Hippocampal Neurons Express GABAA Receptor Insensitive to Diazepam in Hyperexcitable Conditions

Summary: Purpose : To define the properties of γ-aminobutyric acid-type A (GABA_A) receptors expressed on dentate granule cells in neonatal rats and to define the impact of prolonged seizures on GABA_A receptors in 28- to 35-day-old rats.
Methods : Whole GABA_A receptor currents were recorded from acutely isolated dentate granule cells. Cells were isolated from 7- to 14-day-old rats for the first experiment. For the second experiment, cells were isolated from 28- to 35-day-old naive rats and rats that had undergone 45 minutes of status epilepticus. Modulation of GABA_A receptor currents by diazepam (DZP), zinc, and zolpidem was studied.
Results : In 7- to 14-day-old rats, dentate granule cells express DZP- and zolpidem-insensitive, zinc-sensitive GABA_A receptors. In 28- to 35-day-old rats, dentate granule cells express DZP-sensitive GABA_A receptors. At the latter age, prolonged seizures render GABA_A receptors DZP-insensitive.
Conclusion : Hippocampal dentate granule cells express DZP-insensitive receptors in hyperexcitable states. These receptors are likely to contain α4 subunit.     

Activation of the GABAA Receptor Inhibits the Proliferative Effects of bFGF in Cortical Progenitor Cells

Recent studies have localized γ-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABA_A receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABA_A receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the α₁ subunit of the GABA_A receptor but not for the α₂, α₃ or α₅ subunits. The α₁ subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABA_A receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABA_A receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.     

Neuron loss,granule cell axon reorganization,and functional changes in the dentate gyrus of epileptic kainate-treated rats

We sought to describe quantitatively the morphological and functional changes that occur in the dentate gyrus of kainate-treated rats, an experimental model of temporal lobe epilepsy. Adult rats were treated systemically with kainic acid, and, months later, after displaying spontaneous recurrent motor seizures, their dentate gyri were examined. Histological, immunocytochemical, and quantitative stereological techniques were used to estimate numbers of neurons per dentate gyrus of various classes and to estimate the extent of granule cell axon reorganization along the septotemporal axis of the hippocampus in control rats and epileptic kainate-treated rats. Compared with control rats, epileptic kainate-treated rats had fewer Nissl-stained hilar neurons and fewer somatostatin-immunoreactive neurons. There was a correlation between the extent of hilar neuron loss and the extent of somatostatin-immunoreactive neuron loss. However, functional inhibition in the dentate gyrus, assessed with paired-pulse responses to perforant-pathway stimulation, revealed enhanced, and not the expected reduced, inhibition in epileptic kainate-treated rats. Numbers of parvalbumin- and cholecystokinin-immunoreactive neurons were similar in control rats and in most kainate-treated rats. A minority (36%) of the epileptic kainate-treated rats had fewer parvalbumin- and cholecystokinin-immunoreactive neurons than control rats, and those few (8%) with extreme loss in these interneuron classes showed markedly hyperexcitable dentate gyrus field-potential responses to orthodromic stimulation. Compared with control rats, epileptic kainate-treated rats had larger proportions of their granule cell and molecular layers infiltrated with Timm stain. There was a correlation between the extent of abnormal Timm staining and the extent of hilar neuron loss. Granule cell axon reorganization and dentate gyrus neuron loss were more severe in temporal vs. septal hippocampus. These findings from the dentate gyrus of epileptic kainate-treated rats are strikingly similar to those reported for human temporal lobe epilepsy, and they suggest that neuron loss and axon reorganization in the temporal hippocampus may be important in epileptogenesis. J. Comp. Neurol. 385:385–404, 1997. © 1997 Wiley-Liss, Inc.     

Cystatin C expression is associated with granule cell dispersion in epilepsy

Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis, during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater numbers of polysialylated neural cell adhesion molecule-positive newborn cells in the molecular layer, although the overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule cells.     

Linkage analysis between idiopathic generalized epilepsies and the GABAA receptor α5, β3 and γ3 subunit gene cluster on chromosome 15

Introduction - We tested the hypothesis that genetic variants within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). Material and methods - Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA_Aα₅, β₃ and γ₃ subunit gene cluster and three different IGE trait models. Results - Evidence against linkage to the GABA_Aα₅, β₃ and γ₃ subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Z_max=1.40 at θ_max=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (α=0.37; P =0.06) and an autosomal recessive mode of inheritance. Conclusion - The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.     

