Neurosteroid replacement therapy for catamenial epilepsy

Perimenstrual catamenial epilepsy, the cyclical occurrence of seizure exacerbations near the time of menstruation, affects a high proportion of women of reproductive age with drug-refractory epilepsy. Enhanced seizure susceptibility in perimenstrual catamenial epilepsy is believed to be due to the withdrawal of the progesterone-derived GABA_A receptor modulating neurosteroid allopregnanolone as a result of the fall in progesterone at the time of menstruation. Studies in a rat pseudopregnancy model of catamenial epilepsy indicate that after neurosteroid withdrawal there is enhanced susceptibility to chemoconvulsant seizures. There is also a transitory increase in the frequency of spontaneous seizures in epileptic rats that had experienced pilocarpine-induced status epilepticus. In the catamenial epilepsy model, there is a marked reduction in the antiseizure potency of anticonvulsant drugs, including benzodiazepines and valproate, but an increase in the anticonvulsant potency and protective index of neurosteroids such as allopregnanolone and the neurosteroid analog ganaxolone. The enhanced seizure susceptibility and benzodiazepine-resistance subsequent to neurosteroid withdrawal may be related to reduced expression and altered kinetics of synaptic GABA_A receptors and increased expression of GABA_A receptor subunits (such as α4) that confer benzodiazepine insensitivity. The enhanced potency of neurosteroids may be due to a relative increase after neurosteroid withdrawal in the expression of neurosteroid-sensitive δ-subunit-containing perisynaptic or extrasynaptic GABA_A receptors. Positive allosteric modulatory neurosteroids and synthetic analogs such as ganaxolone may be administered to prevent catamenial seizure exacerbations, in what we call neurosteroid replacement therapy.     

Susceptibility of larval zebrafish to the seizurogenic activity of GABA type A receptor antagonists

Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABA_AR) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABA_AR antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABA_AR antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABA_AR antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABA_AR positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABA_AR antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABA_AR antagonism.     

Epilepsy in succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism

Objectives

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a gamma-aminobutyric acid (GABA) degradative defect. Epilepsy affects half of patients. The murine model is associated with a transition from absence to convulsive seizures in the third week, with fatal status epilepticus.

Methods

The clinical phenotype is reported from a patient database. Flumazenil-Positron Emission Topography (FMZ-PET) and Transcranial Magnetic Stimulation (TMS) were used to study GABA neurotransmission. Electrocorticography, single cell electrophysiology, and radioligand binding studies are reported from animal studies.

Results

Generalized seizures predominate, including tonic–clonic, atypical absence, and myoclonic. EEG discharges are typically generalized spike-wave. MRI shows a dentatopallidoluysian pattern. Sudden Unexpected Death in Epilepsy Patients (SUDEP) has occurred and the associated neuropathology reveals chronic excitotoxic injury in gloubus pallidus. Investigations using FMZ-PET and TMS support downregulation of GABA_A and GABA_B activity, respectively, in patients. Gamma-hydroxybutyrate (GHB) induces spike-wave discharges in homozygous null mice via GHB and GABA_B-mediated mechanisms. These resemble absence seizures and are abolished by a GABA_B receptor antagonist. Decreased binding of GABA_A and GABA_B receptor antagonists has been demonstrated in P19 and P14 null mice, respectively. Downregulation of GABA_A and GABA_B receptor subunits is observed by P14. GABA_A and GABA_B mediated potentials are reduced from P8-P14.

Conclusion

Generalized epilepsy and epileptiform discharges are characteristic of SSADH deficiency. Spontaneous absence seizures appear in null mice by the third week, which may be induced by GHB or GABA_B activity. Subsequent overuse dependent downregulation of GABA_A and GABA_B receptor activity may be associated with hyperexcitability concomitant with the transition to generalized seizures.     

