Maternal diet during early gestation influences postnatal taste activity-dependent pruning by microglia.

A key process in central sensory circuit development involves activity-dependent pruning of exuberant terminals. Here, we studied gustatory terminal field maturation in the postnatal mouse nucleus of the solitary tract (NST) during normal development and in mice where their mothers were fed a low NaCl diet for a limited period soon after conception. Pruning of terminal fields of gustatory nerves in controls involved the complement system and is likely driven by NaCl-elicited taste activity. In contrast, offspring of mothers with an early dietary manipulation failed to prune gustatory terminal fields even though peripheral taste activity developed normally. The ability to prune in these mice was rescued by activating myeloid cells postnatally, and conversely, pruning was arrested in controls with the loss of myeloid cell function. The altered pruning and myeloid cell function appear to be programmed before the peripheral gustatory system is assembled and corresponds to the embryonic period when microglia progenitors derived from the yolk sac migrate to and colonize the brain.

Selective Removal of Sodium Salt Taste Disrupts the Maintenance of Dendritic Architecture of Gustatory Relay Neurons in the Mouse Nucleus of the Solitary Tract.

Neuronal activity plays critical roles in the development of sensory circuits in the mammalian brain. Experimental procedures are now available to alter the function of specific taste transduction pathways and have been especially useful in studying how stimulus-specific taste activity influences the development of central gustatory circuits. We previously used a mouse knock-out (KO) model in which the transduction channel necessary for sodium taste is removed from taste bud cells throughout life. In these KO mice, the terminal fields that carry taste information from taste buds into the nucleus of the solitary tract (NST) fail to mature, suggesting that sodium-elicited taste activity is important for the proper development of central gustatory circuits. Here, we tested the hypothesis that the development and maintenance of the dendritic architecture of NST relay cells, the primary postsynaptic partner of gustatory nerve terminal fields, are similarly dependent on sodium-elicited taste activity. The dendritic fields of NST relay cells, from adult male and female mice in which the α-subunit of the epithelial sodium channel (αENaC) was conditionally deleted in taste bud cells throughout life, were up to 2.4× larger and more complex than that of age-matched control mice. Interestingly, these differences in dendritic architecture did not appear until after the age when terminal fields begin "pruning," after postnatal day (P)20. Overall, our results suggest that ENaC-mediated sodium taste activity is necessary for the maintenance of dendritic fields of relay cells in the gustatory NST.

Protective effects of TRPV1 inhibition against sevoflurane-induced cell death.

Sevoflurane is a widely used inhalational anesthetic that can induce developmental neurotoxicity, leading to cognitive dysfunction. In this study, we assessed the role of transient receptor potential vanilloid 1 (TRPV1) in mediating sevoflurane activation and whether the TRPV1 antagonist could prevent anesthesia-induced cell death. Here, we demonstrated that the expression of TRPV1 was increased after sevoflurane treatment, and pretreatment with TRPV1 antagonist SB366791 could attenuate the effect of sevoflurane on TRPV1 expression. Moreover, the inhibition of TRPV1 could prevent sevoflurane-induced cell death. The findings of this study provide novel insights into the treatment of general anesthesia-induced developmental neurotoxicity and even cognitive impairment.

Maintenance of Mouse Gustatory Terminal Field Organization Is Dependent on BDNF at Adulthood.

