The GABA Excitatory/Inhibitory Shift in Brain Maturation and Neurological Disorders

Ionic currents and the network-driven patterns they generate differ in immature and adult neurons: The developing brain is not a "small adult brain." One of the most investigated examples is the developmentally regulated shift of actions of the transmitter GABA that inhibit adult neurons but excite immature ones because of an initially higher intracellular chloride concentration [Cl(-)](i), leading to depolarizing and often excitatory actions of GABA instead of hyperpolarizing and inhibitory actions. The levels of [Cl(-)](i) are also highly labile, being readily altered transiently or persistently by enhanced episodes of activity in relation to synaptic plasticity or a variety of pathological conditions, including seizures and brain insults. Among the plethora of channels, transporters, and other devices involved in controlling [Cl(-)](i), two have emerged as playing a particularly important role: the chloride importer NKCC1 and the chloride exporter KCC2. Here, the authors stress the importance of determining how [Cl(-)](i) is dynamically regulated and how this affects brain operation in health and disease. In a clinical perspective, agents that control [Cl(-)](i) and reinstate inhibitory actions of GABA open novel therapeutic perspectives in many neurological disorders, including infantile epilepsies, autism spectrum disorders, and other developmental disorders.     

Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize E(Cl) during development

In the mature CNS, coincident pre- and postsynaptic activity decreases the strength of gamma-aminobutyric acid (GABA)(A)-mediated inhibition through a Ca2+-dependent decrease in the activity of the neuron-specific K+-Cl- cotransporter KCC2. In the present study we examined whether coincident pre- and postsynaptic activity can also modulate immature GABAergic synapses, where the Na+-K+-2Cl- (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl-](i)). Dual perforated patch-clamp recordings were made from cultured hippocampal neurons prepared from embryonic Sprague-Dawley rats. These recordings were used to identify GABAergic synapses where the reversal potential for Cl- (ECl) was hyperpolarized with respect to the action potential threshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within +/- 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of ECl by 10.03 +/- 1.64 mV, increasing the strength of synaptic inhibition. Blocking the inward transport of Cl- by NKCC1 with bumetanide (10 microm) hyperpolarized ECl by 16.14 +/- 4.8 mV, and prevented this coincident activity-induced shift of ECl. The bumetanide-induced hyperpolarization of ECl occluded furosemide, a K+-Cl- cotransporter antagonist, from producing further shifts in ECl. Together, this indicates that brief coincident pre- and postsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as a mechanism underlying inhibitory synaptic plasticity.     

Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus

Intracellular chloride concentration, [Cl(-)](i), determines the polarity of GABA(A)-induced neuronal Cl(-) currents. In neurons, [Cl(-)](i) is set by the activity of Na(+), K(+), 2Cl(-) cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl(-) in the cell, and Cl(-) extruding K(+), Cl(-) cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex. NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.     

Bumetanide reduces seizure frequency in patients with temporal lobe epilepsy

Alterations in the balance of K‐Na‐2Cl cotransporter (NKCC1) and Na‐Cl cotransporter (KCC2) activity may cause depolarizing effect of γ‐aminobutyric Acid (GABA), and contribute to epileptogenesis in human temporal lobe epilepsy. NKCC1 facilitates accumulation of chloride inside neurons and favors depolarizing responses to GABA. In the current pilot study we provide the first documented look at efficacy of bumetanide, a specific NKCC1 antagonist, on reduction of seizure frequency in adult patients with temporal lobe epilepsy. According to our results, seizure frequency was reduced considerably in these patients. Furthermore, epileptiform discharges decreased in two of our patients. If the efficacy of bumetanide is proven in large scale studies, it can be used as a supplemental therapy in temporal lobe epilepsy.     

KCC2 was downregulated in small neurons localized in epileptogenic human focal cortical dysplasia

Focal cortical dysplasia (FCD), which is characterized histologically by disorganized cortical lamination and large abnormal cells, is one of the major causes of intractable epilepsies. γ-aminobutyric acid (GABA)(A) receptor-mediated synchronous depolarizing potentials have been observed in FCD tissue. Since alterations in Cl(-) homeostasis might underlie these depolarizing actions of GABA, cation-Cl(-) cotransporters could play critical roles in the generation of these abnormal actions. We examined the expression patterns of NKCC1 and KCC2 by in situ hybridization histochemistry and immunohistochemistry in FCD tissue obtained by surgery from patients with intractable epilepsy. KCC2 mRNA and protein were expressed not only in non-dysplastic neurons in histologically normal portions located in the periphery of the excised cortex, but also in dysplastic cells in FCD tissue. The levels of KCC2 mRNA and protein were significantly decreased in the neurons around large abnormal neurons (giant neurons), but not in giant neurons, compared with non-dysplastic neurons. The neurons localized only around giant neurons significantly smaller than non-dysplastic neurons. However NKCC1 expression did not differ among these cell types. These results suggest that the intracellular Cl(-) concentration ([Cl(-)](i)) of small neurons might increase, so that depolarizing GABA actions could occur in the FCD tissue of epileptic foci.     

