The role of HCN channels on the effects of T-type calcium channels and GABAA receptors in the absence epilepsy model of WAG/Rij rats.

In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.

mGlu3 Metabotropic Glutamate Receptors as a Target for the Treatment of Absence Epilepsy: Preclinical and Human Genetics Data.

BACKGROUND: Previous studies suggest that different metabotropic glutamate (mGlu) receptor subtypes are potential drug targets for treating absence epilepsy. However, no information is available on mGlu3 receptors. OBJECTIVE: To examine whether (i) changes of mGlu3 receptor expression/signaling are found in the somatosensory cortex and thalamus of WAG/Rij rats developing spontaneous absence seizures; (ii) selective activation of mGlu3 receptors with LY2794193 affects the number and duration of spikewave discharges (SWDs) in WAG/Rij rats; and (iii) a genetic variant of GRM3 (encoding the mGlu3 receptor) is associated with absence epilepsy. METHODS: Animals: immunoblot analysis of mGlu3 receptors, GAT-1, GLAST, and GLT-1; realtime PCR analysis of mGlu3 mRNA levels; assessment of mGlu3 receptor signaling; EEG analysis of SWDs; assessment of depressive-like behavior. Humans: search for GRM3 and GRM5 missense variants in 196 patients with absence epilepsy or other Idiopathic Generalized Epilepsy (IGE)/ Genetic Generalized Epilepsy (GGE) and 125,748 controls. RESULTS: mGlu3 protein levels and mGlu3-mediated inhibition of cAMP formation were reduced in the thalamus and somatosensory cortex of pre-symptomatic (25-27 days old) and symptomatic (6-7 months old) WAG/Rij rats compared to age-matched controls. Treatment with LY2794193 (1 or 10 mg/kg, i.p.) reduced absence seizures and depressive-like behavior in WAG/Rij rats. LY2794193 also enhanced GAT1, GLAST, and GLT-1 protein levels in the thalamus and somatosensory cortex. GRM3 and GRM5 gene variants did not differ between epileptic patients and controls. CONCLUSION: We suggest that mGlu3 receptors modulate the activity of the cortico-thalamo-cortical circuit underlying SWDs and that selective mGlu3 receptor agonists are promising candidate drugs for absence epilepsy treatment.

Altered GABAA Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

Absence seizures are hyperexcitations within the cortico-thalamocortical (CTC) network, however the underlying causative mechanisms at the cellular and molecular level are still being elucidated and appear to be multifactorial. Dysfunctional feed-forward inhibition (FFI) is implicated as one cause of absence seizures. Previously, we reported altered excitation onto parvalbumin-positive (PV+) interneurons in the CTC network of the stargazer mouse model of absence epilepsy. In addition, downstream changes in GABAergic neurotransmission have also been identified in this model. Our current study assessed whether dysfunctional FFI affects GABAA receptor (GABAAR) subunit expression in the stargazer primary somatosensory cortex (SoCx). Global tissue expression of GABAAR subunits α1, α3, α4, α5, ß2, ß3, γ2 and δ were assessed using Western blotting (WB), while biochemically isolated subcellular fractions were assessed for the α and δ subunits. We found significant reductions in tissue and synaptic expression of GABAAR α1, 18% and 12.2%, respectively. However, immunogold-cytochemistry electron microscopy (ICC-EM), conducted to assess GABAAR α1 specifically at synapses between PV+ interneurons and their targets, showed no significant difference. These data demonstrate a loss of phasic GABAAR α1, indicating altered GABAergic inhibition which, coupled with dysfunctional FFI, could be one mechanism contributing to the generation or maintenance of absence seizures.

Comparison of astrocytes and gap junction proteins in the white matter of genetic absence epileptic and control rats: an experimental study.

