Parvalbumin Interneuron Dysfunction in Neurological Disorders: Focus on Epilepsy and Alzheimer's Disease.

Parvalbumin expressing (PV+) GABAergic interneurons are fast spiking neurons that provide powerful but relatively short-lived inhibition to principal excitatory cells in the brain. They play a vital role in feedforward and feedback synaptic inhibition, preventing run away excitation in neural networks. Hence, their dysfunction can lead to hyperexcitability and increased susceptibility to seizures. PV+ interneurons are also key players in generating gamma oscillations, which are synchronized neural oscillations associated with various cognitive functions. PV+ interneuron are particularly vulnerable to aging and their degeneration has been associated with cognitive decline and memory impairment in dementia and Alzheimer's disease (AD). Overall, dysfunction of PV+ interneurons disrupts the normal excitatory/inhibitory balance within specific neurocircuits in the brain and thus has been linked to a wide range of neurodevelopmental and neuropsychiatric disorders. This review focuses on the role of dysfunctional PV+ inhibitory interneurons in the generation of epileptic seizures and cognitive impairment and their potential as targets in the design of future therapeutic strategies to treat these disorders. Recent research using cutting-edge optogenetic and chemogenetic technologies has demonstrated that they can be selectively manipulated to control seizures and restore the balance of neural activity in the brains of animal models. This suggests that PV+ interneurons could be important targets in developing future treatments for patients with epilepsy and comorbid disorders, such as AD, where seizures and cognitive decline are directly linked to specific PV+ interneuron deficits.

Developmental Inhibitory Changes in the Primary Somatosensory Cortex of the Stargazer Mouse Model of Absence Epilepsy.

Childhood absence epilepsy seizures arise in the cortico-thalamocortical network due to multiple cellular and molecular mechanisms, which are still under investigation. Understanding the precise mechanisms is imperative given that treatment fails in ~30% of patients while adverse neurological sequelae remain common. Impaired GABAergic neurotransmission is commonly reported in research models investigating these mechanisms. Recently, we reported a region-specific reduction in the whole-tissue and synaptic GABAA receptor (GABAAR) α1 subunit and an increase in whole-tissue GAD65 in the primary somatosensory cortex (SoCx) of the adult epileptic stargazer mouse compared with its non-epileptic (NE) littermate. The current study investigated whether these changes occurred prior to the onset of seizures on postnatal days (PN) 17-18, suggesting a causative role. Synaptic and cytosolic fractions were biochemically isolated from primary SoCx lysates followed by semiquantitative Western blot analyses for GABAAR α1 and GAD65. We found no significant changes in synaptic GABAAR α1 and cytosolic GAD65 in the primary SoCx of the stargazer mice at the critical developmental stages of PN 7-9, 13-15, and 17-18. This indicates that altered levels of GABAAR α1 and GAD65 in adult mice do not directly contribute to the initial onset of absence seizures but are a later consequence of seizure activity.

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.

The Impact of Glutamatergic Synapse Dysfunction in the Corticothalamocortical Network on Absence Seizure Generation.

Childhood absence epilepsy (CAE) is the most common pediatric epilepsy affecting 10-18% of all children with epilepsy. It is genetic in origin and the result of dysfunction within the corticothalamocortical (CTC) circuitry. Network dysfunction may arise from multifactorial mechanisms in patients from different genetic backgrounds and thus account for the variability in patient response to currently available anti-epileptic drugs; 30% of children with absence seizures are pharmaco-resistant. This review considers the impact of deficits in AMPA receptor-mediated excitation of feed-forward inhibition (FFI) in the CTC, on absence seizure generation. AMPA receptors are glutamate activated ion channels and are responsible for most of the fast excitatory synaptic transmission throughout the CNS. In the stargazer mouse model of absence epilepsy, the genetic mutation is in stargazin, a transmembrane AMPA receptor trafficking protein (TARP). This leads to a defect in AMPA receptor insertion into synapses in parvalbumin-containing (PV+) inhibitory interneurons in the somatosensory cortex and thalamus. Mutation in the Gria4 gene, which encodes for the AMPA receptor subunit GluA4, the predominant AMPA receptor subunit in cortical and thalamic PV + interneurons, also leads to absence seizures. This review explores the impact of glutamatergic synapse dysfunction in the CTC network on absence seizure generation. It also discusses the cellular and molecular mechanisms involved in the pathogenesis of childhood absence epilepsy.