Traumatic alterations in GABA signaling disrupt hippocampal network activity in the developing brain

Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl(-)](i)) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl(-)](i) correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl(-)](i) demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures.     

The role of Na-K-Cl co-transporter in cerebral ischemia

The electroneutral Na-K-Cl co-transporter (NKCC) protein transports Na(+), K(+) and Cl(-) into cells under physiological conditions with a stoichiometry of 1Na(+) :1K(+) :2Cl(-). NKCC is characteristically inhibited by the sulfamoylbenzoic acid "loop' diuretics, such as bumetanide and furosemide. To date, only two distinct isoforms, NKCC1 and NKCC2, have been identified. NKCC1 has a broad tissue distribution, while the NKCC2 isoform is only found in vertebrate kidney. NKCC serves multiple functions, including ion and fluid movements in secreting or reabsorbing epithelia and cell volume regulation. However, understanding the role of NKCC1 in the central nervous system has just begun. NKCC1 protein is expressed in neurons throughout the brain. Dendritic localization of NKCC1 is found in both pyramidal and non-pyramidal neurons. NKCC1 is important in the maintenance of intracellular Cl(-) in neurons and contributes to GABA-mediated depolarization in immature neurons. Thus, NKCC1 may affect neuronal excitability through regulation of intracellular Cl(-) concentration. Expression of NKCC1 protein has also been found in astrocytes and oligodendrocytes. In addition to its role in the accumulation of Cl(-), NKCC1 may also contribute to K(+) clearance and maintenance of intracellular Na(+) in glia. Our recent studies suggest that NKCC1 activation leads to high [K(+)](o(-)) induced astrocyte swelling and glutamate release, as well as neuronal Na(+) , and Cl(-) influx during acute excitotoxicity. Inhibition of NKCC1 activity significantly reduces infarct volume and cerebral edema following cerebral focal ischemia.     

Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice

The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.     

Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor

Vesicular Zn(2+) regulates postsynaptic neuronal excitability upon its corelease with glutamate. We previously demonstrated that synaptic Zn(2+) acts via a distinct metabotropic zinc-sensing receptor (mZnR) in neurons to trigger Ca(2+) responses in the hippocampus. Here, we show that physiological activation of mZnR signaling induces enhanced K(+)/Cl(-) cotransporter 2 (KCC2) activity and surface expression. As KCC2 is the major Cl(-) outward transporter in neurons, Zn(2+) also triggers a pronounced hyperpolarizing shift in the GABA(A) reversal potential. Mossy fiber stimulation-dependent upregulation of KCC2 activity is eliminated in slices from Zn(2+) transporter 3-deficient animals, which lack synaptic Zn(2+). Importantly, activity-dependent ZnR signaling and subsequent enhancement of KCC2 activity are also absent in slices from mice lacking the G-protein-coupled receptor GPR39, identifying this protein as the functional neuronal mZnR. Our work elucidates a fundamentally important role for synaptically released Zn(2+) acting as a neurotransmitter signal via activation of a mZnR to increase Cl(-) transport, thereby enhancing inhibitory tone in postsynaptic cells.     

Postnatal maturation of Na+, K+, 2Cl- cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl- through GABAA-gated anion channels. The outward Cl- gradient that provides the driving force for Cl- efflux might be generated and maintained by the Na+, K+, 2Cl- cotransporter (NKCC) that keeps intracellular Cl- concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.     

GABAergic signaling in young granule cells in the adult rat and mouse dentate gyrus

Throughout most of the developing brain, including the hippocampus, GABAergic synapses are the first to become functional. Several features of GABAergic signaling change across development, suggesting that this signaling in the immature brain may play important roles in the growth of young neurons and the establishment of networks. To determine whether GABA(A) receptor (GABA(A)R)-containing synapses in new neurons born in the adult dentate gyrus have similar immature features, we examined spontaneous and evoked GABA(A)R-mediated synaptic currents in young (POMC-EGFP or doublecortin-immunostained) granule cells in acute slice preparations from adult mice and rats. Spontaneous inhibitory postsynaptic currents (IPSCs) were observed in nearly all immature granule cells, but their frequency was considerably lower and their decay time constant was nearly two times longer than in neighboring mature (doublecortin-non-immunoreactive or EGFP-non-expressing) granule cells within the sub-granular zone. Evoked IPSCs (eIPSCs) in mature granule cells, but not immature granule cells, were sensitive to zolpidem, suggesting a maturational increase in GABA(A)R alpha1-subunit expression. Perforated-patch recording revealed that eIPSCs depolarized young neurons, but hyperpolarized mature neurons. The early establishment of synaptic GABAergic inputs slow IPSC decay time, and depolarizing action of eIPSCs are remarkably similar to features previously seen in neurons during development, suggesting that they are intrinsic features of immature neurons and not functions of the surrounding circuitry. These developmental features in adult-born granule cells could play a role in maturational processes such as developmental cell death. However, treatment of adult mice with GABA(A)R agonists and an inverse agonist did not significantly alter the number of 4- to 14-day-old BrdU-labeled cells.     