OBJECTIVES: The role of white matter astrocytes in absence epilepsy is unknown. The present study aims to quantify astrocytic markers glial fibrillary acidic protein (GFAP), gap junction's proteins connexin 30 (Cx30) and connexin 43 (Cx43) in the corpus callosum (CC) of genetic absence epileptic rats from Strasbourg (GAERS), Wistar albino glaxo rats from Rijswijk (WAG/Rij)and compare the results with control animals. METHODS: -The density of GFAP, Cx30 and Cx43 positive astrocytes in per unite area were quantified in the CC of GAERS, WAG/Rij and control animals using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR). The quantifications were made from three regions of CC; below the primary somatosensory (S1BF), below the motor (M1) and below the retrosplenial (RSG) cortices. RESULTS: oThe number GFAP, Cx30 and Cx43 immunopositive astrocytes showed heterogeneous distribution within the CC. The GFAP immunopositive astrocytes was significantly high in the S1BF region of the three strains. The immunopositive GFAP and Cx43 showed significant decrease in the S1BF and M1 regions in GAERS and WAG/Rij compared to control animals, however, an increase in the immunopositive Cx30 was observed in the same regions in both GAERS and WAG/Rij compared to control Wistar animals but the increase was significant for GAERS but not for WAG/Rij. The RT-qPCR analysis was corroborated by GFAP immunohistochemistry results. CONCLUSION: The different expression pattern of the two Cx's in the CC of the epileptic strains compared to control animals may indicate a compensatory response or maybe the cause of generalization of absence seizures.

Does astrocyte gap junction protein expression differ during development in absence epileptic rats?

Intercellular communication via gap junctions (GJs) has a wide variety of complex and essential functions in the CNS. In the present developmental study, we aimed to quantify the number of astrocytic GJs protein connexin 30 (Cx30) of genetic model of absence epilepsy rats from Strasbourg (GAERS) at postnatal P10, P30, and P60 days in the epileptic focal areas involved in the cortico-thalamic circuit. We compared the results with Wistar rats using immunohistochemistry and western blotting. The number of Cx30 immunopositive astrocytes per unit area were quantified for the somatosensory cortex (SSCx), ventrobasal (VB), and lateral geniculate (LGN) thalamic nuclei of the two strains and Cx30 western blot was applied to the tissue samples from the same regions. Both immunohistochemical and western blot results revealed the presence of Cx30 in all regions studied at P10 in both Wistar and GAERS animals. The SSCx, VB, and LGN of Wistar animals showed progressive increase in the number of Cx30 immunopositive labeled astrocytes from P10 to P30 and reached a peak at P30; then a significant decline was observed from P30 to P60 for the SSCx and VB. However, in GAERS Cx30 immunopositive labeled astrocytes showed a progressive increase from P10 to P60 for all brain regions studied. The immunohistochemical data highly corresponded with western blotting results. We conclude that the developmental disproportional expression of Cx30 in the epileptic focal areas in GAERS may be related to the onset of absence seizures or may be related to the neurogenesis of absence epilepsy.

AMPK-mediated potentiation of GABAergic signalling drives hypoglycaemia-provoked spike-wave seizures.

Metabolism regulates neuronal activity and modulates the occurrence of epileptic seizures. Here, using two rodent models of absence epilepsy, we show that hypoglycaemia increases the occurrence of spike-wave seizures. We then show that selectively disrupting glycolysis in the thalamus, a structure implicated in absence epilepsy, is sufficient to increase spike-wave seizures. We propose that activation of thalamic AMP-activated protein kinase, a sensor of cellular energetic stress and potentiator of metabotropic GABAB-receptor function, is a significant driver of hypoglycaemia-induced spike-wave seizures. We show that AMP-activated protein kinase augments postsynaptic GABAB-receptor-mediated currents in thalamocortical neurons and strengthens epileptiform network activity evoked in thalamic brain slices. Selective thalamic AMP-activated protein kinase activation also increases spike-wave seizures. Finally, systemic administration of metformin, an AMP-activated protein kinase agonist and common diabetes treatment, profoundly increased spike-wave seizures. These results advance the decades-old observation that glucose metabolism regulates thalamocortical circuit excitability by demonstrating that AMP-activated protein kinase and GABAB-receptor cooperativity is sufficient to provoke spike-wave seizures.

