Gramicidin Perforated-patch Recordings Research Articles

Serotoninergic Modulation of Chloride Homeostasis during Maturation of the Locomotor Network in Zebrafish

During development, neural networks progress through important functional changes such as the generation of spontaneous activity, the expression of a depolarizing chloride gradient, and the appearance of neuromodulation. Little is known about how these processes are integrated to yield mature behaviors. We showed previously that, during the maturation of the locomotor network of the zebrafish, endogenous serotonin (5HT) increased motor activity by reducing intervals of inactivity, without affecting the active swim periods that are the target of 5HT in other and more mature preparations. Because membrane properties were constant during the rest intervals, we examined here whether 5HT modulates chloride homeostasis. We compared the effects of blocking (inward) chloride cotransport with bumetanide to the effects of 5HT and its antagonists, both behaviorally by video imaging and cellularly by whole-cell and gramicidin-perforated patch recordings. Bumetanide mimicked the effects of 5HT antagonists, by prolonging rest intervals without affecting the properties of swim episodes (duration; frequency; extent of depolarization) either behaviorally or during fictive swimming. Furthermore, bumetanide and 5HT antagonists suppressed the amplitude of depolarizing responses evoked by ionophoresis of glycine onto spinal neurons in the presence of tetrodotoxin and transiently suppressed the amplitude of responses to glycine measured after fictive swimming. The effects of bumetanide contrasted with and occluded the effects of 5HT. We suggest that, during development, endogenous 5HT modulates chloride homeostasis during the quiescent intervals and thereby offsets the long periods of quiescence commonly observed in developing networks to allow expression of sustained and behaviorally relevant activity.

Differential Maturation of GABA Action and Anion Reversal Potential in Spinal Lamina I Neurons: Impact of Chloride Extrusion Capacity

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.

Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory Cl− Movements in Salivary Acinar Cells

Elevations of cytoplasmic free calcium concentrations ([Ca2+]i) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl− through Ca2+-activated Cl− channels, while Cl− enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na+-K+-2Cl− cotransporter (NKCC) and/or parallel operations of Cl−-HCO3 − and Na+-H+ exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl− secretion, we analyzed carbachol (CCh)-activated Cl− currents in submandibular acinar cells using the “gramicidin-perforated patch recording configuration.” Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl− currents in the gramicidin-perforated patch recording were carried by Cl− efflux via Cl− channels, dependent upon Cl− entry through Cl− transporters expressed in the acinar cells. CCh-evoked oscillatory Cl− currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl− currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO3 − had significant effects, suggesting that the cotransporter rather than parallel operations of Cl−-HCO3 − and Na+-H+ exchangers is the primary Cl− uptake pathway. Pharmacological manipulation of the activities of the Ca2+-activated Cl− channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl− currents, while adjusting to the rate imposed by the Ca2+-activated Cl− channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl− movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Hyperpolarizing inhibition develops without trophic support by GABA in cultured rat midbrain neurons.

During a limited period of early neuronal development, GABA is depolarizing and elevates [Ca2+]i, which mediates the trophic action of GABA in neuronal maturation. We tested the attractive hypothesis that GABA itself promotes the developmental change of its response from depolarizing to hyperpolarizing (Ganguly et al. 2001). In cultured midbrain neurons we found that the GABA response changed from depolarizing to hyperpolarizing, although GABAA receptors had been blocked throughout development. In immature neurons prolonged exposure of the cells to nanomolar concentrations of GABA or brief repetitive applications of GABA strongly diminished the elevation of [Ca+]i by GABA. As revealed by gramicidin perforated-patch recording, reduced [Ca2+]i responses were due to a diminished driving force for Cl-. This suggests that immature neurons do not have an efficient inward transport that can compensate the loss of cytosolic Cl-resulting from sustained GABAA receptor activation by ambient GABA. Transient increases in external K+, which can induce voltage-dependent Cl- entry, restored GABA-induced [Ca2+]i elevations. In mature neurons, GABA reduced [Ca2+]i provided that background [Ca2+]i was elevated by the application of an L-type Ca2+ channel agonist. This was probably due to a hyperpolarization of the membrane by Cl- currents. K(+)-Cl- cotransport maintained the gradient for hyperpolarizing Cl-currents. We conclude that in immature midbrain neurons an inward Cl- transport is not effective although the GABA response is depolarizing. Further, GABA itself is not required for the developmental switch of GABAergic responses from depolarizing to hyperpolarizing in cultured midbrain neurons.

