Reduced Gene Expression of KCC2 Accelerates Axonal Regeneration and Reduces Motor Dysfunctions after Tibial Nerve Severance and Suturing.

Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-]i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index-indicating lower limb motor function-was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-]i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation.

Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord.

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Slow progression of sciatic nerve degeneration and regeneration after loose ligation through microglial activation and decreased KCC2 levels in the mouse spinal cord ventral horn.

Peripheral nerve injury affects motor functions. To reveal the mechanisms underlying motor dysfunction and recovery after nerve compression, which have not been precisely examined, we investigated the temporal relationship among changes in motor function, nerve histopathology, and marker molecule expression in the spinal cord after loose ligation of the mouse sciatic nerve. After ligation, sciatic motor function suddenly declined, and axons gradually degenerated. During degeneration, galanin was localized in motor neuron cell bodies. Then, in the ventral horn, microglia were activated, and expression of choline acetyltransferase (ChAT), a synthetic enzyme of acetylcholine, and potassium chloride co-transporter 2 (KCC2), which shifts the action of γ-amino butyric acid (GABA) and glycine to inhibitory, decreased. Motor function recovery was insufficient although axonal regeneration was complete. ChAT levels gradually recovered during axonal regeneration. When regeneration was nearly complete, microglial activation declined, and KCC2 expression started to increase. The KCC2 level sufficiently recovered when axonal regeneration was complete, suggesting that the excitatory action of GABA/glycine may participate in axonal regeneration. Furthermore, these changes proceeded slower than those after severance, suggesting that loose ligation, compression, may mediate slower progression of degeneration and regeneration than severance, and these changes may cause the motor dysfunction and its recovery.

Development and persistence of neuropathic pain through microglial activation and KCC2 decreasing after mouse tibial nerve injury.

Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter in the mature brain, but is excitatory during development and after motor nerve injury. This difference in GABAergic action depends on the intracellular chloride ion concentration ([Cl-]i), primarily regulated by potassium chloride co-transporter 2 (KCC2). To reveal precise processes of the neuropathic pain through changes in GABAergic action, we prepared tibial nerve ligation and severance models using male mice, and examined temporal relationships amongst changes in (1) the mechanical withdrawal threshold in the sural nerve area, (2) localization of the molecules involved in GABAergic transmission and its upstream signaling in the dorsal horn, and (3) histology of the tibial nerve. In the ligation model, tibial nerve degeneration disappeared by day 56, but mechanical allodynia, reduced KCC2 localization, and increased microglia density remained until day 90. Microglia density was higher in the tibial zone than the sural zone before day 21, but this result was inverted after day 28. In contrast, in the severance model, all above changes were detected until day 28, but were simultaneously and significantly recovered by day 90. These results suggested that in male mice, allodynia may be caused by reduced GABAergic synaptic inhibition, resulting from elevated [Cl-]i after the reduction of KCC2 by activated microglia. Furthermore, our results suggested that factors from degenerating nerve terminals may diffuse into the sural zone, whereby they induced the development of allodynia in the sural nerve area, while other factors in the sural zone may mediate persistent allodynia through the same pathway.

Allopregnanolone augments epileptiform activity of an in-vitro mouse hippocampal preparation in the first postnatal week.

In the immature brain the neurotransmitter γ-amino butyric acid (GABA) mediates a membrane depolarization and can contribute to both, inhibition and excitation. Therefore the consequences of a positive modulation of GABA(A) receptors by neurosteroids on epileptiform activity are hard to predict. In order to analyze whether neurosteroids attenuate or exaggerate epileptiform activity in the immature brain, we investigated the effect of the neurosteroid allopregnanolone on epileptiform activity in an in-toto hippocampus preparation of early postnatal mice (postnatal days 4-7) using field potential recordings. These in-vitro experiments revealed that 0.5 µmol/L allopregnanolone had no effect on ictal-like epileptiform activity, but increased the occurrence of interictal epileptiform events. The allopregnanolone-induced enhancement of interictal epileptiform activity could be blocked by a selective inhibition of synaptic GABAA receptors. In contrast, allopregnanolone had no effect on interictal epileptiform activity upon enhanced extrasynaptic GABAergic activity. Patch-clamp experiments demonstrated that allopregnanolone prolonged the decay of GABAergic postsynaptic currents, but had no effect on tonic GABAergic currents. We conclude from these results that allopregnanolone can enhance excitability in the immature hippocampus viaprolonged synaptic GABAergic currents. This potential effect of neurosteroids on brain excitability should be considered if they are applied as anticonvulsants to premature or early postnatal babies.