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.

Chemogenetic Activation of Feed-Forward Inhibitory Parvalbumin-Expressing Interneurons in the Cortico-Thalamocortical Network During Absence Seizures.

Parvalbumin-expressing (PV+) interneurons are a subset of GABAergic inhibitory interneurons that mediate feed-forward inhibition (FFI) within the cortico-thalamocortical (CTC) network of the brain. The CTC network is a reciprocal loop with connections between cortex and thalamus. FFI PV+ interneurons control the firing of principal excitatory neurons within the CTC network and prevent runaway excitation. Studies have shown that generalized spike-wave discharges (SWDs), the hallmark of absence seizures on electroencephalogram (EEG), originate within the CTC network. In the stargazer mouse model of absence epilepsy, reduced FFI is believed to contribute to absence seizure genesis as there is a specific loss of excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at synaptic inputs to PV+ interneurons within the CTC network. However, the degree to which this deficit is directly related to seizure generation has not yet been established. Using chemogenetics and in vivo EEG recording, we recently demonstrated that functional silencing of PV+ interneurons in either the somatosensory cortex (SScortex) or the reticular thalamic nucleus (RTN) is sufficient to generate absence-SWDs. Here, we used the same approach to assess whether activating PV+ FFI interneurons within the CTC network during absence seizures would prevent or reduce seizures. To target these interneurons, mice expressing Cre recombinase in PV+ interneurons (PV-Cre) were bred with mice expressing excitatory Gq-DREADD (hM3Dq-flox) receptors. An intraperitoneal dose of pro-epileptic chemical pentylenetetrazol (PTZ) was used to induce absence seizure. The impact of activation of FFI PV+ interneurons during seizures was tested by focal injection of the "designer drug" clozapine N-oxide (CNO) into either the SScortex or the RTN thalamus. Seizures were assessed in PVCre/Gq-DREADD animals using EEG/video recordings. Overall, DREADD-mediated activation of PV+ interneurons provided anti-epileptic effects against PTZ-induced seizures. CNO activation of FFI either prevented PTZ-induced absence seizures or suppressed their severity. Furthermore, PTZ-induced tonic-clonic seizures were also reduced in severity by activation of FFI PV+ interneurons. In contrast, administration of CNO to non-DREADD wild-type control animals did not afford any protection against PTZ-induced seizures. These data demonstrate that FFI PV+ interneurons within CTC microcircuits could be a potential therapeutic target for anti-absence seizure treatment in some patients.

Altered Neurotransmitter Expression in the Corticothalamocortical Network of an Absence Epilepsy Model with impaired Feedforward Inhibition.

The episodes of brief unconsciousness in patients with childhood absence epilepsy are a result of corticothalamocortical circuitry dysfunction. This dysfunction may arise from multifactorial mechanisms in patients from different genetic backgrounds. In previous studies using the epileptic stargazer mutant mouse, which experience frequent absence seizures, we reported a deficit in AMPAR-mediated feed-forward inhibition of parvalbumin-containing (PV+) interneurons. Currently, in order to determine the downstream effects of this impairment on neurotransmitter expression, we performed HPLC of tissue lysates and post-embedding electron microscopy from the cortical and thalamic regions. We report region-specific alterations in GABA expression, but not of glutamate, and most prominently at PV+ synaptic terminals. These results suggest that impaired feed forward inhibition may occur via reduced activation of these interneurons and concomitant decreased GABAergic signaling. Further investigations into GABAergic control of corticothalamocortical network activity could be key in our understanding of absence seizure pathogenesis.

The impact of silencing feed-forward parvalbumin-expressing inhibitory interneurons in the cortico-thalamocortical network on seizure generation and behaviour.