Membrane Potential of CA3 Hippocampal Pyramidal Cells During Postnatal Development

A depolarized resting membrane potential has long been considered to be a universal feature of immature neurons. Despite the physiological importance, the underlying mechanisms of this developmental phenomenon are poorly understood. Using perforated-patch, whole cell, and cell-attached recordings, we measured the membrane potential in CA3 pyramidal cells in hippocampal slices from postnatal rats. With gramicidin perforated-patch recordings, membrane potential was -44 +/- 4 (SE) mV at postnatal days P0-P2, and it progressively shifted to -67 +/- 2 mV at P13-15. A similar developmental change of the membrane potential has been also observed with conventional whole cell recordings. However, the value of the membrane potential deduced from the reversal potential of N-methyl-d-aspartate channels in cell-attached recordings did not change with age and was -77 +/- 2 mV at P2 and -77 +/- 2 mV at P13-14. The membrane potential measured using whole cell recordings correlated with seal and input resistance, being most depolarized in neurons with high, several gigaohms, input resistance and low seal resistance. Simulations revealed that depolarized values of the membrane potential in whole cell and perforated-patch recordings could be explained by a shunt through the seal contact between the pipette and membrane. Thus the membrane potential of CA3 pyramidal cells appears to be strongly negative at birth and does not change during postnatal development.

Differential sensitivity of atrial and ventricular K(ATP) channels to metabolic inhibition.

The aim is to compare the activation of ATP-sensitive potassium channels (K(ATP) channels) in intact and metabolically impaired atrial and ventricular myocytes. The K(ATP) channel current is measured by whole cell and gramicidin-perforated patch clamp recordings in 164 cultured neonate rat cardiomyocytes. In whole cell recordings with 84 micromol/l ADP in pipette, spontaneous activity is significantly higher in atrium than ventricle, and EC(50) for the K(ATP) channel opener diazoxide is 0.13 micromol/l (atrium) versus 3.1 micromol/l (ventricle). With an ATP-regenerating system in pipette, EC(50) for diazoxide is 19.7 micromol/l (atrium) versus 54.9 micromol/l (ventricle). In gramicidin-perforated patch recordings, atrial myocytes respond significantly to 100 nmol/l of the mitochondrial protonophore CCCP, while ventricular myocytes do not. EC(50) for diazoxide is 129 micromol/l (atrium) versus >2500 micromol/l (ventricle) for myocytes exposed to CCCP, and 676 versus >2500 micromol/l, respectively, without CCCP. (1) K(ATP) channels are significantly more sensitive to metabolic inhibition in atrial than ventricular myocytes. (2) Sensitivity of atrium versus ventricle to the channel opener diazoxide increases from 3:1 to > or = 24:1 with ADP or metabolic inhibition. If extended to intact hearts, the results would predict a higher atrial sensitivity to ischemia, and a high sensitivity of the ischemic atrium to K(ATP) channel openers.

Excitatory Actions of GABA in the Cortex

Little is known about how GABAergic inputs interact with excitatory inputs under conditions that maintain physiological concentrations of intracellular anions. Using extracellular and gramicidin perforated-patch recording, we show that somatic and dendritic GABA responses in mature cortical pyramidal neurons are depolarizing from rest and can facilitate action potential generation when combined with proximal excitatory input. Dendritic GABA responses were excitatory regardless of timing, whereas somatic GABA responses were inhibitory when coincident with excitatory input but excitatory at earlier times. These excitatory actions of GABA occur even though the GABA reversal potential is below action potential threshold and largely uniform across the somato-dendritic axis, and arise when GABAergic inputs are temporally or spatially isolated from concurrent excitation. Our findings demonstrate that under certain circumstances GABA will have an excitatory role in synaptic integration in the cortex.

