The Depolarizing Action of GABA Controls Early Network Activity in the Developing Hippocampus

Early in postnatal life γ-aminobutyric acid (GABA), the primary inhibitory transmitter in adults, excites targeted neurons by an outwardly directed flux of chloride which results from the unbalance between the cation–chloride cotransporters NKCC1 and KCC2, involved in chloride uptake and extrusion, respectively. This effect contributes to generate synchronized network activity or giant depolarizing potentials (GDPs) in the developing hippocampus. Here, we review some recent data concerning the mechanisms by which GDPs are generated and their functional role in enhancing synaptic efficacy at poorly developed GABAergic and glutamatergic synapses. In adulthood, reshaping neuronal circuits due to changes in chloride homeostasis and to the shift of GABA from hyperpolarizing to depolarizing, has been implicated in several neurological disorders, including epilepsy. Evidence has been recently provided that in chronically nerve growth factor-deprived mice expressing a progressive age-dependent neurodegenerative pathology resembling that observed in patients with Alzheimer’s disease, the reduced expression of mRNA encoding for the Kcc2 gene and the depolarizing action of GABA lead to the reorganization of the neuronal hippocampal network. This may represent a novel mechanism by which GABAergic signaling counterbalances the loss of synaptic activity in neurodegenerative diseases.     

GABA generates excitement

In the CNS, gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter via ligand-gated GABA(A) receptor channels and G protein-coupled GABA(B) receptors. Both of these receptor types mediate inhibitory postsynaptic transmission throughout the nervous system. For GABA(A) receptors, this inhibitory action is associated with a hyperpolarization due to an increase in conductance to chloride ions. Previous studies show that GABA(A) receptor activation in neonatal neurons and spinal cord neurons can be excitatory. Two papers recently appeared that clearly demonstrate that GABA can have a depolarizing and excitatory action in mature cortical neurons. Here we discuss the evolving story on chloride ion homeostasis in CNS neurons and its role in the bipolar life of the GABA(A) receptor.     

Quaternary structure of the chloride channel ClC-2

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.     

Aging-associated down-regulation of ClC-1 expression in skeletal muscle: phenotypic-independent relation to the decrease of chloride conductance

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.     

Aging reduces the GABA-dependent 36Cl- flux in rat brain membrane vesicles

The function of the chloride channel associated to GABAA receptor complex was analyzed in the brain of aged rats by measuring the chloride flux across the neuronal membrane and its modulation by drugs acting at the level of the GABA receptor complex and 35S-TBPS binding. The basal 36Cl- uptake by brain membrane vesicles of aged rats was higher (22%) than that observed in those of adult rats. The higher 36Cl- uptake found in cortical membrane vesicles of senescent rats was not sensitive to the action of bicuculline indicating that it was not the consequence of a tonic GABAergic modulation. Moreover, the stimulation of 36Cl- uptake induced by GABA was markedly lower in membrane vesicles of aged rats than that observed in those of adult rats. Accordingly, the stimulation of 36Cl- efflux elicited by GABA (18%) and pentobarbital (26%) was higher in membrane vesicles of adult rats with respect to that (8 and 16%, respectively) of old rats. Finally, a significant decrease of 35S-TBPS binding was observed in membrane preparation from the cerebral cortex, cerebellum and hippocampus of aged-rats. Scatchard plot analysis indicated that the decrease was entirely due to a reduction in the total number of binding sites with no change in their affinity. All together the results indicate that in the rat brain the function of the chloride channel coupled to the GABA/benzodiazepine/barbiturate receptor complex is reduced by aging.     

Sex Differences in Glutamic Acid Decarboxylase mRNA in Neonatal Rat Brain: Implications for Sexual Differentiation