Feed-forward inhibition (FFI) is an essential mechanism within the brain, to regulate neuronal firing and prevent runaway excitation. In the cortico-thalamocortical (CTC) network, fast spiking parvalbumin-expressing (PV+) inhibitory interneurons regulate the firing of pyramidal cells in the cortex and relay neurons in the thalamus. PV+ interneuron dysfunction has been implicated in several neurological disorders, including epilepsy. Previously, we demonstrated that loss of excitatory AMPA-receptors, specifically at synapses on PV+ interneurons in CTC feedforward microcircuits, occurs in the stargazer mouse model of absence epilepsy. These mice present with absence seizures characterized by spike and wave discharges (SWDs) on electroencephalogram (EEG) and concomitant behavioural arrest, similar to childhood absence epilepsy. The aim of the current study was to investigate the impact of loss of FFI within the CTC on absence seizure generation and behaviour using new Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology. We crossed PV-Cre mice with Cre-dependent hM4Di DREADD strains of mice, which allowed Cre-recombinase-mediated restricted expression of inhibitory Gi-DREADDs in PV+ interneurons. We then tested the impact of global and focal (within the CTC network) silencing of PV+ interneurons. CNO mediated silencing of all PV+ interneurons by intraperitoneal injection caused the impairment of motor control, decreased locomotion and increased anxiety in a dose-dependent manner. Such silencing generated pathological oscillations similar to absence-like seizures. Focal silencing of PV+ interneurons within cortical or thalamic feedforward microcircuits, induced SWD-like oscillations and associated behavioural arrest. Epileptiform activity on EEG appeared significantly sooner after focal injection compared to peripheral injection of CNO. However, the mean duration of each oscillatory burst and spike frequency was similar, irrespective of mode of CNO delivery. No significant changes were observed in vehicle-treated or non-DREADD wild-type control animals. These data suggest that dysfunctional feed-forward inhibition in CTC microcircuits may be an important target for future therapy strategies for some patients with absence seizures. Additionally, silencing of PV+ interneurons in other brain regions may contribute to anxiety related neurological and psychiatric disorders.

Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments.

There is ample evidence for localization of messenger RNAs (mRNAs) and protein synthesis in neuronal dendrites; however, demonstrations of these processes in presynaptic terminals are limited. We used expansion microscopy to resolve pre- and postsynaptic compartments in rodent neurons. Most presynaptic terminals in the hippocampus and forebrain contained mRNA and ribosomes. We sorted fluorescently labeled mouse brain synaptosomes and then sequenced hundreds of mRNA species present within excitatory boutons. After brief metabolic labeling, >30% of all presynaptic terminals exhibited a signal, providing evidence for ongoing protein synthesis. We tested different classic plasticity paradigms and observed distinct patterns of rapid pre- and/or postsynaptic translation. Thus, presynaptic terminals are translationally competent, and local protein synthesis is differentially recruited to drive compartment-specific phenotypes that underlie different forms of plasticity.

Cortical expression of AMPA receptors during postnatal development in a genetic model of absence epilepsy.

Childhood absence epilepsy has been associated with poor academic performance, behavioural difficulties, as well as increased risk of physical injury in some affected children. The frequent episodes of 'absence' arise from corticothalamocortical network dysfunction, with multifactorial mechanisms potentially involved in genetically different patients. Aberrations in glutamatergic neurotransmission has been implicated in some seizure models, and we have recently reported that reduced cortical AMPA receptor (AMPAR) expression (predominantly GluA4- containing AMPARs) in parvalbumin-containing (PV+) inhibitory interneurons, could underlie seizure generation in the stargazer mutant mouse. In the present study, we investigate AMPA receptor subunit changes occurring during postnatal development in the stargazer mouse, to determine when these changes occur relative to seizure onset and thus could be contributory to seizure generation. Using quantitative western blotting, we analysed the expression of AMPAR GluA1-4 subunits in the somatosensory cortex at three critical time points; two before seizure onset (postnatal days (PN) 7-9 and 13-15), and one at seizure onset (PN17-18) in stargazers. We report that compared to their non-epileptic littermates, in the stargazer somatosensory cortex, there was a significant reduction in expression of AMPARs containing GluA1, 3 and 4 subunits prior to seizure onset, whereas reduction in expression of GluA2-AMPARs appears to be a post-seizure event. Thus, while loss of GluA4-containing AMPARs (likely GluA1/4 and GluA3/4) may be linked to seizure induction, the loss of GluA2-containing AMPARs is a secondary post-seizure mechanism, which is most likely involved in seizure maintenance.

Postnatal expression of thalamic GABAA receptor subunits in the stargazer mouse model of absence epilepsy.