Functional synaptic projections onto subplate neurons in neonatal rat somatosensory cortex.

Subplate neurons (SPn) play an important role in the formation of thalamocortical connections during early development and show glutamatergic and GABAergic spontaneous synaptic activity. We characterized these synaptic inputs by performing whole-cell recordings from SPn in somatosensory cortical slices of postnatal day 0-3 rats. At -70 mV, electrical stimulation of the thalamocortical afferents elicited in 68% of the SPn a monosynaptic CNQX-sensitive postsynaptic current (PSC). These fast PSCs were mediated by AMPA receptors, because they were prolonged by cyclothiazide and blocked by GYKI 52466. On membrane depolarization, thalamocortical stimulation elicited in 50% of the cells an additional slow monosynaptic component mediated by NMDA receptors. Stimulation of the cortical plate evoked in 72% of SPn a monosynaptic AMPA receptor-mediated PSC with an additional NMDA component at depolarized membrane potentials and in 40% of the investigated cells polysynaptic responses, depending on GABA(A) and NMDA receptors. Stimulation of the subplate elicited in 67% of SPn a monosynaptic dual-component PSC mediated by AMPA and NMDA receptors activated at -70 mV and in 12% of SPn a monosynaptic single-component PSC mediated by AMPA receptors with an additional NMDA component activated at depolarized membrane potentials. A monosynaptic GABAergic response could be observed in 68% of SPn after stimulation of the subplate. In gramicidin-perforated patch recordings, bath application of GABA caused membrane depolarization to -40 mV and elicited action potentials. These results demonstrate that SPn receive distinct functional synaptic inputs arising from the thalamus, cortical plate, and subplate, indicating that SPn are capable of integrating and processing information from cortical and subcortical regions.

Functional Synaptic Projections onto Subplate Neurons in Neonatal Rat Somatosensory Cortex

Subplate neurons (SPn) play an important role in the formation of thalamocortical connections during early development and show glutamatergic and GABAergic spontaneous synaptic activity. We characterized these synaptic inputs by performing whole-cell recordings from SPn in somatosensory cortical slices of postnatal day 0–3 rats. At –70 mV, electrical stimulation of the thalamocortical afferents elicited in 68% of the SPn a monosynaptic CNQX-sensitive postsynaptic current (PSC). These fast PSCs were mediated by AMPA receptors, because they were prolonged by cyclothiazide and blocked by GYKI 52466. On membrane depolarization, thalamocortical stimulation elicited in 50% of the cells an additional slow monosynaptic component mediated by NMDA receptors. Stimulation of the cortical plate evoked in 72% of SPn a monosynaptic AMPA receptor-mediated PSC with an additional NMDA component at depolarized membrane potentials and in 40% of the investigated cells polysynaptic responses, depending on GABA_A and NMDA receptors. Stimulation of the subplate elicited in 67% of SPn a monosynaptic dual-component PSC mediated by AMPA and NMDA receptors activated at –70 mV and in 12% of SPn a monosynaptic single-component PSC mediated by AMPA receptors with an additional NMDA component activated at depolarized membrane potentials. A monosynaptic GABAergic response could be observed in 68% of SPn after stimulation of the subplate. In gramicidin-perforated patch recordings, bath application of GABA caused membrane depolarization to –40 mV and elicited action potentials. These results demonstrate that SPn receive distinct functional synaptic inputs arising from the thalamus, cortical plate, and subplate, indicating that SPn are capable of integrating and processing information from cortical and subcortical regions.