Developmental Patterns in the Regulation of Chloride Homeostasis and GABAA Receptor Signaling by Seizures

Summary:  GABA_A receptors have dual functions during development. They depolarize immature neurons but hyperpolarize more mature neurons. This functional switch has been attributed to age-related differences in the relative abundance of cation chloride cotransporters, such as KCC2 and NKCC1, which regulate chloride homeostasis. Certain insults, such as trauma, ischemia, and seizures, if they occur when GABA_Aergic signaling is hyperpolarizing, such as in the adult brain, can lead to reappearance of the immature, depolarizing synaptic responses to GABA_A receptor activation. In certain cases, this has been associated with either reduced expression of KCC2 or increase in NKCC1. In epilepsy, the depolarizing effects of GABA_A receptors have been proposed to be important for the acquisition and/or maintenance of the epileptic state. Using the kainic acid model of status epilepticus, we have studied the effects of repetitive neonatal episodes of status epilepticus on the expression of cation chloride cotransporter KCC2 in the neonatal hippocampus. In contrast to adults, seizures increased KCC2 mRNA expression in the CA3 region of the neonatal hippocampus. The contrasting patterns of regulation of KCC2 by seizures in mature and immature neurons may be one of the age-related factors that protect the neonatal brain against the development of epilepsy.     

Aberrant seizure-induced neurogenesis in experimental temporal lobe epilepsy

Neurogenesis in the hippocampal dentate gyrus persists throughout life and is increased by seizures. The dentate granule cell (DGC) layer is often abnormal in human and experimental temporal lobe epilepsy, with dispersion of the layer and the appearance of ectopic granule neurons in the hilus. We tested the hypothesis that these abnormalities result from aberrant DGC neurogenesis after seizure-induced injury. Bromodeoxyuridine labeling, in situ hybridization, and immunohistochemistry were used to identify proliferating progenitors and mature DGCs in the adult rat pilocarpine temporal lobe epilepsy model. We also examined dentate gyri from epileptic human hippocampal surgical specimens. Prox-1 immunohistochemistry and pulse-chase bromodeoxyuridine labeling showed that progenitors migrate aberrantly to the hilus and molecular layer after prolonged seizures and differentiate into ectopic DGCs in rat. Neuroblast marker expression indicated the delayed appearance of chainlike progenitor cell formations extending into the hilus and molecular layer, suggesting that seizures alter migratory behavior of DGC precursors. Ectopic putative DGCs also were found in the hilus and molecular layer of epileptic human dentate gyrus. These findings indicate that seizure-induced abnormalities of neuroblast migration lead to abnormal integration of newborn DGCs in the epileptic adult hippocampus, and implicate aberrant neurogenesis in the development or progression of recurrent seizures.     

G蛋白偶联内向整流钾通道亚基2 mRNA在颞叶癫疒间大鼠海马内的表达

目的探讨G蛋白偶联内向整流钾通道亚基2(GIRK 2)在颞叶癫癎大鼠海马内的表达变化.方法应用腹腔注射海人酸致癎大鼠,采用原位杂交法检测大鼠海马GIRK 2 mRNA的表达.结果 GIRK 2 mRNA在癫癎大鼠海马齿状回表达增加,与正常对照组相比差异有显著性( P<0.01).结论癫癎大鼠海马内GIRK2增高是机体对神经元网络过度兴奋的代偿反应.     

Computational models of dentate gyrus with epilepsy-induced morphological alterations in granule cells

Temporal lobe epilepsy provokes a number of different morphological alterations in granule cells of the hippocampus dentate gyrus. These alterations may be associated with the hyperactivity and hypersynchrony found in the epileptic dentate gyrus, and their study requires the use of different kinds of approaches including computational modeling. Conductance-based models of both normal and epilepsy-induced morphologically altered granule cells have been used in the construction of network models of dentate gyrus to study the effects of these alterations on epilepsy. Here, we review these models and discuss their contributions to the understanding of the association between alterations in neuronal morphology and epilepsy in the dentate gyrus.This article is part of a Special Issue entitled “NEWroscience 2013”.     

Pharmacological and Electrophysiological Properties of Recombinant GABAA Receptors Comprising the alpha3, beta1 and gamma2 Subunits