The rodent peripheral gustatory system is especially plastic during early postnatal development and maintains significant anatomical plasticity into adulthood. Thus, taste information carried from the tongue to the brain is built and maintained on a background of anatomical circuits that have the capacity to change throughout the animal's lifespan. Recently, the neurotrophin brain-derived neurotrophic factor (BDNF) was shown to be required in the tongue to maintain normal levels of innervation in taste buds at adulthood, indicating that BDNF is a key molecule in the maintenance of nerve/target matching in taste buds. Here, we tested whether maintenance of the central process of these gustatory nerves at adulthood also relies on BDNF by using male and female transgenic mice with inducible CreERT2 under the control of the keratin 14 promoter or under control of the ubiquitin promoter to remove Bdnf from the tongue or from all tissues, respectively. We found that the terminal fields of gustatory nerves in the nucleus of the solitary tract were expanded when Bdnf was removed from the tongue at adulthood and with even larger and more widespread changes in mice where Bdnf was removed from all tissues. Removal of Bdnf did not affect numbers of ganglion cells that made up the nerves and did not affect peripheral, whole-nerve taste responses. We conclude that normal expression of Bdnf in gustatory structures is required to maintain normal levels of innervation at adulthood and that the central effects of Bdnf removal are opposite of those in the tongue.SIGNIFICANCE STATEMENT BDNF plays a major role in the development and maintenance of proper innervation of taste buds. However, the importance of BDNF in maintaining innervation patterns of gustatory nerves into central targets has not been assessed. Here, we tested whether Bdnf removal from the tongue or from all structures in adult mice impacts the maintenance of how taste nerves project to the first central relay. Deletion of Bdnf from the tongue and from all tissues led to a progressively greater expansion of terminal fields. This demonstrates, for the first time, that BDNF is necessary for the normal maintenance of central gustatory circuits at adulthood and further highlights a level of plasticity not seen in other sensory system subcortical circuits.

Maintenance of Mouse Gustatory Terminal Field Organization Is Disrupted following Selective Removal of Peripheral Sodium Salt Taste Activity at Adulthood.

Neural activity plays a critical role in the development of central circuits in sensory systems. However, the maintenance of these circuits at adulthood is usually not dependent on sensory-elicited neural activity. Recent work in the mouse gustatory system showed that selectively deleting the primary transduction channel for sodium taste, the epithelial sodium channel (ENaC), throughout development dramatically impacted the organization of the central terminal fields of three nerves that carry taste information to the nucleus of the solitary tract. More specifically, deleting ENaCs during development prevented the normal maturation of the fields. The present study was designed to extend these findings by testing the hypothesis that the loss of sodium taste activity impacts the maintenance of the normal adult terminal field organization in male and female mice. To do this, we used an inducible Cre-dependent genetic recombination strategy to delete ENaC function after terminal field maturation occurred. We found that removal of sodium taste neural activity at adulthood resulted in significant reorganization of mature gustatory afferent terminal fields in the nucleus of the solitary tract. Specifically, the chorda tympani and greater superficial petrosal nerve terminal fields were 1.4× and 1.6× larger than age-matched controls, respectively. By contrast, the glossopharyngeal nerve, which is not highly sensitive to sodium taste stimulation, did not undergo terminal field reorganization. These surprising results suggest that gustatory nerve terminal fields remain plastic well into adulthood, which likely impacts central coding of taste information and taste-related behaviors with altered taste experience.SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. However, the importance of sensory-driven activity in maintaining these circuits at adulthood, especially in subcortical structures, appears to be much less. Here, we tested whether the loss of sodium taste activity in adult mice impacts the maintenance of how taste nerves project to the first central relay. We found that specific loss of sodium-elicited taste activity at adulthood produced dramatic and selective reorganization of terminal fields in the brainstem. This demonstrates, for the first time, that taste-elicited activity is necessary for the normal maintenance of central gustatory circuits at adulthood and highlights a level of plasticity not seen in other sensory system subcortical circuits.

BDNF is required for taste axon regeneration following unilateral chorda tympani nerve section.