Reduction of KCC2 Expression and GABAAReceptor-Mediated Excitation after In Vivo Axonal Injury

After axotomy, application of muscimol, a GABA_Areceptor agonist, induced an increase in intracellular Ca²⁺([Ca²⁺]_i) in dorsal motor neurons of the vagus (DMV neurons). Elevation of [Ca²⁺]_i by muscimol was blocked by bicuculline, tetrodotoxin, and Ni²⁺. In axotomized DMV neurons measured with gramicidin perforated-patch recordings, reversal potentials of the GABA_A receptor-mediated response, presumably equal to the equilibrium potential of Cl⁻, were more depolarized than that in intact neurons. Thus, GABA_A receptor-mediated excitation is suggested to be attributable to Cl⁻ efflux out of the cell because of increased intracellular Cl⁻ concentration ([Cl⁻]_i) in axotomized neurons. Regulation of [Cl⁻]_i in both control and injured neurons was disturbed by furosemide and bumetanide and by manipulating cation balance across the membrane, suggesting that functional alteration of furosemide-sensitive cation–Cl⁻ cotransporters is responsible for the increase of [Cl⁻]_i after axotomy.<i>In situ</i> hybridization revealed that neuron-specific K⁺-Cl⁻ cotransporter (KCC2) mRNA was significantly reduced in the DMV after axotomy compared with that in control neurons. Similar expression of Na⁺, K⁺-Cl⁻cotransporter mRNA was observed between axotomized and control DMV neurons. Thus, axotomy led to disruption of [Cl⁻]_i regulation attributable to a decrease of KCC2 expression, elevation of intracellular Cl⁻, and an excitatory response to GABA. A switch of GABA action from inhibitory to excitatory might be a mechanism contributing to excitotoxicity in injured neurons.

Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury.

After axotomy, application of muscimol, a GABA(A) receptor agonist, induced an increase in intracellular Ca(2+) ([Ca(2+)](i)) in dorsal motor neurons of the vagus (DMV neurons). Elevation of [Ca(2+)](i) by muscimol was blocked by bicuculline, tetrodotoxin, and Ni(2+). In axotomized DMV neurons measured with gramicidin perforated-patch recordings, reversal potentials of the GABA(A) receptor-mediated response, presumably equal to the equilibrium potential of Cl(-), were more depolarized than that in intact neurons. Thus, GABA(A) receptor-mediated excitation is suggested to be attributable to Cl(-) efflux out of the cell because of increased intracellular Cl(-) concentration ([Cl(-)](i)) in axotomized neurons. Regulation of [Cl(-)](i) in both control and injured neurons was disturbed by furosemide and bumetanide and by manipulating cation balance across the membrane, suggesting that functional alteration of furosemide-sensitive cation-Cl(-) cotransporters is responsible for the increase of [Cl(-)](i) after axotomy. In situ hybridization revealed that neuron-specific K(+)-Cl(-) cotransporter (KCC2) mRNA was significantly reduced in the DMV after axotomy compared with that in control neurons. Similar expression of Na(+), K(+)-Cl(-) cotransporter mRNA was observed between axotomized and control DMV neurons. Thus, axotomy led to disruption of [Cl(-)](i) regulation attributable to a decrease of KCC2 expression, elevation of intracellular Cl(-), and an excitatory response to GABA. A switch of GABA action from inhibitory to excitatory might be a mechanism contributing to excitotoxicity in injured neurons.

Mixed excitatory and inhibitory GABA-mediated transmission in chick cochlear nucleus.