Effect of minocycline on pentylenetetrazol-induced chemical kindled seizures in mice

Inflammation is one of the mechanisms involved in seizure induction. In this study, the effect of minocycline, an anti-inflammatory drug, was investigated on kindling acquisition. Chemical kindling was induced by injection of a subthreshold dose of pentylenetetrazol (PTZ; 37.5 mg/kg) in mice on every other day. Two groups of animals received minocycline (25 mg/kg) at 1 h before or 1 h after PTZ injection. Following the last PTZ injection, the changes in gene expression of TNF-α receptor, γ₂ subunit of GABA_A receptor and NR₂A subunit of NMDA receptor were assessed in the hippocampus and piriform cortex. Injection of minocycline before PTZ increased the latency to stage 4 seizure, and decreased the duration of stages 4 and 5 seizure. It also prevented the increase in the mRNA of NR₂A subunit of NMDA receptor in the hippocampus and removed the PTZ-induced increase in mRNA of γ₂ subunit of GABA_A receptor in piriform cortex of PTZ kindled mice. Minocycline also prevented the increase in TNF-α receptor gene expression in both hippocampus and piriform cortex. Injection of minocycline after PTZ had no significant effect on measured parameters. Therefore, it can be concluded that minocycline may exert an anticonvulsant effect through preventing the increase in GABA_A and NMDA receptor subunits. These effects are accompanied by a reduction in an important inflammation index, TNF-α receptor.     

Molecular mechanisms of antiseizure drug activity at GABAA receptors

The GABA_A receptor (GABA_AR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at GABA_ARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole, actions at the GABA_AR are the primary or only known mechanism of antiseizure action. For topiramate, felbamate, retigabine, losigamone and stiripentol, GABA_AR modulation is one of several possible antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABA_AR function, led to the development of ganaxolone. Other agents modulate GABAergic “tone” by regulating the synthesis, transport or breakdown of GABA. GABA_AR efficacy is also affected by the transmembrane chloride gradient, which changes during development and in chronic epilepsy. This may provide an additional target for “GABAergic” ASDs. GABA_AR subunit changes occur both acutely during status epilepticus and in chronic epilepsy, which alter both intrinsic GABA_AR function and the response to GABA_AR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered receptors may provide a novel approach for seizure prevention.     

Anticonvulsant effect of neural regeneration peptide 2945 on pentylenetetrazol-induced seizures in rats

Neuron regeneration peptides (NRPs) are small synthetic peptides that stimulate neural proliferation, migration, and differentiation with no apparent toxicity and high target specificity in CNS. The aim of this study was to investigate the effect of NRP2945 on seizure activity induced by pentylenetetrazol (PTZ) in rats. Using behavioural assessment and electrocorticographical recordings, the effects of different doses of NRP2945 (5–20 µg/kg) were tested on seizure attacks induced by PTZ injection. In addition, the effect of NRP2945 was evaluated on the production of dark neurons and expression of GABA_A receptor α and β subunits and GAD-65 in the hippocampus and somatosensory cortex of the rat brain. Intraperitoneal injection of NRP2945 at 20 µg/kg prevented seizure attacks after PTZ injection. NRP2945 at doses of 5 and 10 µg/kg significantly decreased the total duration of seizure attacks and reduced the amplitude, duration and latency of epileptiform burst discharges induced by PTZ. In addition, the peptide significantly inhibited the production of dark neurons in the hippocampus and somatosensory cortex of epileptic rats. NRP2945 also significantly increased the expression of GABA_A receptor α and β subunits and GAD-65 in the hippocampus and somatosensory cortex compared with PTZ treated rats. This study indicates that NRP2945 is able to prevent the seizure attacks and neuronal injuries induced by PTZ, likely by stimulating GABA_A and GAD-65 protein expression and/or protecting these components of GABAergic signalling from PTZ-induced alteration. Further studies are needed to elucidate the potential role of NRP2945 as an antiepileptic drug.     

Antisense oligonucleotide to GABAA receptor γ2 subunit induces limbic status epilepticus

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABA_A receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABA_A receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABA_A receptor and epilepsy. We show that selective inhibition of the expression of the GABA_A receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABA_A receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for the involvement of nigral GABAA receptors in seizures of adult rats

Several studies have implicated the substantia nigra GABAergic system in the mediation of seizures in adult rats. The present study examines whether the different GABA receptors (GABA_A and GABA_B), are preferentially involved in this GABAergic seizure suppression mechanism. Adult rats were intranigrally infused with muscimol (GABA_A receptor agonist), bicuculline (GABA_A receptor antagonist) or baclofen (GABA₁ receptor agonist) and were exposed to flurothyl seizures Results indicated that while infusions of muscimol had an anticonvulsant effect, infusions of bicuculline had a proconvulsant effect. Baclofen infusions were found to have no effect on seizures. These findings suggest an involvement of the nigral GABA_A receptors in the mediation of seizures in adult rats.     

