NKCC1 activity modulates formation of functional inhibitory synapses in cultured neocortical neurons

Intracellular Cl(-) concentration ([Cl(-)](i)) in immature neurons is higher than that expected for a passive distribution, therefore the equilibrium potential for chloride is more positive than the resting membrane potential, and the resulting GABA renders immature neurons depolarization. The higher [Cl(-)](i) in immature neurons is thought to be attributed to the uptake of Cl(-) mediated by NKCC1 (Na(+), K(+)-2Cl(-) cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA(A) receptor-mediated pathway. To test this possibility, we examined the effects of a Cl(-)-uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 microM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but not of spontaneous excitatory postsynaptic currents (sEPSCs). Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating decrease in the number of functional GABAergic synapses. Acute treatment with bumetanide (10 microM) decreased neuronal [Cl(-)](i), the amplitude of RDPs, and neuronal excitability, while bumetanide had no effect on RDPs and neuronal excitability in the presence of bicuculline. These results suggest that the uptake of Cl(-) by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response.     

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.     

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.     

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.     

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.     

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.     

Differential development of cation-chloride cotransporters and Cl- homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus

A recent study suggested that gamma-aminobutyric acid (GABA) plays differential roles in activity-dependent plasticity between the visual cortex (VC) and the dorsal lateral geniculate nucleus (dLGN). In the present study, to investigate differential GABAergic functions in postnatal visual system development, the development of [Cl(-)](i), cation-Cl(-) cotransporter expression, and the [Ca(2+)](i) responses evoked by GABA were compared between VC and dLGN during the early stages of development. Using rat brain slices from postnatal days (P) 0-17, GABA-evoked [Ca(2+)](i) responses and resting [Cl(-)](i) were measured by means of optical imaging of Ca(2+) and Cl(-), respectively. Changes in the expression of cation-Cl(-) cotransporters (viz. the outwardly-directed K(+)-Cl(-) cotransporter, KCC2, and the inwardly-directed Na(+),K(+)-2Cl(-) cotransporter, NKCC1) were examined in VC and dLGN by in situ hybridization. At birth, the excitatory actions of GABA were powerful in VC, but missing in dLGN (as indicated by neuronal [Ca(2+)](i) transients), and the resting [Cl(-)](i) was significantly higher in VC than in dLGN. Signals for KCC2 mRNA expression were significantly higher in dLGN than in VC at P0. This suggests that extrusion of Cl(-) from neurons is stronger in dLGN than in VC at P0, so that a GABAergic excitatory effect was not observed in dLGN because of more negative equilibrium potential for Cl(-). The present study indicates clear differences in the molecular and physiological bases of Cl(-) homeostasis and GABA actions between the developing VC and dLGN. Such differential GABAergic actions may underlie the distinct mechanisms involved in VC and dLGN development within the visual system.     

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.     

Increased aquaporin-1 and Na+ -K+ -2Cl- cotransporter 1 expression in choroid plexus leads to blood-cerebrospinal fluid barrier disruption and necrosis of hippocampal CA1 cells in acute rat models of hyponatremia

Hyponatremia is a metabolic disorder characterized by increased cerebrospinal fluid (CSF) volume and pressure, although the site of brain insult is unclear. Specifically, the hippocampus, which is in direct contact with expanding CSF ventricles, may be involved. The present study was undertaken to investigate the possible roles of choroid plexus aquaporin-1 (AQP1) and of cation chloride transporters (Na(+) -K(+) -2Cl(-) cotransporter 1 [NKCC1] and K(+) -Cl(-) cotransporter 4 [KCC4]) in the underlying hippocampal pathophysiology of hyponatremia in acute (6 and 12 hr duration) experimental models. It was found that the expressions of AQP1 and NKCC1 proteins in choroid plexus were significantly increased, whereas the expression of KCC4 protein was unchanged vs. control values after 6 and 12 hr of hyponatremia. Choroid plexuses with increased AQP1 and NKCC1 after 6 hr of hyponatremia showed caspase 3-dependent apoptosis and disruption of the blood-CSF barrier. Furthermore, necrotic changes in CA1 neuronal cells were observed after 6 and 12 hr of hyponatremia. Overall, these data suggest that increases in AQP1 and NKCC1 expression under hyposmotic stress may be one of the molecular mechanisms underlying the pathophysiology of acute hyponatremia, such as the necrotic cell death of hippocampal CA1 region by increasing water transport across the blood-CSF barrier. Furthermore, we suggest that opening of the blood-CSF barrier after acute hyponatremia may be triggered the secondary adverse conditions that are capable of enhancing selective necrosis in hippocampal CA1 cells.     