Activity of the Lateral Hypothalamus during Genetically Determined Absence Seizures.

(1) Background: Absence seizures (ASs) are sudden, transient lapses of consciousness associated with lack of voluntary movements and generalized 2.5-4 Hz spike-wave discharges (SWDs) in the EEG. In addition to the thalamocortical system, where these pathological oscillations are generated, multiple neuronal circuits have been involved in their modulation and associated comorbidities including the serotonergic system. Neuronal activity in one of the major synaptic input structures to the brainstem dorsal raphé nucleus (DRN), the lateral hypothalamus (LH), has not been characterized. (2) Methods: We used viral tract tracing and optogenetics combined with in vitro and in vivo electrophysiology to assess the involvement of the LH in absence epilepsy in a genetic rodent model. (3) Results: We found that a substantial fraction of LH neurons project to the DRN of which a minority is GABAergic. The LH to DRN projection can lead to monosynaptic iGluR mediated excitation in DRN 5-HT neurons. Neuronal activity in the LH is coupled to SWDs. (4) Conclusions: Our results indicate that a brain area involved in the regulation of autonomic functions and heavily innervating the RN is involved in ASs. The decreased activity of LH neurons during SWDs could lead to both a decreased excitation and disinhibition in the DRN. These results support a long-range subcortical regulation of serotonergic neuromodulation during ASs and further our understanding of the state-dependence of these seizures and some of their associated comorbidities.

Vasoactive intestinal peptide (VIP) conducts the neuronal activity during absence seizures: GABA seems to be the main mediator of VIP.

Absence epilepsy is classified as a childhood generalized epilepsy syndrome with distinctive electroencephalographic patterns. The Wistar Albino Glaxo originating from Rijswijk (WAG/Rij) strain is a very well validated animal model of absence epilepsy that also shows behavioral deficits. In addition to the gastrointestinal system, VIP is highly expressed throughout numerous brain regions, and it plays crucial roles as a neurotransmitter and as a neuromodulatory, neurotrophic and neuroprotective factor in both the central and peripheral nervous systems. In this study, adult WAG/Rij rats were divided into two groups (n = 10): a group that was administered VIP (25 ng/kg i.p.) every 2 days for 15 days and an age-matched control group that was administered physiological saline. Electrical brain activity and behavior (depressive- like behavior, learning and memory and anxiety) were investigated in both groups. In addition, the extracellular concentrations of GABA and glutamate and the GABA/glutamate ratio were measured by high-performance liquid chromatography in microdialysate samples collected from the somatosensorial cortex of WAG/Rij rats. Our results demonstrated that VIP treatment significantly suppressed the total duration and number of spike wave discharges in WAG/Rij rats. However, VIP had no significant effect on behavior. VIP increased the extracellular concentration of GABA and the GABA/glutamate ratio in the somatosensory cortex. In conclusion, VIP has suppressive effects on absence seizures, possibly by increasing the GABA concentration and inducing the transformation of glutamate to GABA in the somatosensory cortex of WAG/Rij rats.

Group I metabotropic glutamate receptor-mediated long term depression is disrupted in the hippocampus of WAG/Rij rats modelling absence epilepsy.

Absence epilepsy is frequently associated with cognitive dysfunction, although the underlying mechanisms are not well understood. Here we report that some forms of hippocampal synaptic plasticity are abnormal in symptomatic Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Metabotropic Glu 1/5 receptor-mediated long term depression (LTD) at Schaffer collateral CA1 synapses is significantly reduced in symptomatic, 5-6 months old WAG/Rij rats compared to age-matched non epileptic control rats. There were no significant changes in mGlu1/5-dependent LTD in pre-symptomatic, 4-6 weeks old WAG/Rij rats compared to age matched controls. The changes in LTD found in symptomatic WAG/Rij forms are not indicative of general deficits in all forms of synaptic plasticity as long term potentiation (LTP) was unchanged. Immunoblot analysis of hippocampal tissue showed a significant reduction in mGlu5 receptor expression, a trend to an increase in pan Homer protein levels and a decrease in GluA1 receptor expression in the hippocampus of symptomatic WAG/Rij rats vs non-epileptic control rats. There were no changes in mGlu1α receptor or GluA2 protein levels. These findings suggest that abnormalities in hippocampal mGlu5 receptor-dependent synaptic plasticity are associated with the pathological phenotype of WAG/Rij rats. This lays the groundwork for the study of mGlu5 receptors as a candidate drug target for the treatment of cognitive dysfunction linked to absence epilepsy.