Comparative Study of the Anticonvulsant Effect of γ-Aminobutyric Acid Agonists in the Feline Kindling Model of Epilepsy

We made a comparative study of the anticon-vulsant effect of GABA agonists on feline amygdala or hippocampal kindled seizures. Progabide (PGB) [γ-ami-nobutyric acid (GABA) receptor agonist 25–100 mg/kg intraperitoneally, i.p.] significantly reduced both the kindled seizure stage and after discharge (AD) duration in a dose-dependent manner. SKF89976A (GABA uptake inhibitor 0.5–2.0 mg/kg i.p.) also significantly reduced the kindled seizure stage. Toxic doses of SKF89976A caused generalized paroxysmal EEG discharges and myoclonus, but AD generation in the kindled focus was suppressed completely. Furthermore, γ-vinyl GABA (GABA cata-bolic enzyme inhibitor, GVG 50–200 mg/kg i.p.) significantly reduced the seizure stage, while causing prolongation of the AD duration. In contrast, baclofen (selective GABA_B receptor agonist, 1 or 5 mg/kg) did not show anticonvulsant effects on any parameters of kindled seizures. Therefore, these GABA agonists, which potentiate the inhibitory function of the GABA_A systems, have potent anticonvulsant effects on partial onset and secondarily generalized limbic seizures.     

The expression of GABAA receptor subunits in the substantia nigra is developmentally regulated and region-specific

The substantia nigra pars reticulata (SNR) controls the spread of seizures. GABA_Aergic drug (agonist or antagonist) infusions into the SNR have age-specific and site-specific effects on flurothyl-induced seizures. Developmental and cell-specific regulation of GABA_A receptor subunit expression may be responsible for these specific effects. To test this hypothesis,in situ hybridization was used to examine regional expression of α1 and γ2L GABA_A receptor subunit mRNAs in the SNR during development. Distinct temporal and spatial patterns of expression were observed. In rats at postnatal days (PN) 21–60, fewer neurons were labeled with probes directed to α1 and γ2L subunits in SNR_anterior compared with SNR_posterior. In addition, neurons in SNR_anterior contained higher amounts of hybridization grains than in SNR_posterior. In PN 15 rats, the labeling of neurons was relatively diffuse throughout the anterior and posterior SNR regions with moderate amounts of hybridization grains for both subunits. The finding of age-related differential distribution of α1 and γ2L subunit mRNAs in the SNR suggests that GABA_A receptor heterogeneity may play a role in the age-specific and site-specific effects of GABA_Aergic agents on seizures in the SNR.     

Effects of temperature elevation on neuronal inhibition in hippocampal neurons of immature and mature rats

Febrile seizures are the most common seizure type in children, and hyperthermia may contribute to seizure generation during fever. We have previously demonstrated that hyperthermia suppressed γ‐aminobutyric acid (GABA)‐ergic synaptic transmission in CA1 neurons of immature rats. However, whether this suppression is age‐dependent is unknown. Moreover, it is unclear whether hyperthermia has differential effects on neuronal inhibition in CA1 pyramidal cells (PCs) and dentate gyrus granule cells (GCs). In this study, we investigated the effects of hyperthermia on GABA_A and GABA_B receptor‐mediated inhibitory postsynaptic currents (IPSCs) in CA1 and DG neurons from immature (11–17 days old) and mature (6–8 weeks old) rats using whole‐cell recordings in vitro. In immature rats, hyperthermia decreased the peak amplitude of GABA_A receptor‐mediated IPSCs (GABA_A IPSCs) in PCs but not in GCs. However, hyperthermia decreased the decay time constant of GABA_A IPSCs to a similar extent in both PCs and GCs. In mature rats, hyperthermia decreased the peak amplitude but not the decay time constant of GABA_A IPSCs in both PCs and GCs. Hyperthermia decreased charge transfer (area) of the GABA_A IPSC of PCs more in immature than in mature rats. In contrast, hyperthermia decreased the GABA_B receptor‐mediated IPSCs to the same degree in immature and mature rats, for either CA1 or DG neurons. Because the hippocampus has been found to be involved in hyperthermia‐induced behavioral seizures in immature rats, we suggest that the higher sensitivity of CA1 inhibitory synaptic function to hyperthermia in immature compared with mature rats might partially explain the higher susceptibility for febrile seizures in immature animals. © 2009 Wiley‐Liss, Inc.     