Absence seizures are known to originate from disruptions within the corticothalamocortical network; however, the precise underlying cellular and molecular mechanisms that induce hypersynchronicity and hyperexcitability are debated and likely to be complex and multifactorial. Recent studies implicate impaired thalamic GABAergic inhibition as a common feature in multiple animal models of absence epilepsy, including the well-established stargazer mouse model. Recently, we demonstrated region-specific increases in the whole tissue and synaptic levels of GABAA receptor (GABAAR) subunits α1 and ß2, within the ventral posterior region of the thalamus in adult epileptic stargazer mice compared with nonepileptic control littermates. The objective of this study was to investigate whether such changes in GABAAR subunits α1 and ß2 can be observed before the initiation of seizures, which occur around postnatal (PN) days 16-18 in stargazers. Semiquantitative western blotting was used to analyze the relative tissue level expression of GABAAR α1 and ß2 subunits in the thalamus of juvenile stargazer mice compared with their nonepileptic control littermates at three different time points before the initiation of seizures. We show that there is a statistically significant increase in the expression of α1 and ß2 subunits in the thalamus of stargazer mice, at the PN7-9 stage, compared with the control littermates, but not at PN10-12 and PN13-15 stages. These results suggest that an aberrant expression of GABAAR subunits α1 and ß2 in the stargazers does not occur immediately before seizure onset and therefore is unlikely to directly contribute to the initiation of absence seizures.

Characterisation of early changes in ovine CLN5 and CLN6 Batten disease neural cultures for the rapid screening of therapeutics.

Batten disease (neuronal ceroid lipofuscinosis) refers to a group of neurodegenerative lysosomal storage diseases predominantly affecting children. There are currently no effective treatments, and the functions of many of the associated gene products are unknown. Here we characterise fetal neural cultures from two genetically distinct sheep forms of Batten disease, with mutations in the lysosomal protein encoding gene CLN5 and endoplasmic reticulum membrane protein encoding gene CLN6, respectively. We found similar reductions in autophagy, acidic organelles and synaptic recycling in both forms compared to unaffected cells. We then developed a high-throughput screen and tested for correction of deficient cells with lentiviral-mediated CLN5 or CLN6 gene transfer and fibrate drugs, gemfibrozil and fenofibrate in CLN6 deficient neural cultures. These assays provide a simple system to rapidly screen candidate therapies or libraries of drugs prior to in vivo testing.

Synaptic Changes in AMPA Receptor Subunit Expression in Cortical Parvalbumin Interneurons in the Stargazer Model of Absence Epilepsy.

Feedforward inhibition is essential to prevent run away excitation within the brain. Recent evidence suggests that a loss of feed-forward inhibition in the corticothalamocortical circuitry may underlie some absence seizures. However, it is unclear if this aberration is specifically linked to loss of synaptic excitation onto local fast-spiking parvalbumin-containing (PV+) inhibitory interneurons, which are responsible for mediating feedforward inhibition within cortical networks. We recently reported a global tissue loss of AMPA receptors (AMPARs), and a specific mistrafficking of these AMPARs in PV+ interneurons in the stargazer somatosensory cortex. The current study was aimed at investigating if cellular changes in AMPAR expression were translated into deficits in receptors at specific synapses in the feedforward inhibitory microcircuit. Using western blot immunolabeling on biochemically isolated synaptic fractions, we demonstrate a loss of AMPAR GluA1-4 subunits in the somatosensory cortex of stargazers compared to non-epileptic control mice. Furthermore, using double post-embedding immunogold-cytochemistry, we show a loss of GluA1-4-AMPARs at excitatory synapses onto cortical PV+ interneurons. Altogether, these data indicate a loss of synaptic AMPAR-mediated excitation of cortical PV+ inhibitory neurons. As the cortex is considered the site of initiation of spike wave discharges (SWDs) within the corticothalamocortical circuitry, loss of AMPARs at cortical PV+ interneurons likely impairs feed-forward inhibitory output, and contributes to the generation of SWDs and absence seizures in stargazers.

Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse.