To assess the role of subunits for channel function and drug modulation in recombinant GABA_A receptors, the α3β1γ2 subunits and the dual combinations α3β1, β1γ2 and α3γ2 were expressed by transfection of human embryonic kidney cells and by RNA injection in Xenopus oocytes (α3β1γ2 combination). GABA-induced chloride currents were recorded using the whole-cell configuration of the patch-clamp technique (transfected cells) or the voltage-clamp technique (oocytes). The currents recorded from the α3β1Γ2 subunit combination in transfected cells were reduced by bicuculline and picrotoxin, enhanced by flunitrazepam in a flumazenil-sensitive manner and reduced by β-carboline-3-carboxylic acid methyl ester (β-CCM). The GABA-induced current was reduced by β-CCM in all combinations containing the γ2 subunit, but potentiation by flunitrazepam was only obtained when the 72 subunit was coexpressed in the presence of the α3 subunit (α3β1γ2 or α3γ2). The GABA sensitivities of the receptors were similar when the α3β1γ2 combination was expressed in oocytes (half-maximum effective concentration = 240 μM) or in the kidney cell line (270 μM). However, the currents were less potentiated by flunitrazepam in oocytes (129% of controls) than in transfected cells (189%). These results suggest that the α3β1γ2 subunit combination, which is coexpressed in various brain regions as shown by in situ hybridization histochemistry, may represent a building block of functional GABA_A receptors in situ.     

Status epilepticus in epileptogenesis

There has been direct evidence of gamma-aminobutyric acidA receptor modification during status epilepticus. Neuropeptides galanin and neuropeptide Y were demonstrated to play a role in terminating status epilepticus. Many of the CA3 pyramidal neurons destined to die as a consequence of status epilepticus were demonstrated to diminish expression of the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors. It was demonstrated that the pattern of cell loss due to status epilepticus is distinct in immature pups compared with adult rats. The genetic basis for susceptibility to neuronal loss during status epilepticus was described. There was increasing evidence of unique receptors and ion channels in the epileptic brain. The molecular studies of epileptic gamma-aminobutyric acidA receptors present on dentate granule cells of rats with temporal lobe epilepsy revealed altered gene and receptor expression before onset of recurrent spontaneous seizures. They also revealed insertion of new gamma-aminobutyric acidA receptors in the inhibitory synapses present on soma and proximal dendrites of dentate granule cells.     

GABAA Receptor Internalization during Seizures

Summary:  A rapid modification in the postsynaptic γ-aminobutyric acid (GABA_A) receptor population occurs during the prolonged seizures of status epilepticus (SE). This rapid modification contributes to a reduction in GABA-mediated inhibition and the development of benzodiazepine pharmacoresistance. Previous hypotheses to explain the modification have included an alteration in the structural composition or posttranslational modification of the receptors. In a cultured hippocampal neuron model, we found that there was differential subcellular distribution of GABA_A receptor subunits and that the constitutive internalization of GABA_A receptors containing a β2/3 subunit was rapid and activity-dependent. Based on this finding, we posit that an activity-dependent increase in the rate of internalization of synaptic GABA_A receptors during SE contributes to the reduction in inhibitory transmission and the development of benzodiazepine pharmacoresistance.     

Pharmacology and Biophysical Properties of α7 and α7 - α8 α-Bungarotoxin Receptor Subtypes Immunopurified from the Chick Optic Lobe

Two chick optic lobe α-bungarotoxin receptor subtypes (α7 and α7 - α8) were immunopurified using polyclonal antibodies raised against synthetic peptides of chick α7 and α8 α-bungarotoxin receptor subunits. The α7 subtype contained the M _r 57 000 α7 subunit, and represented 60 - 70% of the α-bungarotoxin receptors; the α7-α8 subtype contained the M _r 57 000 α7 and α8 subunits, and represented only 20 - 25% of the receptors. Both subtypes also had an additional M _r 52 000 subunit. The affinity of these subtypes for α-bungarotoxin as well as antagonists was similar. However, the α7 - α8 subtype displayed consistently higher affinities for agonists. When reconstituted in planar lipid bilayers, the α7 - α8 subtype displayed several conductance states of 10 - 50 pS; the α7 subtype had only one conductance state of 45 pS. The α7 -α8 subtype was activated by lower agonist concentrations than the α7 subtype. When expressed in Xenopus oocytes, the α8 subunit formed functional homomeric receptors that desensitized rapidly. These channels were blocked by α-bungarotoxin and displayed a higher affinity for agonists than the α7 homomeric receptor. Taken together, these data indicate that at least two α-bungarotoxin subtypes are present in the chick optic lobe. They operate as ligand-gated channels and display different agonist sensitivities and kinetics/conductance properties.     