Cocaine: evidence for NMDA-, β-carboline- and dopaminergic-mediated seizures in mice

The present study was undertaken to examine the role of the benzodiazepine/GABA and N-methyl-d-aspartate (NMDA) systems in the convulsive effect of cocaine in mice. When cocaine (3.5 mg/ml) solution was infused into the tail vein at a rate of 0.3 ml/min, mice showed clonic and tonic convulsions. These seizures were not affected by low doses of bicuculline or picrotoxin, a GABA_A receptor antagonist and a Cl ion channel blocker, respectively. Aminooxyacetic acid (AOAA), a GABA deaminase inhibitor, and phenobarbital, a Cl ion channel activator, and baclofen, a GABA_B receptor agonist, also had no effect on these convulsions. Benzodiazepine inverse agonist β-DMCM, at a dose which by itself had no convulsive effect lowered the convulsive threshold of cocaine. This lowered convulsive threshold was reversed by flumazenil, a benzodiazepine inverse antagonist, and diazepam, a benzodiazepine full agonist, which by themselves did not inhibit cocaine seizure. It is likely that cocaine seizure involves a benzodiazepine (β-carboline) recognition site other than the benzodiazepine/GABA_A receptor–Cl ionophore complex system. CPP and MK-801, competitive and noncompetitive NMDA receptor antagonists, respectively, inhibited cocaine seizures. The inhibitory effects of CPP on cocaine convulsion were reversed by a low dose of NMDA, which by itself did not induce seizure. A dopamine D₁ receptor agonist SKF38393 enhanced both clonic and tonic convulsions, while a dopamine D₂ receptor agonist bromocriptine inhibited these convulsions. These stimulatory and inhibitory effects were reversed by the D₁ and D₂ receptor antagonists, SCH23390 and haloperidol, respectively. These results suggest that the cocaine-induced convulsion may involve an activation of the NMDA–Ca ionophore complex system, which is mediated by the dopaminergic system, and a β-carboline recognition site other than the benzodiazepine/GABA_A receptor–Cl ionophore complex system.     

Decreased GABABR expression and increased neuronal cell death in developing rat brain after PTZ-induced seizure

The objective of this study was to evaluate the PTZ-induced seizures effects on GABA_B receptor (R) expression and to observe its neurodegenerative effect in hippocampal part of developing rat brain. In the present study, high dose of pentylenetetrazol (PTZ 40 mg/kg) was injected in developing rats of age 5 weeks having average weight of 60–65 g for 4 days. Further, baclofen (B 3 mg/kg i.p) agonist and phaclofen (P 30 μg/rat) antagonist of GABA_BR were injected along with PTZ. Western blot analysis was used to elucidate expression of GABA_BR protein upon PTZ, baclofen and phaclofen exposure in the developing rat brain. Furthermore, PTZ-induced apoptotic neurodegeneration was also observed through the release of caspase-3 antibody and propidium iodide (PI) staining using confocal microscopy. Seizure was confirmed using electroencephalography (EEG) data obtained from the Laxtha EEG-monitoring device in the EEG recording room and EEG was monitored 5–15 min after PTZ injection. The results of the present study showed that PTZ-induced seizure significantly decreased GABA_BR expression and induced neuronal apoptosis in cortical and hippocampal part of brain. While, baclofen reverse the effect of PTZ by increasing the expression of GABA_BR as compared to the PTZ- , PTZ plus B- and PTZ plus P-treated groups. Our findings indicated that PTZ-induced seizure showed not only decrease in GABA_BR expression but also cause neuronal apoptosis in the developing rat brain.     