1. Neurons of the chick nucleus magnocellularis (NM) receive depolarizing GABAergic input from the superior olivary nucleus (SON). We examined the response to exogenous GABA or to stimulation of GABAergic fibres in order to identify the ionic basis of GABAergic synaptic transmission and its physiological implications. 2. Reversal potentials of GABA responses (E(GABA)) were determined exclusively by the Cl(-) gradient, measured using whole-cell recording. With gramicidin-perforated patch recording, E(GABA) was -25 +/- 5 mV (mean +/- S.D.), and was stable between embryonic day 17 and post-hatch day 10. With normal intracellular Cl(-), GABA depolarized neurons by 12 mV. 3. In current clamp, repetitive activation of the GABAergic axons reduced the probability of spiking in response to simultaneous stimulation of excitatory axons. However, IPSPs could themselves elicit action potentials, and facilitation of IPSPs by repetitive activation could lead to a characteristic pattern of spiking. 4. These data indicate that IPSPs with reversal potentials positive to spike threshold may have dual functions, depending on the context of their activation.

Post-episode depression of GABAergic transmission in spinal neurons of the chick embryo.

Whole cell recordings were obtained from ventral horn neurons in spontaneously active spinal cords isolated from the chick embryo [embryonic days 10 to 11 (E10-E11)] to examine the post-episode depression of GABAergic transmission. Spontaneous activity occurred as recurrent, rhythmic episodes approximately 60 s in duration with 10- to 15-min quiescent inter-episode intervals. Current-clamp recording revealed that episodes were followed by a transient hyperpolarization (7 +/- 1.2 mV, mean +/- SE), which dissipated as a slow (0.5-1 mV/min) depolarization until the next episode. Local application of bicuculline 8 min after an episode hyperpolarized spinal neurons by 6 +/- 0.8 mV and increased their input resistance by 13%, suggesting the involvement of GABAergic transmission. Gramicidin perforated-patch recordings showed that the GABAa reversal potential was above rest potential (E(GABAa) = -29 +/- 3 mV) and allowed estimation of the physiological intracellular [Cl(-)] = 50 mM. In whole cell configuration (with physiological electrode [Cl(-)]), two distinct types of endogenous GABAergic currents (I(GABAa)) were found during the inter-episode interval. The first comprised TTX-resistant, asynchronous miniature postsynaptic currents (mPSCs), an indicator of quantal GABA release (up to 42% of total mPSCs). The second (tonic I(GABAa)) was complimentary to the slow membrane depolarization and may arise from persistent activation of extrasynaptic GABAa receptors. We estimate that approximately 10 postsynaptic channels are activated by a single quantum of GABA release during an mPSC and that about 30 extrasynaptic GABAa channels are required for generation of the tonic I(GABAa) in ventral horn neurons. We investigated the post-episode depression of I(GABAa) by local application of GABA or isoguvacine (100 microM, for 10-30 s) applied before and after an episode at holding potentials (V(hold)) -60 mV. The amplitude of the evoked I(GABA) was compared after clamping the cell during the episode at one of three different V(hold): -60 mV, below E(GABAa) resulting in Cl(-) efflux; -30 mV, close to E(GABAa) with minimal Cl(-) flux; and 0 mV, above E(GABAa) resulting in Cl(-) influx during the episode. The amplitude of the evoked I(GABA) changed according to the direction of Cl(-) flux during the episode: at -60 mV a 41% decrease, at -30 mV a 4% reduction, and at 0 mV a 19% increase. These post-episode changes were accompanied by shifts of E(GABAa) of -10, -1.2, and +7 mV, respectively. We conclude that redistribution of intracellular [Cl(-)] during spontaneous episodes is likely to be an important postsynaptic mechanism involved in the post-episode depression of GABAergic transmission in chick embryo spinal neurons.

Correlation between electrophysiological and morphological characteristics during maturation of rat supraoptic neurons.