Absence seizures arise from disturbances within the corticothalamocortical network, however the precise cellular and molecular mechanisms underlying seizure generation arising from different genetic backgrounds are not fully understood. While recent experimental evidence suggests that changes in inhibitory microcircuits in the cortex may contribute to generation of the hallmark spike-wave discharges, it is still unclear if altered cortical inhibition is a result of interneuron dysfunction due to compromised glutamatergic excitation and/or changes in cortical interneuron number. The stargazer mouse model of absence epilepsy presents with a genetic deficit in stargazin, which is predominantly expressed in cortical parvalbumin-positive (PV+) interneurons, and involved in the trafficking of glutamatergic AMPA receptors. Hence, in this study we examine changes in (1) the subunit-specific expression of AMPA receptors which could potentially result in a loss of excitation onto cortical PV+ interneurons, and (2) PV+ neuron density that could additionally impair cortical inhibition. Using Western blot analysis we found subunit-specific alterations in AMPA receptor expression in the stargazer somatosensory cortex. Further analysis using confocal fluorescence microscopy revealed that although there are no changes in cortical PV+ interneuron number, there is a predominant loss of GluA1 and 4 containing AMPA receptors in PV+ neurons in stargazers compared to non-epileptic controls. Taken together, these data suggest that the loss of AMPA receptors in PV+ neurons could impair their feed-forward inhibitory output, ultimately altering cortical network oscillations, and contribute to seizure generation in stargazers. As such the feed-forward inhibitory interneurons could be potential targets for future therapeutic intervention for some absence epilepsy patients.

AMPA Receptors as Therapeutic Targets for Neurological Disorders.

Almost every neurological disease directly or indirectly affects synapse function in the brain. However, these diseases alter synapses through different mechanisms, ultimately resulting in altered synaptic transmission and/or plasticity. Glutamate is the major neurotransmitter that mediates excitatory synaptic transmission in the brain through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. These receptors have therefore been identified as a target for the development of therapeutic treatments for neurological disorders including epilepsy, neurodegenerative diseases, autism, and drug addiction. The fact that AMPA receptors play a dominant role throughout the brain raises the significant challenge of selectively targeting only those regions affected by disease, and clinical trials have raised doubt regarding the feasibility of specifically targeting AMPA receptors for new therapeutic options. Benzamide compounds that act as positive allosteric AMPA receptor modulators, known as AMPAkines, can act on specific brain regions and were initially proposed to revolutionize the treatment of cognitive deficits associated with neurological disorders. Their therapeutic potential has since declined due to inconsistent results in clinical trials. However, recent advances in basic biomedical research are significantly increasing our knowledge of AMPA receptor structure, binding sites, and interactions with auxiliary proteins. In particular, the large complex of postsynaptic proteins that interact with AMPA receptor subunits have been shown to control AMPA receptor insertion, location, pharmacology, synaptic transmission, and plasticity. These proteins are now being considered as alternative therapeutic target sites for modulating AMPA receptors in neurological disorders.

Altered thalamic GABAA-receptor subunit expression in the stargazer mouse model of absence epilepsy.

PURPOSE: Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression. METHODS: Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy. KEY FINDINGS: Immunofluorescence confocal microscopy revealed that GABAAR α1 and ß2 subunits are predominantly expressed in the VP, whereas α3 and ß3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and ß2 subunit expression levels in the VP were significantly increased (α1: 33%, ß2: 96%) in epileptic stargazers, whereas α3 and ß3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates. SIGNIFICANCE: These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy.

Selective loss of AMPA receptor subunits at inhibitory neuron synapses in the cerebellum of the ataxic stargazer mouse.

AMPA receptor subunits (GluA1-4) are trafficked to membrane synaptic sites by transmembrane AMPA receptor regulatory proteins (TARPs). In the stargazer mutant mouse, expression of TARP-γ2 (stargazin) is severely reduced, resulting in cerebellar ataxia. Stargazer granule cells (GCs) have a complete loss of functional AMPARs, as γ2 is their main TARP; hence mossy fiber (MF)-GC synapses are silent. The aim of the current study was to investigate how the stargazin deficit affects expression levels of AMPAR subunits at output synapses from GC parallel fibers (PF) onto inhibitory neurons in the molecular layer. Cerebella from male litter-pairs of stargazer and control mice were analyzed by post-embedding immunogold-microscopy. Levels of GluA2/3 and GluA4 were evaluated by measuring relative density of immunogold at PF-Purkinje cell (PF-PC) and PF-interneuron (PF-In) synapses respectively. In total, 100 synapses were analyzed in each pair of stargazer and control littermates. GluA2/3 and GluA4 expression was significantly reduced throughout the stargazer cerebellar cortex. GluA2/3 levels were reduced by 52% (p<0.001) at PF-PC synapses, and GluA4 levels by 31% (p<0.001) at PF-In synapses in stargazers. In neither case, however, was there a total loss of synaptic AMPAR subunits as occurs at MF-GC synapses. As the inhibitory neurons of the molecular layer express other TARPs in addition to stargazin, TARP compensation may limit the loss of GluA subunits at these synapses and explain why they are not silent like the MF-GC synapses. These data suggest that the ataxic phenotype in stargazers is primarily due to absence of AMPARs at cerebellar MF-GC synapses.