Ganaxolone

Ganaxolone (3α-hydroxy-3β-methyl-5α-pregnan-20-one) (GNX) is the 3β-methylated synthetic analog of allo-pregnanolone; it belongs to a class of compounds referred to as neurosteroids. GNX is an allosteric modulator of GABA_A receptors acting through binding sites which are distinct from the benzodiazepine binding site. It has activity in a broad range of animal models of epilepsy. GNX has been shown to be well tolerated in adults and children. In early phase II studies, GNX has been shown to have activity in adult patients with partialonset seizures and epileptic children with history of infantile spasms. It is currently undergoing further development in infants with newly diagnosed infantile spasms, in women with catamenial epilepsy, and in adults with refractory partial-onset seizures.     

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

γ‐Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABA_A receptor. Nineteen different subunits (α1‐6, β1‐3, γ1‐3, δ, ε, π, θ, ρ1‐3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABA_A receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure‐ and age‐characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3‐immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure‐ and age‐characteristic colocalization between subunits during development suggests differences in GABA_A receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. J. Comp. Neurol. 524:1805–1824, 2016. © 2015 Wiley Periodicals, Inc.     

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress,inflammation, and cognitive impairment in rats: Possible mechanisms of neuroprotection

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5 mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic–clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABA_A receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABA_A receptor to produce anticonvulsant effects and to ameliorate cognitive impairment.     

Effects of angiotensin III and angiotensin IV on pentylenetetrazol seizure susceptibility (threshold and kindling): interaction with adenosine A1 receptors

The effects of angiotensin (ANG) III and ANG IV on pentylenetetrazol (PTZ) seizure susceptibility—threshold and kindling in mice—as well as the influence of adenosine A₁ receptor agents (agonist and antagonist) on these effects were studied. It was found that ANG III and ANG IV increased dose-dependently the PTZ seizure threshold and decreased the seizure intensity in PTZ kindled mice. Cyclohexyladenosine (CHA), an adenosine A₁ receptor agonist, potentiated the effects of ANG III and ANG IV on the seizure threshold and kindling, whereas DPCPX (an A₁ receptor antagonist) reversed peptide-induced effects on the PTZ kindling. Taken together, ANG III and ANG IV decrease the PTZ seizure susceptibility. We could suggest that these effects are realized in part through interaction with adenosine A₁ receptors.     

Hippocampal loss of the GABAA receptor α1 subunit in patients with chronic pharmacoresistant epilepsies

Alterations of gamma aminobutyric acid (GABA)-mediated neurotransmission have been implicated in the pathogenesis of epilepsies. Here we examine the distribution of the GABA_A receptor in the hippocampus of 78 surgical specimens from patients with chronic pharmacoresistant focal epilepsies. The receptor was localized immunohistochemically with the monoclonal antibody bd-24 which selectively recognizes the ₁ subunit of the GABA_A receptor. The results were compared with the receptor distribution of 28 normal hippocampal specimens obtained at autopsy. In the great majority of the surgical specimens a loss of GABA_A receptor immunoreactivity was present in CA1 (92.3%), CA4 (78.2%), the dentate granular cell layer (70.5%) and the molecular layer of the dentate gyrus (65.4%). The subiculum revealed a normal staining pattern in all but 4 cases. In no instance did we observe an increase of immunoreactivity in any region or cell population. The decrease of GABA_A receptor immunoreactivity was closely related to neuronal loss in the respective specimen and to Ammon's horn sclerosis. There was no correlation between GABA_A receptor loss and the patient's age at surgery, duration of seizures, age at onset of seizures and to the presence or absence of secondary generalized tonic clonic seizures. The data suggest that the observed loss of GABA_A receptor immunoreactivity is a secondary phenomenon rather than an event that is relevant for the pathogenesis of epileptic seizures.     