Changes in the expression and localization of signaling molecules in mouse facial motor neurons during regeneration of facial nerves.

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.

Distinct development of the glycinergic terminals in the ventral and dorsal horns of the mouse cervical spinal cord.

In the spinal cord, glycine and γ-amino butyric acid (GABA) are inhibitory neurotransmitters. However, the ontogeny of the glycinergic network remains unclear. To address this point, we examined the developmental formation of glycinergic terminals by immunohistochemistry for glycine transporter 2 (GlyT2), a marker of glycinergic terminals, in developing mouse cervical spinal cord. Furthermore, the developmental localization of GlyT2 was compared with that of glutamic acid decarboxylase (GAD), a marker of GABAergic terminals, and vesicular GABA transporter (VGAT), a marker of inhibitory terminals, by single and double immunolabeling. GlyT2-positive dots (glycinergic terminals) were first detected in the marginal zone on embryonic day 14 (E14). In the ventral horn, they were detected at E16 and increased in observed density during postnatal development. Until postnatal day 7 (P7), GAD-positive dots (GABAergic terminals) were dominant and GlyT2 immunolabeling was localized at GAD-positive dots. During the second postnatal week, GABAergic terminals markedly decreased and glycinergic terminals became dominant. In the dorsal horn, glycinergic terminals were detected at P0 in lamina IV and P7 in lamina III and developmentally increased. GlyT2 was also localized at GAD-positive dots, and colocalizing dots were dominant at P21. VGAT-positive dots (inhibitory terminals) continued to increase until P21. These results suggest that GABAergic terminals first appear during embryonic development and may often change to colocalizing terminals throughout the gray matter during development. The colocalizing terminals may remain in the dorsal horn, whereas in the ventral horn, colocalizing terminals may give rise to glycinergic terminals.

KCC2-mediated regulation of respiration-related rhythmic activity during postnatal development in mouse medulla oblongata.

GABA acts as inhibitory neurotransmitter in the adult central nervous system but as excitatory neurotransmitter during early postnatal development. This shift in GABA's action from excitation to inhibition is caused by a decrease in intracellular chloride concentration ([Cl(-)]i), which in turn is caused by changes in the relative expression levels of the K(+)-Cl(-) co-transporter (KCC2) and the Na(+), K(+)-2Cl(-) co-transporter (NKCC1) proteins. Previous studies have used slices containing the medullary pre-Bötzinger complex (pre-BötC) to record respiration-related rhythmic activity (RRA) from the hypoglossal nucleus (12 N). The role of GABAergic transmission in the regulation of medullary RRA neonatally, however, is yet to be determined. Here, we examined how GABA and chloride co-transporters contribute to RRA during development in the 12 N where inspiratory neurons reside. We recorded extracellular RRA in medullary slices obtained from postnatal day (P) 0-7 mice. RRA was induced by soaking slices in artificial cerebrospinal fluid (aCSF) containing 8mM-K(+). Application of GABA significantly increased the frequency of RRA after P3, whereas application of a KCC2 blocker (R (+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl)oxy]acetic acid (DIOA)) significantly decreased the frequency of RRA after P1. In addition, dense KCC2 immunolabeling was seen in the superior longitudinalis (SL) of the 12 N, which is responsible for retraction of the tongue, from P0 and P7. These results indicate that GABA administration can increase RRA frequency during the first week following birth. This in turn suggests that decreasing [Cl(-)]i levels caused by increasing KCC2 levels in the 12 N could play important roles in regulating the frequency of RRA during development.

Embryonic development of GABAergic signaling in the mouse spinal trigeminal nucleus interpolaris.

In the mature central nervous system, γ-amino butyric acid (GABA) is an inhibitory neurotransmitter, whereas during development, GABA induces depolarization. To examine the embryonic development of GABAergic transmission in the mouse spinal trigeminal nucleus interpolaris (SpVi), which receives sensory input from the face and is important in survival of rodents, we performed immunohistochemistry for three related molecules: glutamic acid decarboxylase (GAD), a marker of GABAergic neurons; vesicular GABA transporter (VGAT), a marker of GABAergic and glycinergic vesicles; and potassium chloride co-transporter 2 (KCC2), which shifts GABA action from excitatory to inhibitory. GAD-positive longitudinal projection fibers, where VGAT-positive dots were localized, were clearly discernible until embryonic day (E)17, and were markedly decreased in number on postnatal day 0. GAD-positive neurons were detected after E15, and GAD- and VGAT-positive axon varicosities were observed after E17. KCC2 immunolabeling was first localized in the dendrites and cell bodies of several neurons in the lateral part of the SpVi on E13 and throughout the nucleus on E17. These results suggest that the SpVi may first receive GABAergic projection fibers from extra-nuclear area before birth, and GABAergic interneurons may form synapses within the SpVi after E17. In addition, GABA action may gradually shift from excitatory to inhibitory between E13 and E17.

Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus.

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 µM taurine or 10 µM glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 µM 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 µM strychnine or after incubation with the loop diuretic bumetanide (10 µM), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (≥ 1 mM) or glycine (100 µM) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 µM) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

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.

Ontogeny of Cl- homeostasis in mouse hypoglossal nucleus.

We tested the immunoreactivity of KCC2 using KCC2 antibody in the developmental mouse medulla. Age-dependent changes in immunoreactivity were remarkable in the hypoglossal nucleus, and interestingly, the immunoreactivity in the hypoglossal nucleus relative to the dorsal vagal nucleus at P0 appeared to be higher than that of P7. Thus Cl(-) homeostasis in the hypoglossal nucleus might be differentially regulated in the developmental stage.

Cellular mechanisms of subplate-driven and cholinergic input-dependent network activity in the neonatal rat somatosensory cortex.

Early coordinated network activity promotes the development of cortical structures. Although these early activity patterns have been recently characterized with respect to their developmental, spatial and dynamic properties, the cellular mechanisms by which specific neuronal populations trigger coordinated activity in the neonatal cerebral cortex are still poorly understood. Here we characterize the cellular and molecular processes leading to generation of network activity during early postnatal development. We show that the somatosensory cortex of newborn rats expresses cholinergic-driven calcium transients which are synchronized within the deeply located subplate. Correspondingly, endogenous or agonist-induced activation of predominantly m1/m5-assembled muscarinic acetylcholine receptors elicits bursts of action potentials (up states) as a result of suprathreshold activation of the subplate. Tonic activation by ambient nonsynaptically released gamma-amino butyric acid (GABA) facilitates the generation of up states in the neonatal cortex. Additionally, this network activity critically depends on neuronal gap junctions but not on glutamatergic or GABAergic synaptic transmission. Thus, an early circuit relying on the integrative function of the subplate as well as on cholinergic-driven tonic GABA depolarization and tight electrical coupling is able to generate coordinated network activity, which may shape the architecture and control the function of the developing cerebral cortex.

Respiratory neuron group in the high cervical spinal cord discovered by optical imaging.

Spontaneous breathing occurs transiently in spinalized animals suggesting that the cervical spinal cord generates respiratory rhythm. We recorded optical signals from isolated brainstem-spinal cord preparations of neonatal rats using a voltage-sensitive dye, and visualized respiratory-related activity by processing the optical data with a correlation coefficient imaging technique. We identified a novel region that extended from the medullo-spinal junction to the C2 segment, approximately 100-300 mum deep from the ventral surface, and termed the high cervical respiratory group. We recorded inspiratory and preinspiratory-inspiratory neurons in this region. High cervical respiratory group neurons are distinct from motoneurons, because they are small, spindle in shape, and have only two or three long processes, and thus may be interneurons that are involved in respiratory rhythmogenesis in the cervical spinal cord.

Changes in the expression of cation-Cl- cotransporters, NKCC1 and KCC2, during cortical malformation induced by neonatal freeze-lesion.

Focal cortical malformations comprise a heterogeneous group of disturbances in brain development, often associated with intractable epilepsy. A focal freeze-lesion of cerebral cortex in newborn rat produces a cortical malformation that resembles human polymicrogyria, clinical conditions that results from abnormal neuronal migration. The change in GABAergic functions that occurs during early brain development is induced by an alteration in Cl(-) homeostasis and plays important roles in neocortical development by modulating such events as laminar organization and synaptogenesis. We therefore investigated the relationship between pathogenesis of polymicrogyria and ontogeny of Cl(-) homeostasis in developing parietal cortex after creation of a freeze-lesion at P0. We demonstrated, by in situ hybridization histochemistry for cation-Cl(-) cotrtansporters, that NKCC1 mRNA expression was upregulated and KCC2 mRNA expression downregulated at P4 in "bridge" structure (formed in lesion site across the gap in intact exofocal cortex) as compared to exfocal cortex. Immunohistochemical investigation revealed a colocalization of NKCC1 and neuron specific enolase (NSE) within this structure, while BrdU-positive cells express GFAP and NKCC1 appeared beneath it. These results suggest that immature cortical plate neurons might produce "bridge" structure during formation of microgyrus, and that altered neuronal Cl(-) homeostasis might be involved in neuronal migration disorder that ultimately results in cortical malformations.