Prolongation and enhancement of gamma-aminobutyric acid receptor mediated excitation by chronic treatment with estradiol in developing rat hippocampal neurons

GABA(A) receptor activation during brain development is a critical source of excitation. This is due to the positive equilibrium potential for chloride relative to resting membrane potential, resulting in membrane depolarization sufficient to open voltage sensitive calcium channels. The gonadal steroid estradiol has pronounced trophic effects on the developing hippocampus, promoting cell survival and synaptogenesis. In the current study, we investigated the effect of estradiol on GABA(A) receptor-mediated calcium transients in cultured neonatal hippocampal neurons, from Sprague-Dawley rats, using the calcium sensitive dye, Fura-2-AM. Treatment of hippocampal neurons with physiological levels of estradiol significantly increased the peak amplitude of calcium transients, increased the number of cells responding to the GABA(A) agonist muscimol with membrane depolarization, and delayed the rate of clearance of free intracellular calcium. These effects were significantly attenuated by pretreatment with the oestrogen receptor antagonist ICI-182,780. This suggests that estradiol, via its action on the oestrogen receptor, prolongs the developmental duration of depolarizing GABA. Estradiol likely maintains GABA-mediated excitation by promoting increased protein levels of the active/phosphorylated form of the chloride cotransporter Na+K+2CL- and L-type voltage sensitive calcium channels containing the alpha1C subunit. We propose that a component of the trophic effects of estradiol on hippocampal development results from enhanced calcium influx subsequent to GABA(A) receptor activation.     

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.     

Intracellular acidification in neurons induced by ammonium depends on KCC2 function

The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.     

Roles of the cation-chloride cotransporters in neurological disease

In the nervous system, the intracellular chloride concentration ([Cl(-)](i)) determines the strength and polarity of gamma-aminobutyric acid (GABA)-mediated neurotransmission. [Cl(-)](i) is determined, in part, by the activities of the SLC12 cation-chloride cotransporters (CCCs). These transporters include the Na-K-2Cl cotransporter NKCC1, which mediates chloride influx, and various K-Cl cotransporters--such as KCC2 and KCC3-that extrude chloride. A precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult CNS, and for excitatory GABAergic signaling in the developing CNS and the adult PNS. Altered chloride homeostasis, resulting from mutation or dysfunction of NKCC1 and/or KCC2, causes neuronal hypoexcitability or hyperexcitability; such derangements have been implicated in the pathogenesis of seizures and neuropathic pain. [Cl(-)](i) is also regulated to maintain normal cell volume. Dysfunction of NKCC1 or of swelling-activated K-Cl cotransporters has been implicated in the damaging secondary effects of cerebral edema after ischemic and traumatic brain injury, as well as in swelling-related neurodegeneration. CCCs represent attractive therapeutic targets in neurological disorders the pathogenesis of which involves deranged cellular chloride homoestasis.     

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.     

Bumetanide administration attenuated traumatic brain injury through IL-1 overexpression

OBJECTIVE: To examine the effects of administration of bumetanide, a specific NKCC1 inhibitor, on traumatic brain injury (TBI)-induced interleukin-1 (IL-1) expression. METHODS: TBI model was induced by the calibrated weight drop device (450 g in weight, 2.0 m in height) in adult rats based on procedures previously reported. One hundred and sixty Wistar rats were divided into sham-control group and experimental group for time course works of TBI. The expression of IL-1beta brain edema and neuronal damage were determined in these animals after TBI. RESULTS: We found that both mRNA and protein of IL-1beta were up-regulated in the hippocampus 3-24 hours after TBI. Animals displayed severe brain edema and neuron damage after TBI. Bumetanide (15 mg/kg), a specific Na(+) -K(+) -2Cl(-) cotransporter inhibitor, significantly attenuated the TBI-induced neuronal damage by IL-1beta overexpression. The present study suggests that administration of bumetanide could significantly decreased TBI-induced inflammatory response and neuronal damage.     