Taste nerves readily regenerate to reinnervate denervated taste buds; however, factors required for regeneration have not yet been identified. When the chorda tympani nerve is sectioned, expression of brain-derived neurotrophic factor (BDNF) remains high in the geniculate ganglion and lingual epithelium, despite the loss of taste buds. These observations suggest that BDNF is present in the taste system after nerve section and may support taste nerve regeneration. To test this hypothesis, we inducibly deleted Bdnf during adulthood in mice. Shortly after Bdnf gene recombination, the chorda tympani nerve was unilaterally sectioned causing a loss of both taste buds and neurons, irrespective of BDNF levels. Eight weeks after nerve section, however, regeneration was differentially affected by Bdnf deletion. In control mice, there was regeneration of the chorda tympani nerve and taste buds reappeared with innervation. In contrast, few taste buds were reinnervated in mice lacking normal Bdnf expression such that taste bud number remained low. In all genotypes, taste buds that were reinnervated were normal-sized, but non-innervated taste buds remained small and atrophic. On the side of the tongue contralateral to the nerve section, taste buds for some genotypes became larger and all taste buds remained innervated. Our findings suggest that BDNF is required for nerve regeneration following gustatory nerve section.

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. SIGNIFICANCE STATEMENT: Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role in the maturation of the terminal fields in the mouse brainstem. We found that the specific deletion of sodium salt taste during development produced terminal fields in adults that were dramatically larger than in control mice, demonstrating for the first time that sodium salt taste-elicited activity is necessary for the normal maturation of gustatory inputs into the brain.

Expanded terminal fields of gustatory nerves accompany embryonic BDNF overexpression in mouse oral epithelia.

Brain-derived neurotrophic factor (BDNF) is expressed in gustatory epithelia and is required for gustatory neurons to locate and innervate their correct target during development. When BDNF is overexpressed throughout the lingual epithelium, beginning embryonically, chorda tympani fibers are misdirected and innervate inappropriate targets, leading to a loss of taste buds. The remaining taste buds are hyperinnervated, demonstrating a disruption of nerve/target matching in the tongue. We tested the hypothesis here that overexpression of BDNF peripherally leads to a disrupted terminal field organization of nerves that carry taste information to the brainstem. The chorda tympani, greater superficial petrosal, and glossopharyngeal nerves were labeled in adult wild-type (WT) mice and in adult mice in which BDNF was overexpressed (OE) to examine the volume and density of their central projections in the nucleus of the solitary tract. We found that the terminal fields of the chorda tympani and greater superficial petrosal nerves and overlapping fields that included these nerves in OE mice were at least 80% greater than the respective field volumes in WT mice. The shapes of terminal fields were similar between the two groups; however, the density and spread of labels were greater in OE mice. Unexpectedly, there were also group-related differences in chorda tympani nerve function, with OE mice showing a greater relative taste response to a concentration series of sucrose. Overall, our results show that disruption in peripheral innervation patterns of sensory neurons have significant effects on peripheral nerve function and central organization of their terminal fields.

Temporal signatures of taste quality driven by active sensing.

Animals actively acquire sensory information from the outside world, with rodents sniffing to smell and whisking to feel. Licking, a rapid motor sequence used for gustation, serves as the primary means of controlling stimulus access to taste receptors in the mouth. Using a novel taste-quality discrimination task in head-restrained mice, we measured and compared reaction times to four basic taste qualities (salt, sour, sweet, and bitter) and found that certain taste qualities are perceived inherently faster than others, driven by the precise biomechanics of licking and functional organization of the peripheral gustatory system. The minimum time required for accurate perception was strongly dependent on taste quality, ranging from the sensory-motor limits of a single lick (salt, ∼100 ms) to several sampling cycles (bitter, >500 ms). Further, disruption of sensory input from the anterior tongue significantly impaired the speed of perception of some taste qualities, with little effect on others. Overall, our results show that active sensing may play an important role in shaping the timing of taste-quality representations and perception in the gustatory system.

Loss of cholecystokinin-containing terminals in temporal lobe epilepsy.