Cerebellar contribution to absence epilepsy.

The new aggregate data analyses revealed the earlier missing role of cerebellum long-term electrical stimulation in the absence epilepsy. Neurophysiologic data gained by authors favor that cerebellar serial deep brain stimulation (DBS) (100 Hz) causes the transformation of penicillin-induced cortical focal discharges into prolonged 3,5-3,75 sec oscillations resembling spike-wave discharges (SWD) in cats. Such SWDs were not organized in the form of bursts and persisted continuously after stimulation. Therefore, the appearance of prolonged periods of SWD is regarded as a tonic cerebellar influence upon pacemaker of SWD and might be caused by the long-lasting DBS-induced increase of GABA-ergic extrasynaptic inhibition in the forebrain networks. The absence seizure facilitation caused by cerebellar DBS was discussed with the reviewed data on optogenetic stimulation, neuronal activity of cerebellar structures, and imaging data.

Impact of Dysfunctional Feed-Forward Inhibition on Glutamate Decarboxylase Isoforms and γ-Aminobutyric Acid Transporters.

Absence seizures are associated with generalised synchronous 2.5-4 Hz spike-wave discharges causing brief and sudden alteration of awareness during childhood, which is known as childhood absence epilepsy (CAE). CAE is also associated with impaired learning, psychosocial challenges, and physical danger. Absence seizures arise from disturbances within the cortico-thalamocortical (CTC) network, including dysfunctional feed-forward inhibition (FFI); however, the precise mechanisms remain unclear. In epileptic stargazers, a genetic mouse model of CAE with chronic seizures, levels of γ-aminobutyric acid (GABA), and expression of GABA receptors are altered within the CTC network, implicating altered GABAergic transmission in absence seizures. However, the expression of GABA synthesising enzymes (GAD65 and GAD67) and GABA transporters (GAT-1 and 3) have not yet been characterised within absence seizure models. We found a specific upregulation of GAD65 in the somatosensory cortex but not the thalamus of epileptic stargazer mice. No differences were detected in GAD67 and GAT-3 levels in the thalamus or somatosensory cortex. Then, we assessed if GAD65 upregulation also occurred in Gi-DREADD mice exhibiting acute absence seizures, but we found no change in the expression profiles of GAD65/67 or GAT-3. Thus, the upregulation of GAD65 in stargazers may be a compensatory mechanism in response to long-term dysfunctional FFI and chronic absence seizures.

Neurogenesis is Enhanced in Young Rats with Genetic Absence Epilepsy: An Immuno-electron Microscopic Study.

AIM: To investigate neurogenesis in both adult and 3-week-old genetic absence epilepsy rats from Strasbourg (GAERS) to determine if newly formed neurons within the dentate gyrus (DG) form synaptic contacts with GABAergic (gamma aminobutyric acid) and glutamatergic nerve terminals and compared to the control (non-GAERS) Wistar rats. MATERIAL AND METHODS: Brain tissue was processed for electron microscopic assessment. Thin sections from the hippocampal DG were double-labelled for anti-GABA or anti-VGLUT1 (vesicular glutamate transporter 1) and anti-doublecortin (DCX) antibodies using immunogold methodology and examined with the transmission electron microscope for morphological changes and to quantify the density of gold labeling. RESULTS: DCX immunoreactivity was demonstrated within axon terminals, dendrites and somata in all groups. DCX and GABA or VGLUT1 were found to be co-localized in the axon terminals in all groups. We observed that DCX-immunoreactive (-ir) profiles formed synaptic contacts with GABAergic and glutamatergic terminals. The percentage of DCX labeling in dendrites, compared to axons, and the percentage of DCX-ir terminal profiles forming asymmetrical synapses, compared to those forming symmetrical synapses, were increased in all groups compared to the control group. DCX immunoreactivity in the 21-day-old GAERS group was found to be increased compared to the Wistar group. CONCLUSION: We conclude that newly born neurons are incorporated into the local hippocampal network in both the GAERS and the control Wistar rats. The results suggest that the neurogenesis taking place in the hippocampus may also be involved in the mechanism underlying absence seizures in GAERS.