Kinetic properties of Cl uptake mediated by Na+-dependent K+-2Cl cotransport in immature rat neocortical neurons.

GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl- concentration ([Cl-]i), the mechanisms of Cl- accumulation in immature neurons are still a matter of debate. Using patch-clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl- uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 (P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady-state [Cl-]i of approximately 30 mM that was reduced to values close to passive distribution by bumetanide or Na+-free solutions, suggesting a participation of Na+-K+-2Cl- cotransport isoform 1 (NKCC1) in maintaining elevated [Cl-]i. Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl-]i homeostasis in detail, Cl- uptake rates were analyzed after artificial [Cl-]i depletion. Active Cl- uptake was relatively slow (47.2 +/- 5.0 microM/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl- conductance in CR cells. The low capacity of NKCC1-mediated Cl- uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl- uptake mediated by this transporter is sufficient to maintain high [Cl-]i required to render GABA responses excitatory.

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.

Neuroprotective effects of melatonin against anoxia/aglycemia stress, as assessed by synaptic potentials and superoxide production in rat hippocampal slices.

Melatonin, which plays an important role in circadian rhythm regulation, is highly potent endogenous free radical scavenger and antioxidant. To clarify the neuroprotective effects of melatonin as a free radical scavenger, we recorded changes in synaptic potentials and monitored the generation of superoxide (O)(2)(-) (using hydroethidine) in the CA1 pyramidal layers of rat hippocampal slices exposed to anoxia/aglycemia ('ischemic') stress. Synaptic responses evoked by stimulation of Schaffer collateral/commissural afferents were suppressed by ischemic stress. When the duration of the stress was 8 min, the suppression was reversible, irrespective of the presence or absence of melatonin treatment, while the amount of O(2)(-) generated was reduced by the presence of melatonin. When stress duration was 12 min, the suppression of synaptic responses lasted more than 90 min, but melatonin significantly improved the recovery. The amount of O(2)(-) generated in the 'recirculation' phase after a 12 min ischemic stress was less in the ischemic alone group than in the melatonin-treated group. This probably reflects that the number of viable cells with the ability to generate O(2)(-) had been reduced by the more severe ischemic stress. Other radical scavengers (ascorbic acid and alpha-tocopherol) had similar effects. These results show that melatonin has the potential to protect the functions of neurons against an ischemic insult by reducing O(2)(-) generation.

Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1.

GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl(-)](i) in immature neocortical neurones causes GABA(A) receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl(-) accumulation remain controversial. Therefore, the GABA reversal potential (E(GABA)) or [Cl(-)](i) in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation-Cl(-) cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl(-)](i). The mRNA expression levels were positively (the Cl(-) accumulating Na(+),K(+)-2Cl(-) cotransporter NKCC1) or negatively (the Cl(-) extruding K(+)-Cl(-) cotransporter KCC2) correlated with [Cl(-)](i). NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl(-)](i) and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on E(GABA) were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex.

Experience-dependent changes in intracellular Cl- regulation in developing auditory neurons.

A developmental change in GABA and glycine responses, from a depolarization to a hyperpolarization, have been reported for a range of CNS neurons, and has been demonstrated to be due to a developmental decrease in the intracellular Cl- concentration ([Cl-](i)). We examined [Cl-](i) in isolated rat lateral superior olive (LSO) neurons using patch-clamp recordings of glycine gated Cl- currents and by measuring intracellular Cl- -fluorescence. In neurons from 14-16-day-old rats (P14-P16), which had previously received unilateral or bilateral cochlear ablations before the onset of hearing, there was no developmental decrease in [Cl-](i). No significant differences in [Cl-](i) were observed amongst rats with either ipsi- and contralateral ablations. Implanted strychnine pellets also prevented the decrease in [Cl-](i) in most neurons. In some of these neurons in which [Cl-](i) remained high, there was a lack of expression of the K+-Cl- cotransporter 2 (KCC2) mRNA. These results demonstrate that the developmental decrease in [Cl-](i) in LSO neurons is dependent on neuronal activity and that both GABAergic/glycinergic and glutamatergic afferent activity contribute to this maturation of the Cl- regulatory mechanisms.