Disruption of the neurogenic potential of the dentate gyrus in a mouse model of temporal lobe epilepsy with focal seizures

Adult hippocampal neurogenesis is enhanced in response to multiple stimuli including seizures. However, the relationship between neurogenesis and the development of temporal lobe epilepsy (TLE) remains unclear. Unilateral intrahippocampal injection of kainate in adult mice models the morphological characteristics (e.g. neuronal loss, gliosis, granule cell dispersion and hypertrophy) and occurrence of chronic, spontaneous recurrent partial seizures observed in human TLE. We investigated the influence of a kainate-induced epileptogenic focus on hippocampal neurogenesis, comparing neural stem cell proliferation following status epilepticus and spontaneous recurrent partial seizures. Cell proliferation in the subgranular zone was transiently increased bilaterally after kainate treatment. As a result, neurogenesis was stimulated in the contralateral dentate gyrus. In contrast, the epileptic hippocampus exhibited a strongly reduced neurogenic potential, even after onset of spontaneous recurrent partial seizures, possibly due to an alteration of the neurogenic niche in the subgranular zone. These results show that neurogenesis does not contribute to the formation of the epileptic focus and may be affected when dispersion of dentate gyrus granule cells occurs. Therefore, in patients with TLE, hippocampal sclerosis and granule cell dispersion may play a significant role in disrupting the potential for hippocampal neurogenesis.     

Dentate granule cell GABA(A) receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology

The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure-prone hippocampus. This filter or 'gatekeeper' attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABAA receptors (GABAARs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABAARs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc-sensitive GABAARs is coupled with delayed, epilepsy-induced development of a zinc delivery system provided by aberrant sprouting of zinc-containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal 'gatekeeper' function of the DG through dynamic zinc-induced failure of inhibition, predisposing the hippocampal circuit to generate seizures.     

Strong paired pulse depression of dentate granule cells in slices from patients with temporal lobe epilepsy

Summary. Recurrent, feedback excitation by sprouted mossy fibers may contribute to the hyperexcitability observed in human temporal lobe epilepsy. Observations in rodent models of epilepsy mimic the findings in human tissue and reveal that dentate granule cells sprout axons which innervate fibers in their own dendritic layer. However, recent evidence in rodents suggest that these sprouted fibers may form connections which cause inhibition of dentate granule cells, not excitation. Thus, the net effect of sprouting in human epileptic tissue may not only be recurrent excitation. We analyzed paired pulse depression in dentate slices from 9 patients with temporal lobe epilepsy and found evidence for strong feedback inhibition. We also noted failure of high frequency stimulation induced inhibition in our human specimens. These data challenge the concept that human epileptic dentate granule cells are excited by recurrent mossy fiber sprouting. Accepted February 20, 1998; received December 1997     

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2‐specific markers Purkinje cell protein 4 (PCP4) and regulator of G‐Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin‐expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE‐associated pathological restructuring. © 2015 Wiley Periodicals, Inc.     

Distribution of Nicotinic Receptors in the Human Hippocampus and Thalamus

Neuronal nicotinic acetylcholine receptors consist of different subunits, α and β, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of α3, α7 and β2 mRNA in the human brain was studied by in situ hybridization and compared to [³H]nicotine, [³H]cytisine and [¹²⁵l]α-bungarotoxin binding in contiguous sections. The β2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The α3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of α7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [³H]Nicotine and [³H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [¹²⁵l]α-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.     

Rapid and long-term alterations of hippocampal GABAB receptors in a mouse model of temporal lobe epilepsy

Alterations of gamma-aminobutyric acid (GABA)B receptor expression have been reported in human temporal lobe epilepsy (TLE). Here, changes in regional and cellular expression of the GABAB receptor subunits R1 (GBR1) and R2 (GBR2) were investigated in a mouse model that replicates major functional and histopathological features of TLE. Adult mice received a single, unilateral injection of kainic acid (KA) into the dorsal hippocampus, and GABAB receptor immunoreactivity was analysed between 1 day and 3 months thereafter. In control mice, GBR1 and GBR2 were distributed uniformly across the dendritic layers of CA1-CA3 and dentate gyrus. In addition, some interneurons were labelled selectively for GBR1. At 1 day post-KA, staining for both GBR1 and GBR2 was profoundly reduced in CA1, CA3c and the hilus, and no interneurons were visible anymore. At later stages, the loss of GABAB receptors persisted in CA1 and CA3, whereas staining increased gradually in dentate gyrus granule cells, which become dispersed in this model. Most strikingly, a subpopulation of strongly labelled interneurons reappeared, mainly in the hilus and CA3 starting at 1 week post-KA. In double-staining experiments, these cells were selectively labelled for neuropeptide Y. The number of GBR1-positive interneurons also increased contralaterally in the hilus. The rapid KA-induced loss of GABAB receptors might contribute to epileptogenesis because of a reduction in both presynaptic control of transmitter release and postsynaptic inhibition. In turn, the long-term increase in GABAB receptors in granule cells and specific subtypes of interneurons may represent a compensatory response to recurrent seizures.