GABAA-receptor heterogeneity in the adult rat brain: Differential regional and cellular distribution of seven major subunits

GABA_A-receptors display an extensive structural heterogeneity based on the differential assembly of a family of at least 15 subunits (α1–6, β1–3, γ1–3, θ, ρl–2) into distinct heteromeric receptor complexes. The subunit composition of receptor subtypes is expected to determine their physiological properties andipharmacological profiles, thereby contributing to flexibility in signal transduction and allosteric modulation. In heterologous expression systems, functional receptors require a combination of α-, β-, and γ-subunit variants, the γ2-subunit being essential to convey a classical benzodiazepine site to the receptor. The subunit composition and stoichiometry of native GABA_A-receptor subtypes remain unknown. The aim of this study was to identify immunohistochemically the main subunit combinations expressed in the adult rat brain and to allocate them to identified neurons. The regional and cellular distribution of seven major subunits (α1, α2, α3, α5, β2,3, γ2, δ) was visualized by immunoperoxidase staining with subunit-specific antibodies (the β2- and β3-subunits were covisualized with the monoclonal antibody bd-17). Putative receptor subtypes were identified on the basis of colocalization of subunits within individual neurons, as analyzed by confocal laser microscopy in double- and triple-immunofluoreseence staining expeximents. The results reveal an extraordinary heterogeneity in the distribution of GABA_A-receptor subunits, as evidenced by abrupt changes in immunoreactivity along well-defined cytoarchitectonic boundaries and by pronounced differences in the cellular distribution of subunits among various types of neurons. Thus, functionally and morphologically diverse neurons were characterized by a distinct GABA_A-receptor subunit repertoire. The pultiple staining experiments identified 12 subunit combinations in defined neurons. The most prevalent combination was the triplet α1/β2,3/γ2, detected in numerous cell types throughout the brain. An additional subunit (α2, α3, or δ) sometimes was associated with this triplet, pointing to the existence of receptors containing four subunits. The triplets α2/β2,3/γ2, α3/β2,3/γ2, and α5/β2,3/γ2 were also identified in discrete cell populations. The prevalence of these seven combinations suggest that they represent major GABAA-receptor subtypes. Five combinations also apparently lacked the β2,3-subunits, including one devoid of γ2-subunit (α1/α2/γ2, α2/γ2, α3/γ2, α2/α3/γ2, α2/α5/δ). These combinations were selectively associated with small neuron populations, thereby representing minor GABA_A receptor subtypes. These results provide the basis for a functional analysis of GABA_A-receptor subtypes of known subunit composition and may open the way for unproved therapeutic approaches based on the development of subtype-selective drugs. © 1995 Wiley-Liss, Inc.     

Increased number of GABAB receptors in the lethargic (lh/lh) mouse model of absence epilepsy

This study begins to explore mechanisms underlying the role of GABA_B receptors in absence seizures in lethargic (lh/lh) mice. To test the hypothesis that alterations intrinsic to the GABA_B receptor underlie enhanced synaptic activation of these receptors in absence seizures, we measured GABA-displaceable [³H]baclofen binding to neocortical plasma membranes prepared from lh/lh and wild (+/+) age-matched congenic mice. The number (B_max) of binding sites was significantly greater (20%) in lh/lh (4.2pmol/mg protein, n = 43 pairs, P < 0.02) than in +/+ mice (3.3 pmol/mg protein) in an age-dependent manner. Interestingly, the subject of lh/lh mice with greater seizure frequency (40–70 seizures/15 min, measured by bipolar electrodes implanted into neocortex; n = 11) had a significantly greater B_max (P < 0.003) than the subset with lower seizure frequency (1–20 seizures/15 min; n = 11). The equilibrium dissociation constant (K_d) was unchanged (60 nM in both). The K_d of both strains was inhibited to an equal degree by the nonhydrolysable GTP analogue 5′-guanylimido-diphosphate [Gpp(NH)p]. The increased number of GABA_B binding sites was selective, because binding to NMDA sites ([³H]glutamate binding) and to GABA_A sites ([³H]muscimol binding) was not significantly different in the two strains. These data suggest that the increased number of GABA_B receptors in lh/lh mice underlies enhanced synaptic activation of these receptors. Together with evidence that GABA_B receptor activation can produce disinhibition, our data support a role for GABA_B receptors in the expression of absence seizures in lh/lh mice.