Sexual differentiation of rodent brain is dependent upon hormonal exposure during a “critical period” beginning in late gestation and ending in early neonatal life. Steroid hormone action at this time results in anatomical and physiological sexual dimorphisms in adult brain, but the mechanism mediating these changes is essentially unknown. The inhibitory neurotransmitter, GABA, is involved in regulation of sexually dimorphic patterns of behavior and gonadotropin secretion in the adult. Recent evidence suggests that during development GABA is excitatory and provides critical neurotrophic and neuromodulatory influences. We hypothesized that steroid-induced changes in GABAergic neurotransmission during this critical period are important mediators of sexual differentiation in brain. Therefore, we quantified levels of mRNA for GAD, the rate-limiting enzyme in GABA synthesis. On Postnatal Day 1, males had significantly higher levels of GAD mRNA in the dorsomedial nucleus, arcuate nucleus, and CA1 region of hippocampus. On Postnatal Day 15, after the critical period for sexual differentiation has ended, these differences were no longer present. We examined the role of gonadal steroids in regulating GAD by removing testes of males and administering testosterone to females at birth. Exposure to testosterone was correlated with increased GAD mRNA in the dorsomedial nucleus. A sex difference in GAD mRNA was also observed in the medial preoptic area, but the influence of testosterone was inconclusive. We conclude that sex differences in the GABAergic system during development are partially hormonally mediated, and that these differences may contribute to the development of sexually dimorphic characteristics in adult brain.     

Chloride distribution in the CA1 region of newborn and adult hippocampus by light microscopic histochemistry

GABA, the main inhibitory neurotransmitter in the central nervous system, exerts its effect by rendering the postsynaptic GABAA receptors permeable to chloride ions. Thus, depolarizing or excitatory effects of GABA, experienced in early postnatal life or in certain regions and/or conditions of the adult brain, is thought to be associated with a reversed transmembrane chloride gradient. However, there is only limited direct information about the correlation of the actual excitatory versus inhibitory effects of GABA and the local chloride distribution. Precipitation of chloride with silver is a potential way to immobilize and visualize chloride ions in biological tissue. We examined the applicability of light microscopic histochemistry, based on trapping tissue chloride with silver ions during freeze-substitution or aldehyde fixation, to visualize the chloride distribution in hippocampal slices. The freeze-substitution procedure yielded better chloride retention while with aldehyde fixation tissue preservation was more appropriate. Both methods were qualitative only, had limited applicability to the superficial 20-30 microns of slices, but were able to demonstrate a reduced extracellular-to-intracellular chloride gradient in the CA1 pyramidal neurons of the newborn hippocampus as compared to adult animals. In the 4-aminopyridine model of epilepsy, redistribution of chloride from extracellular to intracellular space could also be demonstrated.     

The depolarizing action of GABA in cultured hippocampal neurons is not due to the absence of ketone bodies

Two recent reports propose that the depolarizing action of GABA in the immature brain is an artifact of in vitro preparations in which glucose is the only energy source. The authors argue that this does not mimic the physiological environment because the suckling rats use ketone bodies and pyruvate as major sources of metabolic energy. Here, we show that availability of physiologically relevant levels of ketone bodies has no impact on the excitatory action of GABA in immature cultured hippocampal neurons. Addition of β-hydroxybutyrate (BHB), the primary ketone body in the neonate rat, affected neither intracellular calcium elevation nor membrane depolarizations induced by the GABA-A receptor agonist muscimol, when assessed with calcium imaging or perforated patch-clamp recording, respectively. These results confirm that the addition of ketone bodies to the extracellular environment to mimic conditions in the neonatal brain does not reverse the chloride gradient and therefore render GABA hyperpolarizing. Our data are consistent with the existence of a genuine "developmental switch" mechanism in which GABA goes from having a predominantly excitatory role in immature cells to a predominantly inhibitory one in adults.     

ATP depletion inhibits the endocytosis of ClC-2

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel.     

Receptors for gamma-aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants

The function of chloride (Cl-) channel proteins is to regulate the transport of Cl- across membranes. There are two major kinds of Cl- channels: 1) those activated by binding of a transmitter such as gamma-aminobutyric acid (GABA), glycine, or glutamate, and thus are receptors; and 2) those activated by membrane depolarization or by calcium. There are two kinds of GABA receptors: GABAA is the major inhibitory receptor of vertebrate brain and the one that operates a Cl- channel, and the GABAB receptor, which is proposed to regulate cAMP production that is stimulated by other receptors. Except for binding of GABA, these two GABA receptors differ completely in their drug specificities. However, there are many similarities among the GABAA receptor, the glycine receptor, and the voltage-dependent Cl- channel. The two receptors and Cl- channels bind avermectin, whereas bicuculline binds only to mammalian GABAA and glycine receptors, not to the insect brain GABAA receptor. Barbiturates bind to GABAA and voltage-dependent Cl- channels, possibly directly activating them. Benzodiazepines potentiate both the glycine and GABAA receptors. Several insecticides act on the GABAA receptor and voltage-dependent Cl- channel. It is suggested that the GABAA receptor is the primary target for the action of toxaphene and cyclodiene insecticides but a secondary target for lindane and type II pyrethroids. On the other hand, the Cl- channel may be a primary target for avermectin and lindane but a secondary one for cyclodienes. The similarity in certain drug specificities and the operation of Cl- channels suggest a degree of homology between the subunits of GABAA and glycine receptors and the voltage-dependent Cl- channels.     

Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.     

Regulation of spinal interneuron differentiation by the paracrine action of glycine

Glycine and γ-aminobutyric acid (GABA) are depolarizing during early development but the purpose is unclear. We tested the effect of altering glycine signaling in zebrafish embryos by overexpressing the potassium-chloride co-transporter type 2 (KCC2) to reverse the chloride gradient or by blocking glycine receptors with strychnine or by selectively knocking down the embryonic glycine receptor (GlyR KD). Using a variety of markers we observed in all three cases a reduction of all types of spinal interneuron populations examined, indicating that glycine modulates their overall differentiation rather than choice of cell fate. Other cell populations (motor, sensory, and glial cells) were unaffected. As glycine appeared to act preceding neural and synaptic development, we examined the bandoneon (beo) mutant in which glycine receptors are functional but not clustered at synapses. Neural populations in beo embryos appeared normal, suggesting a paracrine action of circulating glycine in promoting interneuron differentiation.     

Nonsynaptic glycine release is involved in the early KCC2 expression

The cation‐chloride co‐transporters are important regulators of the cellular Cl^‐ homeostasis. Among them the Na⁺‐K⁺?2Cl^? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K⁺‐Cl^? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na⁺ action potentials but was mimicked by a Ca²⁺‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016     

Regional changes in the concentrations of glutamate,glycine, taurine,and GABA in the vitamin B-6 deficient developing rat brain: Association with neonatal seizures

It is well known that a dietary restriction of vitamin B-6 during gestation and lactation produces spontaneous seizures in neonatal animals. Since pyridoxal phosphate, one of the biologically active forms of vitamin B-6, is the cofactor for GAD the neonatal seizures have been attributed to low levels of brain GABA as a result of cofactor depletion. Although GABA levels are significantly lower in B-6 restricted neonatal rats with spontaneous seizures, seizure activity is not present in B-6 deficient adult rats or 28 day old rats in the present study, despite significantly low levels of brain GABA. These facts suggest that depletion of GABA is not the only biochemical alteration essential for the emergence of seizures. In the present study, the effect of vitamin B-6 undernutrition on the concentrations of the neuroactive amino acids, Glu, Gly, Tau, and GABA was determined in selected regions of the developing rat brain. The results show that the concentrations of Glu, Tau, and GABA were significantly lower and GLY significantly higher in selected brain regions of the B-6 restricted 14 day old rat compared to control tissue. Most of these changes were unique to 14 days of age, the time when spontaneous seizures are observed, and not present at 28 or 56 days of age when seizures are absent. This pattern of amino acid changes in the brain and the magnitude of the changes was consistent with those measured in a variety of chemically-induced animal models of epilepsy and in human epileptic foci. The regional distribution of amino acid changes was associated with brain regions which have been suggested to be responsible for the initiation and propagation of seizure activity. Two unique findings were also made in this study. First, there was a regional brain heterogeneity in the age-associated loss of brain Tau concentrations with the pons/medulla and substantia nigra appearing to be highly vulnerable and the hippocampus quite resistant to the loss of Tau. A second finding was the normalization of the neonatal GABA deficit in most brain regions by 56 days of age. The normalization of brain GABA was present in the face of continued dietary vitamin B-6 restriction. In summary, this study shows that the neuroactive amino acids Glu, Gly, Tau, and GABA are markedly altered in the seizure-prone vitamin B-6 restricted neonatal rat brain. The alterations in the brain concentration of Glu, Gly, and Tau may play an equally important role as GABA in the underlying mechanism of seizures associated with this condition.Abbreviations GAD Glutamic acid decarboxylase - GABA gamma-aminobutyric acid - Glu glutamate - Gly glycine - Tau taurine - CNS central nervous system - CTX cortex - HIPP hippocampus - C/P caudate/putamen - SN substantia nigra - Cb cerebellum - P/M pons/medulla