Spatial learning-induced increase in agmatine levels at hippocampal CA1 synapses.

Agmatine, a metabolite of L-arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training-induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold-labeling and electron-microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague-Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning-induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter.

Loss of calcium channels in the cerebellum of the ataxic and epileptic stargazer mutant mouse.

The stargazer mouse displays cerebellar ataxia and absence epilepsy as a result of a single, recessive mutation on chromosome 15 which silences the expression of the voltage-dependent calcium channel (VDCC) subunit gamma2, termed stargazin. Stargazin is the predominant gamma-subunit expressed in the cerebellum and is essential for correct assembly and trafficking of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-subtype of glutamate receptors (AMPARs) to postsynaptic membranes. As a functional association between AMPARs and VDCCs has been reported, and loss of stargazin results in a loss of AMPA receptors at cerebellar synapses, we investigated whether the loss of stargazin might also change the expression levels of calcium channels at cerebellar synapses. We present data showing that the stargazin mutation affects the expression of postsynaptic L-type Ca(v)1.2 (alpha(1C)-class) but not presynaptic P/Q-type Ca(v)2.1 (alpha(1A)-class) calcium channel proteins at cerebellar synapses. Both Western blot and immunogold analyses demonstrated a significant reduction in the levels of L-type calcium channel Ca(v)1.2 at stargazer cerebellar synapses compared to their non-ataxic littermates. This is in contrast to stargazer hippocampal synapses where no differences were detected in Ca(v)1.2 and 2.1 levels compared to controls, likely due to compensation by subunit gamma8. The loss of L-type calcium channel Ca(v)1.2 at stargazer cerebellar synapses suggests that stargazin mutation may contribute to the loss of VDCCs at postsynaptic sites. It is therefore possible that stargazin is involved in the trafficking of both AMPARs and VDCCs or in the formation of a functional AMPA receptor-calcium channel complex in the postsynaptic membrane.

Selective reduction in synaptic proteins involved in vesicle docking and signalling at synapses in the ataxic mutant mouse stargazer.

The spontaneous recessive mutant mouse stargazer has a specific and pronounced deficit in brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. Cerebellar granule cells, in particular, show a selective and near-total loss of BDNF. The mutation involves a defect in the calcium channel subunit Cacng2. This severely reduces expression of stargazin. A stargazin-induced failure in BDNF expression is thought to underlie the cerebellar ataxia with which the mutant presents. BDNF is known to regulate plasticity at cerebellar synapses. However, relatively little is known about the mechanism involved. We previously demonstrated that the stargazer mutation affects the phenotype of cerebellar glutamatergic neurons. Stargazer neurons have less glutamate and proportionally fewer docked vesicles at presynaptic sites than controls. In the current study, we investigate the mechanism underlying BDNF-induced synaptic changes by analyzing alterations in synaptic signalling proteins in the stargazer cerebellum. Expression levels of synaptic proteins were evaluated by measuring relative density of immunogold label over granule cell terminals in ultrathin sections from ataxic stargazer mutants compared with matched nonataxic littermates. We show that there is a selective and marked depletion in the levels of vesicle-associated proteins (synaptobrevin, synaptophysin, synaptotagmin, and Rab3a) but not of plasma membrane-associated protein (SNAP-25) in the terminals of the BDNF-deficient granule cells. Changes are restricted to the cerebellum; levels in the hippocampus are unaltered. These data suggest that the BDNF deficits in the cerebellum of stargazer affect synaptic vesicle docking by selectively altering synaptic-protein distribution and abundance.