Plasticity of spinal cord locomotor networks and contribution of cation-chloride cotransporters

Locomotor burst activity in the mature intact spinal cord alternates between the left and right sides of a segment through reciprocal inhibition. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. These data reveal the plasticity of spinal cord locomotor networks. This review describes recent evidence suggesting that regulation of cation-chloride cotransporter expression and activity may underlie this plasticity. GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. This post-synaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)) which is regulated by a protein in the plasma membrane: the K(+)-Cl(-) cotransporter (KCC2) extruding both K(+) and Cl(-) ions. No or a reduced KCC2 expression leads to a depolarizing (excitatory) action of GABA and glycine. This latter situation is observed early during development and in several pathological conditions, such as epilepsy, neuronal injury and chronic pain.     

Gain-of-function missense variant in SLC12A2, encoding the bumetanide-sensitive NKCC1 cotransporter,identified in human schizophrenia

Perturbations of γ-aminobutyric acid (GABA) neurotransmission in the human prefrontal cortex have been implicated in the pathogenesis of schizophrenia (SCZ), but the mechanisms are unclear. NKCC1 (SLC12A2) is a Cl^–-importing cation-Cl^– cotransporter that contributes to the maintenance of depolarizing GABA activity in immature neurons, and variation in SLC12A2 has been shown to increase the risk for schizophrenia via alterations of NKCC1 mRNA expression. However, no disease-causing mutations or functional variants in NKCC1 have been identified in human patients with SCZ. Here, by sequencing three large French-Canadian (FC) patient cohorts of SCZ, autism spectrum disorders (ASD), and intellectual disability (ID), we identified a novel heterozygous NKCC1 missense variant (p.Y199C) in SCZ. This variant is located in an evolutionarily conserved residue in the critical N-terminal regulatory domain and exhibits high predicted pathogenicity. No NKCC1 variants were detected in ASD or ID, and no KCC3 variants were identified in any of the three neurodevelopmental disorder cohorts. Functional experiments show Y199C is a gain-of-function variant, increasing Cl^–-dependent and bumetanide-sensitive NKCC1 activity even in conditions in which the transporter is normally functionally silent (hypotonicity). These data are the first to describe a functional missense variant in SLC12A2 in human SCZ, and suggest that genetically encoded dysregulation of NKCC1 may be a risk factor for, or contribute to the pathogenesis of, human SCZ.     

Differential functional expression of cation-Cl- cotransporter mRNAs (KCC1, KCC2, and NKCC1) in rat trigeminal nervous system

GABA is the main inhibitory neurotransmitter in the adult brain, which causes Cl- influx into the cell via GABAA receptors. The direction of Cl- inflow is dependent on the Cl- gradient across the membrane. Cation-Cl- cotransporters have been considered to play pivotal roles in controlling intracellular Cl- concentration ([Cl-]i) of neurons; hence, they modulate the GABAergic function. To elucidate how these cotransporters are distributed in the trigeminal nuclei, we investigated the expressions of K+-Cl- cotransporters (KCC1 and KCC2) and Na+-K+-2Cl- cotransporter (NKCC1) mRNAs by using in situ hybridization histochemistry. KCC2 mRNA was expressed in the motor trigeminal nucleus (Mo5), the principal trigeminal nucleus (Pr5), and the spinal trigeminal nucleus (Sp5), but not in the trigeminal ganglion (TG) and the mesencephalic trigeminal nucleus (Me5). On the other hand, KCC1 and NKCC1 mRNAs were expressed in all the trigeminal nuclei. The resting [Cl-]i of Me5 neurons was significantly higher than that of Mo5 neurons. Thus, in primary sensory neurons such as the TG and the Me5, [Cl-]i would be higher than those in the other trigeminal nuclei because of the lack of KCC2 mRNA expression. Since Me5 neurons, but not Mo5 neurons, responded to GABA by depolarization, GABA would have differential physiological functions among trigeminal nuclei and TG.     

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.