The neurohypophysial peptides oxytocin (OT) and vasopressin (AVP) are well known for their role in reproductive functions and fluid balance regulation, respectively. During development, these peptides are thought to act as trophic factors on both peripheral and central structures. However, despite this early developmental function, the maturation of their secreting neurons remains poorly investigated. In this study, we have characterized the electrical and morphological characteristics displayed by OT and AVP supraoptic (SO) neurons between embryonic day 21 and postnatal day 20. Transient changes in passive membrane properties, correlated with a transient increase in the dendritic arborization, were observed at the beginning of the second postnatal week (PW2). The action potential matured mostly during PW1 and its threshold progressively hyperpolarized in parallel with the resting membrane potential. During PW1, SO neurons displayed unique characteristics with a low-threshold Ca(2+)-dependent depolarizing potential and a prominent hyperpolarization-activated current (I(h) ). This latter is involved in a depolarizing sag during hyperpolarization and an after hyperpolarizing potential following a depolarization. During this period, maintaining E(Cl) unchanged by the use of gramicidin-perforated patch recordings revealed excitatory GABAergic potentials, that became inhibitory during PW2, whilst glutamatergic potential appeared. The electrical activity was very erratic in young neurons and progressively differentiated in the typical firing observed in mature neurons (tonic and phasic for OT and AVP neurons, respectively) during PW2--3. These results show that the development of electrical properties of SO neurons is correlated with the maturation of their dendritic arborization.

An Excitatory GABAergic Plexus in Developing Neocortical Layer 1

Layer 1 of the developing rodent somatosensory cortex contains a dense, transient GABAergic fiber plexus. Axons arising from the zona incerta (ZI) of the ventral thalamus contribute to this plexus, as do axons of intrinsic GABAergic cells of layer 1. The function of this early-appearing fiber plexus is not known, but these fibers are positioned to contact the apical dendrites of most postmigratory neurons. Here we show that electrical stimulation of layer 1 results in a GABA(A)-mediated postsynaptic current (PSC) in pyramidal neurons. Gramicidin perforated patch recording demonstrates that the GABAergic layer 1 synapse is excitatory and can trigger action potentials in cortical neurons. In contrast to electrical stimulation, activation of intrinsic layer 1 neurons with a glutamate agonist fails to produce PSCs in pyramidal cells. In addition, responses can be evoked by stimulation of layer 1 at relatively large distances from the recording site. These findings are consistent with a contribution of the widely projecting incertocortical pathway, the only described GABAergic projection to neonatal cortex. Recording of identified neonatal incertocortical neurons reveals a population of active cells that exhibit high frequencies of spontaneous action potentials and are capable of robustly activating neonatal cortical neurons. Because the fiber plexus is confined to layer 1, this pathway provides a spatially restricted excitatory GABAergic innervation of the distal apical dendrites of pyramidal neurons during the peak period of cortical synaptogenesis.

Intracellular [Cl −] modulates synchronous electrical activity in rat neocortical neurons in culture by way of GABAergic inputs

The influence of GABAergic neurons on spontaneous electrical activities of neocortical neurons in culture, which was estimated to be about 9.5% of the total neurons by immunohistochemistry, was examined using dual whole-cell recording. Synchronized depolarization or hyperpolarization was observed in recorded neurons with pipettes containing low [Cl −] solution, while synchronized bursting of action potentials (APs) was observed with pipettes containing high [Cl −] solution. Spontaneous currents (SCs) were synchronous in all pairs tested with either pipettes containing low or high [Cl −] solution and spontaneous outward currents (SOCs) observed at around −30 mV were sensitive to the GABA-A receptor antagonist, bicuculline. Their reversal potential ( V rev) was linearly related to the logarithm of Cl − activity in the pipette (−56.9 mV/decade). The intracellular chloride concentration was estimated from the V rev of SCs with gramicidin perforated-patch recordings and was between 5.9 and 28.1 mM (mean: 13.0 mM). These results suggest that GABA depolarized some neurons and hyperpolarized others, depending on the E Cl. Bicuculline decreased the frequency of periodic depolarized potentials and increased their amplitudes. However, perfusion with low [Cl −] bath solution did not decrease the frequency. Our data indicate that recurrent subthreshold electrical activities by GABAergic inputs along with glutamatergic inputs take part in deterring synchronized bursting and that intracellular [Cl −] can modulate this bursting.