Altered GABA-mediated inhibition is proposed to play a role in the pathogenesis of epilepsy. Previous studies have demonstrated a loss of somatostatin-containing GABAergic interneurons innervating granule cells in epileptic animals. However, the reorganization of synapses between interneurons and granule cells has not been investigated. We studied synapse organization in an animal model of temporal lobe epilepsy (TLE) using continuous hippocampal stimulation. The distribution of axon terminals and inhibitory synapses on granule cell dendrites was studied using a combination of immunohistochemistry and pre-embedding electron microscopy techniques. A whole-cell patch-clamp technique was applied to study the functional changes in GABAergic input from different interneurons. In epileptic animals, the density of cholecystokinin (CCK)-immunoreactive (IR) fibers and α2 subunit containing GABAA receptors in the inner molecular layer of the dentate gyrus was reduced. Quantitative immuno-electron microscopy study revealed that the ratio of CCK-containing symmetric synapses to the total symmetric synapses was reduced. The frequency of GABAergic synaptic currents (sIPSC) was decreased and their amplitude was increased. The inhibitory effect of the activation of cannabinoid 1 (CB1) receptors was also reduced in epileptic animals. Isolation of CCK- and parvalbumin (PV)-containing GABAergic inputs by N- and P/Q-type calcium channel blockers respectively suggested that GABA release from CCK-containing interneurons was selectively reduced in epileptic rats. This study found that there was a loss of CCK-containing GABAergic synapses to granule cells both morphologically and functionally. These studies add to our understanding of the mechanisms that contribute to altering GABAergic inhibition of granule cells in TLE.

Characterization of status epilepticus induced by two organophosphates in rats.

Organophosphates (OPs) inhibit the enzyme cholinesterase and cause accumulation of acetylcholine, and are known to cause seizures and status epilepticus (SE) in humans. The animal models of SE caused by organophosphate analogs of insecticides are not well characterized. SE caused by OPs paraoxon and diisopropyl fluorophosphate (DFP) in rats was characterized by electroencephalogram (EEG), behavioral observations and response to treatment with the benzodiazepine diazepam administered at various stages of SE. A method for SE induction using intrahippocampal infusion of paraoxon was also tested. Infusion of 200nmol paraoxon into the hippocampus caused electrographic seizures in 43/52 (82.7%) animals tested; and of these animals, 14/43 (30%) had self-sustaining seizures that lasted 4-18h after the end of paraoxon infusion. SE was also induced by peripheral subcutaneous injection of diisopropyl fluorophosphate (DFP, 1.25mg/kg) or paraoxon (1.00mg/kg) to rats pretreated with atropine (2mg/kg) and 2-pralidoxime (2-PAM, 50mg/kg) 30min prior to OP injection. SE occurred in 78% paraoxon-treated animals and in 79% of DFP-treated animals. Diazepam (10mg/kg) was administered 10min and 30min after the onset of continuous EEG seizures induced by paraoxon and it terminated SE in a majority of animals at both time points. DFP-induced SE was terminated in 60% animals when diazepam was administered 10min after the onset of continuous EEG seizure activity but diazepam did not terminate SE in any animal when it was administered 30min after the onset of continuous seizures. These studies demonstrate that both paraoxon and DFP can induce SE in rats but refractoriness to diazepam is a feature of DFP induced SE.

A mouse monoclonal antibody against the γ2 subunit of GABAA receptors.

A mouse monoclonal antibody directed against the N terminal extracellular epitope of rat γ amino butyric acid (GABA) type-A (GABA(A)R) receptor γ2 subunit was generated. This antibody identified a protein of approximately 42 kDa in Western blot assays using rat and mouse hippocampal proteins. The antibody also detected the expression of γ2 subunit by immunohistochemistry and could immunoprecipitate the γ2 subunit.

Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals.