GABA is excitatory in adult vasopressinergic neuroendocrine cells

Neuronal excitability in the adult brain is controlled by a balance between synaptic excitation and inhibition mediated by glutamate and GABA, respectively. While generally inhibitory in the adult brain, GABA(A) receptor activation is excitatory under certain conditions in which the GABA reversal potential is shifted positive due to intracellular Cl(-) accumulation, such as during early postnatal development and brain injury. However, the conditions under which GABA is excitatory are generally either transitory or pathological. Here, we reveal GABAergic synaptic inputs to be uniformly excitatory in vasopressin (VP)-secreting magnocellular neurons in the adult hypothalamus under normal conditions. The GABA reversal potential (E(GABA)) was positive to resting potential and spike threshold in VP neurons, but not in oxytocin (OT)-secreting neurons. The VP neurons lacked expression of the K(+)-Cl(-) cotransporter 2 (KCC2), the predominant Cl(-) exporter in the adult brain. The E(GABA) was unaffected by inhibition of KCC2 in VP neurons, but was shifted positive in OT neurons, which express KCC2. Alternatively, inhibition of the Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1), a Cl(-) importer expressed in most cell types mainly during postnatal development, caused a negative shift in E(GABA) in VP neurons, but had no effect on GABA currents in OT neurons. GABA(A) receptor blockade caused a decrease in the firing rate of VP neurons, but an increase in firing in OT neurons. Our findings demonstrate that GABA is excitatory in adult VP neurons, suggesting that the classical excitation/inhibition paradigm of synaptic glutamate and GABA control of neuronal excitability does not apply to VP neurons.     

GABAergic synaptic transmission triggers action potentials in thalamic reticular nucleus neurons

GABAergic neurons in the thalamic reticular nucleus (TRN) form powerful inhibitory connections with several dorsal thalamic nuclei, thereby controlling attention, sensory processing, and synchronous oscillations in the thalamocortical system. TRN neurons are interconnected by a network of GABAergic synapses, but their properties and their role in shaping TRN neuronal activity are not well understood. Using recording techniques aimed to minimize changes in the intracellular milieu, we show that synaptic GABA(A) receptor activation triggers postsynaptic depolarizations in mouse TRN neurons. Immunohistochemical data indicate that TRN neurons express very low levels of the Cl(-) transporter KCC2. In agreement, perforated-patch recordings show that intracellular Cl(-) levels are high in TRN neurons, resulting in a Cl(-) reversal potential (E(Cl)) significantly depolarized from rest. Additionally, we find that GABA(A) receptor-evoked depolarizations are amplified by the activation of postsynaptic T-type Ca(2+) channels, leading to dendritic Ca(2+) increases and the generation of burst firing in TRN neurons. In turn, GABA-evoked burst firing results in delayed and long-lasting feedforward inhibition in thalamic relay cells. Our results show that GABA-evoked depolarizations can interact with T-type Ca(2+) channels to powerfully control spike generation in TRN neurons.     

NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.     

Inflammation alters cation chloride cotransporter expression in sensory neurons

Cation chloride cotransporters have been proposed to play a role in the modulation of neuronal responses to gamma-aminobutyric acid (GABA). In conditions of neuronal damage, where neuronal excitability is increased, the expression of the KCC2 transporter is decreased. This is also seen in spinal cord in models of neuropathic pain. We have investigated the expression of the Na-K-Cl, and K-Cl cotransporters NKCC1 and KCC2, in dorsal root ganglion (DRG) and spinal sensory neurons during arthritis, a condition in which neuronal excitability is also increased. NKCC1 was expressed in control DRG neurons, and its expression was decreased in arthritis. Both NKCC1 and KCC2 were expressed in sensory neurons in the spinal cord. In acute arthritis, both NKCC1 and KCC2 mRNA increased in superficial but not deep dorsal horn, and this was accompanied by an increase in protein expression. In chronic arthritis, NKCC1 expression remained raised, but KCC2 mRNA and protein expression returned to control levels. Altered KCC2 and NKCC1 expression in arthritis may contribute to the control of spinal cord excitability and may represent novel therapeutic targets in the treatment of inflammatory pain.     