Afferent Regulation of Inhibitory Synaptic Transmission in the Developing Auditory Midbrain

To determine whether afferent innervation regulates the strength of inhibitory connections in the gerbil auditory midbrain, both cochleas were surgically removed in postnatal day 7 animals, before sound-driven activity is first observed. Inhibitory synaptic currents were measured in a brain slice preparation 1-7 d after the ablations. Whole-cell and gramicidin-perforated patch recordings were obtained from inferior colliculus neurons, and IPSCs were evoked by stimulation of the commissure of the inferior colliculus (CIC) or the ipsilateral lateral lemniscus (LL) in the presence of kynurenic acid. Deafferentation led to a 24 mV depolarizing shift in the IPSC equilibrium potential within 1 d of deafferentation. As a consequence, there was a large reduction of IPSC amplitude at a holding potential of -20 mV in neurons from bilaterally ablated animals. Furthermore, both afferent pathways displayed a 50% reduction of the inhibitory synaptic conductance after deafferentation, indicating that driving force was not solely responsible for the decline in IPSC amplitude. When paired pulses were delivered to the LL or CIC pathway in control neurons, the evoked IPSCs exhibited facilitation. However, paired pulse facilitation was nearly eliminated after deafferentation. Thus, normal innervation affects inhibitory synaptic strength by regulating postsynaptic chloride homeostasis and presynaptic transmitter release properties.

Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation.

1. The inhibitory neurotransmitter glycine can elicit depolarizing responses in immature neurones. We investigated the changes in glycine responses and their ionic mechanism in developing neurones of the rat lateral superior olive (LSO), an auditory brainstem nucleus involved in sound localization. 2. Whole-cell and gramicidin perforated-patch recordings were performed from visually identified LSO neurones in brain slices and glycine was pressure applied for 3-100 ms to the soma. Glycine-evoked currents were reversibly blocked by strychnine. They were mostly monophasic, but biphasic responses occurred in approximately 30 % of P8-11 neurones in perforated-patch recordings. 3. In whole-cell recordings from P2-11 neurones, the reversal potential of glycine-evoked currents (EGly) was determined by the transmembranous Cl- gradient and corresponded closely to the Nernst potential for Cl-, regardless of age. This indicates that Cl- is the principle ion permeating glycine receptors, but is also consistent with a low relative (10-20 %) permeability for HCO3-. The Cl- gradient also determined the polarity and amplitude of glycine-evoked membrane potential changes. 4. Leaving the native intracellular [Cl-] undisturbed with gramicidin perforated-patch recordings, we found a highly significant, age-dependent change of EGly from -46.8 +/- 1.8 mV (P1-4, n = 28) to -67.6 +/- 3.3 mV (P5-8, n = 10) to -82.2 +/- 4.1 mV (P9-11, n = 18). The majority of P1-4 neurones were depolarized by glycine ( approximately 80 %) and spikes were evoked in approximately 30 %. In contrast, P9-11 neurones were hyperpolarized. 5. In perforated-patch recordings, EGly was influenced by the voltage protocol and the glycine application interval; it could be shifted in the positive and negative direction. For a given application interval, these shifts were always larger in P1-4 than in P8-11 neurones, pointing to less effective Cl- regulation mechanisms in younger neurones. 6. Furosemide (frusemide), a blocker of cation-Cl- cotransporters, reversibly shifted EGly in the negative direction in P2-4 neurones, yet in the positive direction in P8-10 neurones, suggesting the blockade of net inward and net outward Cl- transporters, respectively. 7. Taken together, age-dependent changes in active Cl- regulation are likely to cause the developmental shift from depolarizing to hyperpolarizing glycine responses. A high intracellular [Cl-] is generated in neonatal LSO neurones which decreases during maturation.

Neurotrophin-3 potentiates excitatory GABAergic synaptic transmission in cultured developing hypothalamic neurones of the rat.