Neurosteroid sensitivity of GABA(A) receptor mediated inhibition of the hippocampal dentate granule cells (DGCs) is reduced in animal models of temporal lobe epilepsy. However, the properties and subunit composition of GABA(A) receptors mediating tonic inhibition in DGCs of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-655708 sensitivity of holding current was diminished and δ subunit was retained in the endoplasmic reticulum and its surface expression was decreased the in the hippocampus. Ro15-4513 and lanthanum had distinct effects on holding current recorded from DGCs of control and epileptic animals suggesting that the pharmacological properties of GABA(A) receptors maintaining tonic inhibition in DGCs of epileptic animals were similar to those containing the α4ßxγ2 subunits. Furthermore, surface expression of the α4 subunit increased and a larger fraction of the subunit co-immunoprecipitated with theγ2 subunit in hippocampi of epileptic animals. Together, these studies revealed that functional α4ßxδ and α5ßxγ2 receptors were reduced in the hippocampi of epileptic animals and that novel α4bxγ2 receptors contributed to the maintenance of tonic inhibition. The presence of α4ßxγ2 receptors resulted in low GABA affinity and neurosteroid sensitivity of tonic currents in the DGCs of epileptic animals that could potentially increase seizure vulnerability. These receptors may represent a novel therapeutic target for anticonvulsant drugs without sedative actions.

Endogenous neurosteroid synthesis modulates seizure frequency.

Inhibitory neurosteroids, molecules generated in glia from circulating steroid hormones and de novo from cholesterol, keep seizures in check in epileptic animals. They can enhance inhibitory transmission mediated by gamma-aminobutyric acid receptors and have anticonvulsant action.

Homeostatic regulation of synaptic excitability: tonic GABA(A) receptor currents replace I(h) in cortical pyramidal neurons of HCN1 knock-out mice.

Homeostatic control of synaptic efficacy is often mediated by dynamic regulation of excitatory synaptic receptors. Here, we report a novel form of homeostatic synaptic plasticity based on regulation of shunt currents that control dendritosomatic information transfer. In cortical pyramidal neurons from wild-type mice, HCN1 channels underlie a dendritic hyperpolarization-activated cationic current (I(h)) that serves to limit temporal summation of synaptic inputs. In HCN1 knock-out mice, as expected, I(h) is reduced in pyramidal neurons and its effects on synaptic summation are strongly diminished. Unexpectedly, we found a markedly enhanced bicuculline- and L-655,708-sensitive background GABA(A) current in these cells that could be attributed to selective upregulation of GABA(A) alpha5 subunit expression in the cortex of HCN1 knock-out mice. Strikingly, despite diminished I(h), baseline sublinear summation of evoked EPSPs was unchanged in pyramidal neurons from HCN1 knock-out mice; however, blocking tonic GABA(A) currents with bicuculline enhanced synaptic summation more strongly in pyramidal cells from HCN1 knock-out mice than in those cells from wild-type mice. Increasing tonic GABA(A) receptor conductance in the context of reduced I(h), using computational or pharmacological approaches, restored normal baseline synaptic summation, as observed in neurons from HCN1 knock-out mice. These data indicate that upregulation of alpha5 subunit-mediated GABA(A) receptor tonic current compensates quantitatively for loss of dendritic I(h) in cortical pyramidal neurons from HCN1 knock-out mice to maintain normal synaptic summation; they further imply that dendritosomatic synaptic efficacy is a controlled variable for homeostatic regulation of cortical neuron excitability in vivo.

Altered pharmacology and GABA-A receptor subunit expression in dorsal midline thalamic neurons in limbic epilepsy.

The mediodorsal (MD) and paraventricular (PV) thalamic nuclei play a significant role in limbic epilepsy, and previous reports have shown changes in GABA-A receptor (GABAAR) mediated synaptic function. In this study, we examined changes in the pharmacology of GABAergic drugs and the expression of the GABAAR subunits in the MD and PV neurons in epilepsy. We observed nucleus specific changes in the sensitivity of sIPSCs to zolpidem and phenobarbital in MD and PV neurons from epileptic animals. In contrast, the magnitude of change in electrically evoked response (eIPSC) to zolpidem and phenobarbital were uniformly diminished in both MD and PV neurons in epilepsy. Immunohistochemical studies revealed that in epilepsy, there was a reduction in GAD65 expression and NeuN positive neurons in the MD neurons. Also, there was a decrease in immunoreactivity of the alpha1 and beta2/3 subunit of GABAARs, but not the gamma2 of the GABAAR in both MD and PV in epilepsy. These findings demonstrate significant alterations in the pharmacology of GABA and GABAARs in a key region for seizure generation, which may have implications for the physiology and pharmacology of limbic epilepsy.

Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the alpha4 subunit of GABA(A) receptors in an animal model of epilepsy.

In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABA(A) receptors on dentate granule cells (DGCs) is diminished; the molecular mechanism underlying this phenomenon remains unclear. The current study investigated a mechanism for loss of neurosteroid sensitivity of synaptic GABA(A) receptors in TLE. Synaptic currents recorded from DGCs of epileptic animals (epileptic DGCs) were less frequent, larger in amplitude, and less sensitive to allopregnanolone modulation than those recorded from DGCs of control animals (control DGCs). Synaptic currents recorded from epileptic DGCs were less sensitive to diazepam and had altered sensitivity to benzodiazepine inverse agonist RO 15-4513 (ethyl-8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) and furosemide than those recorded from control DGCs. Properties of synaptic currents recorded from epileptic DGCs appeared similar to those of recombinant receptors containing the alpha4 subunit. Expression of the alpha4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the alpha4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The alpha4 immunogold labeling was present more commonly within the synapse in epileptic DGCs compared with control DGCs, in which the subunit was extrasynaptic. These studies demonstrate that, in epileptic DGCs, the neurosteroid modulation of synaptic currents is diminished and alpha4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

GABA(A) receptor internalization during seizures.

A rapid modification in the postsynaptic gamma-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 beta2/3 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.

Selective loss of dentate hilar interneurons contributes to reduced synaptic inhibition of granule cells in an electrical stimulation-based animal model of temporal lobe epilepsy.

Neuropeptide-containing hippocampal interneurons and dentate granule cell inhibition were investigated at different periods following electrical stimulation-induced, self-sustaining status epilepticus (SE) in rats. Immunohistochemistry for somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), cholecystokinin (CCK), and Fluoro-Jade B was performed on sections from hippocampus contralateral to the stimulated side and studied by confocal laser scanning microscopy. Compared to paired age-matched control animals, there were fewer SOM and NPY-immunoreactive (IR) interneurons in the hilus of the dentate gyrus in animals with epilepsy (40-60 days after SE), and 1, 3, and 7 days following SE. In the hilus of animals that had recently undergone SE, some SOM-IR and NPY-IR interneurons also stained for Fluoro-Jade B. Furthermore, there was electron microscopic evidence of the degeneration of SOM-IR interneurons following SE. In contrast, the number of CCK and PV-IR basket cells in epileptic animals was similar to that in controls, although it was transiently diminished following SE; there was no evidence of degeneration of CCK or PV-IR interneurons. Patch-clamp recordings revealed a diminished frequency of inhibitory postsynaptic currents in dentate granule cells (DGCs) recorded from epileptic animals and animals that had recently undergone SE compared with controls. These results confirm the selective vulnerability of a particular subset of dentate hilar interneurons after prolonged SE. This loss may contribute to the reduced GABAergic synaptic inhibition of granule cells in epileptic animals.

Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAA Receptors.

We combined a study of the subcellular distribution of the alpha1, alpha2, alpha4, beta1, beta2/3, gamma2, and delta subunits of the GABAA receptor with an electrophysiological analysis of GABAA receptor currents determine the to types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that alpha1, alpha2, beta2/3, and gamma2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, alpha4, beta1, and delta subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA receptor currents were moderately sensitive to GABAA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn2+, furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABAA receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The alpha1, alpha2, beta2/3, and gamma2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The alpha4, beta1, and delta subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing alpha4, beta1, and delta subunits.