Enhancement of Ketone Supplements-Evoked Effect on Absence Epileptic Activity by Co-Administration of Uridine in Wistar Albino Glaxo Rijswijk Rats.

Both uridine and exogenous ketone supplements decreased the number of spike-wave discharges (SWDs) in a rat model of human absence epilepsy Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. It has been suggested that alleviating influence of both uridine and ketone supplements on absence epileptic activity may be modulated by A1 type adenosine receptors (A1Rs). The first aim was to determine whether intraperitoneal (i.p.) administration of a specific A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.2 mg/kg) and a selective adenosine A2A receptor antagonist (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine) (SCH 58261; 0.5 mg/kg) have a modulatory influence on i.p. 1000 mg/kg uridine-evoked effects on SWD number in WAG/Rij rats. The second aim was to assess efficacy of a sub-effective dose of uridine (i.p. 250 mg/kg) combined with beta-hydroxybutyrate salt + medium chain triglyceride (KSMCT; 2.5 g/kg, gavage) on absence epilepsy. DPCPX completely abolished the i.p. 1000 mg/kg uridine-evoked alleviating effect on SWD number whereas SCH 58261 was ineffective, confirming the A1R mechanism. Moreover, the sub-effective dose of uridine markedly enhanced the effect of KSMCT (2.5 g/kg, gavage) on absence epileptic activity. These results demonstrate the anti-epilepsy benefits of co-administrating uridine and exogenous ketone supplements as a means to treat absence epilepsy.

Ultrastructural GABA immunogold labeling in the substantia nigra pars reticulata of kindled genetic absence epilepsy rats.

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

IL-6 Receptor Blockade by Tocilizumab Has Anti-absence and Anti-epileptogenic Effects in the WAG/Rij Rat Model of Absence Epilepsy.

Increased expression of interleukin-6 (IL-6) both in cerebrospinal fluid (CSF) and plasma is closely associated with convulsive epilepsy and symptom severity of depression. By comparison, at present, little is known about the role of this cytokine in childhood (non-convulsive) absence epilepsy. The aim of this work was to investigate the potential effects of acute and chronic treatment with tocilizumab (TCZ, 10 and 30 mg/kg/day), on absence seizures, their development, and related psychiatric comorbidity in WAG/Rij rats. It is known that lipopolysaccharide (LPS)-induced changes in inflammatory processes increase absence epileptic activity. In order to study the central effects of TCZ, we investigated whether administration of this anti-IL-6R antibody could modulate the lipopolysaccharide (LPS) or IL-6-evoked changes in absence epileptic activity in WAG/Rij rats. Our results demonstrate that TCZ, at both doses, significantly reduced the development of absence seizures in adult WAG/Rij rats at 6 months of age (1 month after treatment suspension) compared with untreated controls, thus showing disease-modifying effects. Decreased absence seizure development at 6 months of age was also accompanied by reduced comorbid depressive-like behavior, whereas no effects were observed on anxiety-related behavior. Acute treatment with TCZ, at 30 mg/kg, had anti-absence properties lasting ~25 h. The co-administration TCZ with i.c.v. LPS or IL-6 showed that TCZ inhibited the worsening of absence seizures induced by both proinflammatory agents in the WAG/Rij rats, supporting a central anti-inflammatory-like protective action. These results suggest the possible role of IL-6 and consequent neuroinflammation in the epileptogenic process underlying the development and maintenance of absence seizures in WAG/Rij rats. Accordingly, IL-6 signaling could be a promising pharmacological target in absence epilepsy and depressive-like comorbidity.