1. Neurotrophin-3 (NT-3) supports the survival and differentiation of neurones in the central and peripheral nervous systems through a number of mechanisms that occur in a matter of hours or days. NT-3 may also have a more rapid mode of action that influences synaptic activity in mature neurones. In the present study, the effect of NT-3 on developing GABAergic synapses was investigated in 3- to 7-day-old cultures of rat hypothalamic neurones with whole-cell patch-clamp recording. 2. NT-3 induced a substantial dose-dependent potentiation of the frequency of spontaneous postsynaptic currents (sPSCs; 160 %) in developing neurones during a period when GABA evoked inward (depolarizing) current, as determined with gramicidin-perforated patch recordings. The NT-3 effect was long lasting; continued enhancement was found > 30 min after NT-3 wash-out. NT-3 evoked a substantial 202 % increase in total GABA-mediated inward current, measured as the time-current integral. Action potential frequency was also increased by NT-3 (to 220 %). 3. The frequency of GABA-mediated miniature postsynaptic currents in developing neurones in the presence of tetrodotoxin was potentiated (to 140%) by NT-3 with no change in the mean amplitude, suggesting a presynaptic locus of the effect. 4. In striking contrast to immature neurones, when more mature neurones were studied, NT-3 did not enhance the frequency of GABA-mediated spontaneous postsynaptic currents (sPSCs), but instead evoked a slight (16%) decrease. The frequency of miniature post-synaptic currents was also slightly decreased (16%) by the NT-3, with no change in amplitude. These results were recorded during a later period of neuronal maturity when GABA would evoke outward (hyperpolarizing) currents. NT-3 had no effect on the mean amplitude of GABA-evoked postsynaptic currents in either developing or mature neurones. 5. Intracellular application of K252a, a non-selective tyrosine kinase inhibitor, did not block the NT-3 effect postsynaptically. In contrast, bath application of K252a prevented the enhancement of sPSCs by NT-3, consistent with NT-3 acting through presynaptic induction of tyrosine kinase. Decreasing extracellular calcium with BAPTA or inhibiting calcium channels with Cd2+ blocked the augmentation of sPSC frequency by NT-3, suggesting that an increase of calcium entry may be required for the facilitation of NT-3. 6. Together, our results suggest NT-3 enhances GABA release during the developmental period when GABA is depolarizing and calcium elevating, but not later when GABA is inhibitory, suggesting that one mechanism through which NT-3 may influence neuronal development is via presynaptic potentiation of GABA excitation.

Regulation of Intracellular Chloride by Cotransporters in Developing Lateral Superior Olive Neurons

The regulatory mechanisms of intracellular Cl- concentration ([Cl-]i) were investigated in the lateral superior olive (LSO) neurons of various developmental stages by taking advantage of gramicidin perforated patch recording mode, which enables neuronal [Cl-]i measurement. Responses to glycine changed from depolarization to hyperpolarization during the second week after birth, resulting from [Cl-]i decrease. Furosemide equally altered the [Cl-]i of both immature and mature LSO neurons, indicating substantial contributions of furosemide-sensitive intracellular Cl- regulators; i.e., K+-Cl- cotransporter (KCC) and Na+-K+-Cl- cotransporter (NKCC), throughout this early development. Increase of extracellular K+ concentration and replacement of intracellular K+ with Cs+ resulted in [Cl-]i elevation at postnatal days 13-15 (P13-P15), but not at P0-P2, indicating that the mechanism of neuronal Cl- extrusion is sensitive to both furosemide and K+-gradient and poorly developed in immature LSO neurons. In addition, removal of extracellular Na+ decreased [Cl-]i at P0-P2, suggesting the existence of extracellular Na+-dependent and furosemide-sensitive Cl- accumulation in immature LSO neurons. These data show clearly that developmental changes of Cl- cotransporters alter [Cl-]i and are responsible for the switch from the neonatal Cl- efflux to the mature Cl- influx in LSO neurons. Such maturational changes in Cl- cotransporters might have the important functional roles for glycinergic and GABAergic synaptic transmission and the broader implications for LSO and auditory development.