of cortical activity in individual Kcnt1m/m mice predicted their epileptic activity burden. This suggests that cortical areas with excessive interictal activity are vulnerable to seizures, but epilepsy is not an inevitable outcome. Global scaling of the intensity of cortical activity below levels found in the healthy brain may provide a natural mechanism of seizure protection.SIGNIFICANCE STATEMENT Defining the scope and structure of an epilepsy-causing gene variant's effects on mesoscale brain activity constitutes a major contribution to our understanding of how epileptic brains differ from healthy brains, and informs the development of precision epilepsy therapies. We provide a clear roadmap for measuring how severely brain activity deviates from normal, not only in pathologically active areas, but across large portions of the brain and outside of epileptic activity. This will indicate where and how activity needs to be modulated to holistically restore normal function. It also has the potential to reveal unintended off-target treatment effects and facilitate therapy optimization to deliver maximal benefit with minimal side-effect potential.

Increased thalamocortical synaptic response and decreased layer IV innervation in GAP-43 knockout mice.

The growth-associated protein, GAP-43, is an axonally localized neuronal protein with high expression in the developing brain and in regenerating neurites. Mice that lack GAP-43 (GAP-43 -/-) fail to form a whisker-related barrel map. In this study, we use GAP-43 -/- mice to examine GAP-43 synaptic function in the context of thalamocortical synapse development and cortical barrel map formation. Examination of thalamocortical synaptic currents in an acute brain slice preparation and in autaptic thalamic neurons reveals that GAP-43 -/- synapses have larger alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-mediated currents than controls despite similar AMPAR function and normal probability of vesicular release. Interestingly, GAP-43 -/- synapses are less sensitive to blockade by a competitive glutamate receptor antagonist, suggesting higher levels of neurotransmitter in the cleft during synaptic transmission. Field excitatory postsynaptic potentials (EPSPs) from GAP-43 -/- thalamocortical synapses reveal a reduced fiber response, and anatomical analysis shows reduced thalamic innervation of barrel cortex in GAP-43 -/- mice. Despite this fact synaptic responses in the field EPSPs are similar in GAP-43 -/- mice and wild-type littermate controls, and the ratio of AMPAR-mediated to N-methyl-d-aspartate receptor (NMDAR)-mediated currents (AMPAR:NMDAR ratio) is larger than normal. This suggests that GAP-43 -/- mice form fewer thalamocortical synapses in layer IV because of decreased anatomical innervation of the cortex, but the remaining contacts are individually stronger possibly due to increased neurotransmitter concentration in the synaptic cleft. Together, these results indicate that in addition to its well known role in axonal pathfinding GAP-43 plays a functional role in regulating neurotransmitter release.

Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain.

Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2.

Initially recognized for their importance in control of appetite, orexins (also called hypocretins) are neuropeptides that are also involved in regulating sleep, arousal, and cardiovascular function. Loss of orexin appears to be the primary cause of narcolepsy. Cells expressing the orexins are restricted to a discrete region of the hypothalamus, but their terminal projections are widely distributed throughout the brain. With the diversity of function and broad distribution of orexin terminals, it is not known whether the orexin cells constitute a homogeneous population. Because orexins produce neuroexcitatory effects, we hypothesized that orexin-containing neurons are glutamatergic. In the present study we used digoxigenin-labeled cRNA probes for the vesicular glutamate transporters, VGLUT1 and VGLUT2, for in situ hybridization studies in combination with immunohistochemical detection of orexin cell bodies in the hypothalamus. In general, cells in the hypothalamus expressed low levels of the vesicular glutamate transporters relative to other areas of the forebrain, such as the cortex and thalamus. Light labeling for VGLUT2 mRNA was detected in about 50% of the orexin-immunoreactive neurons, and a much smaller percentage (approximately 13%) of orexin-immunoreactive cells was found to express VGLUT1. Despite the fact that intense labeling for GAD67 mRNA was found in a large number of cells throughout the hypothalamus, none of the orexin-immunoreactive cells was found to be GABAergic. These findings, showing that many of the orexin neurons are glutamatergic, are consistent with the neuroexcitatory effects of orexin but suggest that another neurochemical phenotype may define the remaining subset of orexin neurons.