Neuroligin 2 regulates absence seizures and behavioral arrests through GABAergic transmission within the thalamocortical circuitry.

Epilepsy and autism spectrum disorders (ASD) are two distinct brain disorders but have a high rate of co-occurrence, suggesting shared pathogenic mechanisms. Neuroligins are cell adhesion molecules important in synaptic function and ASD, but their role in epilepsy remains unknown. In this study, we show that Neuroligin 2 (NLG2) knockout mice exhibit abnormal spike and wave discharges (SWDs) and behavioral arrests characteristic of absence seizures. The anti-absence seizure drug ethosuximide blocks SWDs and rescues behavioral arrests and social memory impairment in the knockout mice. Restoring GABAergic transmission either by optogenetic activation of the thalamic reticular nucleus (nRT) presynaptic terminals or postsynaptic NLG2 expression in the thalamic neurons reduces the SWDs and behavioral arrests in the knockout mice. These results indicate that NLG2-mediated GABAergic transmission at the nRT-thalamic circuit represents a common mechanism underlying both epileptic seizures and ASD.

Evidence for altered insulin signalling in the brains of genetic absence epilepsy rats from Strasbourg.

Insulin-mediated signalling in the brain is critical for neuronal functioning. Insulin resistance is implicated in the development of some neurological diseases, although changes associated with absence epilepsy have not been established yet. Therefore, we examined the major components of PI3K/Akt-mediated insulin signalling in cortical, thalamic, and hippocampal tissues collected from Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic Control (NEC) rats. Insulin levels were also measured in plasma and cerebrospinal fluid (CSF). For the brain samples, the nuclear fraction (NF) and total homogenate (TH) were isolated and investigated for insulin signalling markers including insulin receptor beta (IRß), IR substrate-1 and 2 (IRS1 & 2), phosphatase and tensin homologue (PTEN), phosphoinositide 3-kinase phospho-85 alpha (PI3K p85α), phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate, protein kinase B (PKB/Akt1/2/3), glucose transporter-1 and 4 (GLUT1 & 4) and glycogen synthase kinase-3ß (GSK3ß) using western blotting. A significant increase in PTEN and GSK3ß levels and decreased PI3K p85α and pAkt1/2/3 levels were observed in NF of GAERS cortical and hippocampal tissues. IRß, IRS1, GLUT1, and GLUT4 levels were significantly decreased in hippocampal TH of GAERS compared to NEC. A non-significant increase in insulin levels was observed in plasma and CSF of GAERS rats. An insulin sensitivity assay showed decreased p-Akt level in cortical and hippocampal tissues. Together, altered hippocampal insulin signalling was more prominent in NF and TH compared to cortical and thalamic regions in GAERS. Restoring insulin signalling may improve the pathophysiology displayed by GAERS, including the spike-and-wave discharges that relate to absence seizures in patients.

Adult loss of Cacna1a in mice recapitulates childhood absence epilepsy by distinct thalamic bursting mechanisms.

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.

Cortical Somatosensory Neurons in WAG/Rij Rats Transform Firing Evoked by Simulation of Posterior Thalamic Nucleus from Tonic to Phasic at Age of 6 Months.

Functional peculiarities of paralemniscal subdivision of the thalamocortical system were examined in normal Wistar and in WAG/Rij rats genetically prone to absence epilepsy. In 6-7-month-old WAG/Rij characterized by developed epileptic activity, the response of cortical somatosensory neurons to single electrical stimulation of the posterior thalamic nucleus was phasic, whereas in normal Wistar rats, similar reaction was tonic. The study views this phasic response as neural equivalent of spike-wave discharges known as typical EEG symptom of absence epilepsy.