Whole-cell patch study of GABAergic inhibition in CA1 neurons of immature rat hippocampal slices

Inhibitory processes mediated by gamma-aminobutyric acid (GABA) were studied in immature rat hippocampal slices using the whole-cell patch clamp technique. Orthodromically evoked hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were observed in CA1 neurons of postnatal 2-5 (P2-5) and 7-13 (P7-13) day old rats under conditions of low internal [Cl-]. In the whole-cell voltage-clamp mode, applications of GABA evoked outwards currents which reversed at -55 mV and -62 mV in P2-5 and P7-13 CA1 neurons, respectively, with comparable reversal potential for the IPSPs for each age. An increase in internal [Cl-] caused a depolarizing shift of the GABA reversal potential which followed the Nernst equation. In both groups of neurons, the IPSPs and GABA currents were blocked with the bath applications of bicuculline (10 microM) and picrotoxin (100 microM). We conclude that the GABAA-mediated inhibitory synaptic process exists in P2-5 CA1 neurons and hypothesize that the absence of such IPSPs noted in previous studies of immature CA1 neurons was likely due to higher internal [Cl-] in the more immature neurons.     

Role of bicarbonate ion in mediating decreased synaptic conductance in benzodiazepine tolerant hippocampal CA1 pyramidal neurons

Chronic flurazepam treatment substantially impairs the function of GABAergic synapses on hippocampal CA1 pyramidal cells. Previous findings included a significant decrease in the synaptic and unitary conductance of CA1 pyramidal neuron GABA(A) receptor channels and the appearance of a GABA(A)-receptor mediated depolarizing potential. To investigate the ionic basis of the decreased conductance, whole-cell voltage-clamp techniques were used to record evoked, GABA(A) receptor-mediated IPSCs carried by HCO(3)(-)-Cl(-) or Cl(-) alone. Hippocampal slices were prepared from rats administered flurazepam orally for 1 week, 2 days after ending drug treatment. Slices were superfused with HCO(3)(-)-aCSF or with HEPES-aCSF (without HCO(3)(-)) plus 50 microM APV and 10 microM DNQX. The micropipette contained 130 mM CsCl and 1 microM QX-314. GABA(A) receptors located on pyramidal cell somata or dendrites were activated monosynaptically by maximal stimulation of GABAergic terminals at the stratum oriens-pyramidale (SO-SP) or stratum lacunosum-molecular (S-L-M) border, respectively. In HCO(3)(-)-aCSF, there was a significant reduction in synaptic-conductance in flurazepam-treated neurons following both SO-SP (control: 1058 pS, flurazepam: 226 pS, P<0.01) and S-L-M (control 998 pS, flurazepam: 179 pS, P<0.01) stimulation, as well as the total charge transfer, indicating a decreased HCO(3)(-)-Cl(-) flux. In HEPES-aCSF, the synaptic conductance and total charge transfer, and thus Cl(-) flux, was unchanged in flurazepam-treated neurons (SO-SP: control 588 pS, flurazepam: 580 pS, P>0.05; S-L-M: control 595 pS, flurazepam: 527 pS, P>0.05). Taken together, these findings suggest that a reduction in HCO(3)(-) flux may play a prominent role in mediating the action of GABA and that a loss of HCO(3)(-) conductance may significantly contribute to impaired GABA(A) receptor function after chronic benzodiazepine treatment.     

Long-term maintenance of mature hippocampal slices in vitro

Cultures of primary neurons or thin brain slices are typically prepared from immature animals. We introduce a method to prepare hippocampal slice cultures from mature rats aged 20-30 days. Mature slice cultures retain hippocampal cytoarchitecture and synaptic connections up to 3 months in vitro. Spontaneous epileptiform activity is rarely observed suggesting long-term retention of normal neuronal excitability and of excitatory and inhibitory synaptic networks. Picrotoxin, a GABAergic Cl(-) channel antagonist, induced characteristic interictal-like bursts that originated in the CA3 region, but not in the CA1 region. These data suggest that mature slice cultures displayed long-term retention of GABAergic inhibitory synapses that effectively suppressed synchronized burst activity via recurrent excitatory synapses of CA3 pyramidal cells. Mature slice cultures lack the reactive synaptogenesis, spontaneous epileptiform activity, and short life span that limit the use of slice cultures isolated from immature rats. Mature slice cultures are anticipated to be a useful addition for the in vitro study of normal and pathological hippocampal function.     

Chronic hyperosmotic stress converts GABAergic inhibition into excitation in vasopressin and oxytocin neurons in the rat

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.