Mean Decay Time Constant Research Articles

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering.

Lateral olivocochlear (LOC) neurons of the mouse LSO receive excitatory and inhibitory synaptic inputs with slower kinetics than LSO principal neurons

We examined membrane properties and synaptic responses of neurons in the mouse lateral superior olivary nucleus (LSO). Two clear populations were identified consistent with: principal neurons which are involved in detecting interaural intensity differences (IIDs) and efferent neurons of the lateral olivocochlear (LOC) system which project to the cochlea. Principal neurons fired a short latency action potential (AP) often followed by an AP train during maintained depolarization. They possessed sustained outward K + currents, with little or no transient K + current (I A) and a prominent hyperpolarization-activated non-specific cation conductance, I H. On depolarization, LOC neurons exhibited a characteristic delay to the first AP. These neurons possessed a prominent transient outward current I A, but had no I H. Both LOC and principal neurons received glutamatergic and glycinergic synaptic inputs. LOC synaptic responses decayed more slowly than those of principal neurons; the mean decay time constant of AMPA receptor-mediated EPSCs was around 1 ms in principal neurons and 4 ms in LOC neurons. Decay time constants for glycinergic IPSCs were around 5 ms in principal neurons and 10 ms in LOC neurons. We conclude that principal cells receive fast synaptic responses appropriate for integration of IID inputs, while the LOC cells possess excitatory and inhibitory receptors with much slower kinetics.

Glycinergic input of widefield, displaced amacrine cells of the mouse retina

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRalpha1 (Glra1(spd-ot), 'oscillator' mouse), the GlyRalpha2 (Glra2(-/-)) and the GlyRalpha3 subunit (Glra3(-/-)). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, tau approximately 25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1(spd-ot) and Glra3(-/-) mice (tau approximately 25 ms); however, glycinergic currents were absent in group II cells of Glra2(-/-) mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (tau approximately 50-70 ms). We propose that GlyRs of group II cells are dominated by the alpha2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the alpha4 subunit.

Small-Conductance Calcium-Activated Potassium Channels Control Excitability and Firing Dynamics in Gonadotropin-Releasing Hormone (GnRH) Neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.

Platelet-derived growth factor (PDGF)-BB inhibits AMPA receptor-mediated synaptic transmission via PDGF receptor-β in murine nucleus tractus solitarius

Although platelet-derived growth factor (PDGF)-BB activates PDGF receptor-β (PDGFR-β) and, in turn, inhibits the glutamate N-methyl- d-aspartate (NMDA) receptor function, whether PDGF-BB modulates the CNS function mediated by another glutamate receptors, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, remains poorly understood. Here we now report the inhibitory effect of PDGF-BB on the AMPA receptor function in the nucleus tractus solitarius (NTS) by using slice patch-clamp techniques. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the tractus solitarius in mouse NTS second-order neurons. EPSCs were nearly completely eliminated by CNQX but not by MK-801, implying mediation through non-NMDA receptors. PDGF-BB significantly decreased the amplitude of EPSCs without affecting the mean decay time constant. This inhibitory effect was transient and reversible after removing PDGF-BB. Furthermore, PDGF-BB significantly reduced the amplitude of AMPA-induced currents in NTS neurons, which showed that PDGF-BB could suppress the AMPA receptor-mediated excitatory input via the postsynaptic mechanism. The inhibitory effect of PDGF-BB on EPSCs was not observed in mutant mice with conditional deletion of the PDGFR-β gene in neurons. Together, these studies suggest that the PDGF-B/PDGFR-β axis inhibits the AMPA receptor-mediated synaptic transmission that comprises the major part of the primary afferent to the NTS second-order neuron. The detected inhibitory action may be involved in the CNS regulation of the respiratory response.

CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Rapid Signaling at Inhibitory Synapses in a Dentate Gyrus Interneuron Network

Mutual synaptic interactions between GABAergic interneurons are thought to be of critical importance for the generation of network oscillations and for temporal encoding of information in the hippocampus. However, the functional properties of synaptic transmission between hippocampal interneurons are largely unknown. We have made paired recordings from basket cells (BCs) in the dentate gyrus of rat hippocampal slices, followed by correlated light and electron microscopical analysis. Unitary GABA(A) receptor-mediated IPSCs at BC-BC synapses recorded at the soma showed a fast rise and decay, with a mean decay time constant of 2.5 +/- 0.2 msec (32 degrees C). Synaptic transmission at BC-BC synapses showed paired-pulse depression (PPD) (32 +/- 5% for 10 msec interpulse intervals) and multiple-pulse depression during repetitive stimulation. Detailed passive cable model simulations based on somatodendritic morphology and localization of synaptic contacts further indicated that the conductance change at the postsynaptic site was even faster, decaying with a mean time constant of 1.8 +/- 0.6 msec. Sequential triple recordings revealed that the decay time course of IPSCs at BC-BC synapses was approximately twofold faster than that at BC-granule cell synapses, whereas the extent of PPD was comparable. To examine the consequences of the fast postsynaptic conductance change for the generation of oscillatory activity, we developed a computational model of an interneuron network. The model showed robust oscillations at frequencies >60 Hz if the excitatory drive was sufficiently large. Thus the fast conductance change at interneuron-interneuron synapses may promote the generation of high-frequency oscillations observed in the dentate gyrus in vivo.

γ-Aminobutyrate, α-carboxy-2-nitrobenzyl ester selectively blocks inhibitory synaptic transmission in rat dentate gyrus

γ-Aminobutyrate, α-carboxy-2-nitrobenzyl ester (cGABA) is a stable photoactivatable probe used to study γ-aminobutyrate (GABA) receptors. GABA is released from this compound when it is exposed to ultraviolet light, but little is known about the electrophysiological effects of the compound itself. Whole cell patch clamp recordings on rat hippocampal slices demonstrated that cGABA blocked polysynaptic inhibitory postsynaptic currents (IPSCs) evoked in dentate granule cells by antidromic stimulation of the mossy fibers. It also reduced monosynaptically evoked IPSCs with an IC 50 of 28 μM. In contrast, cGABA had no effect on excitatory postsynaptic currents (EPSCs) evoked by perforant path stimulation. The effect of cGABA was not mediated by depression of GABA release through activation of presynaptic GABA B receptors. cGABA inhibited muscimol-evoked currents by only 15% at a concentration of 40 μM. At this same concentration, it reduced the mean frequency of miniature inhibitory postsynaptic potentials by 71%, their mean peak amplitude by 44%, their mean decay time constant by 26% and the mean charge transfer per event by 52%. These effects may be explained by a phenothiazine-like modification of GABA A receptor kinetics and/or a selective block of somatic GABA synapses.

Restrictions on inhibitory circuits contribute to limited recruitment of fast inhibition in rat neocortical pyramidal cells.

To further define the operational boundaries on fast inhibition in neocortex, whole cell recordings were made from layer V pyramidal neurons in neocortical slices to evaluate evoked inhibitory postsynaptic currents (IPSCs) and spontaneous miniature IPSCs (mIPSCs). Stimulating electrodes were placed in layers VI and I/II to determine whether simultaneous stimulation of deep and superficial laminae could extend the magnitude of maximal IPSCs evoked by deep-layer stimulation alone. The addition of superficial-layer stimulation did not increase maximal IPSC amplitude, confirming the strict limit on fast inhibition. Spontaneous miniature IPSCs were recorded in the presence of tetrodotoxin. The frequency of spontaneous mIPSCs ranged from 10.0 to 33.1 Hz. mIPSC amplitude varied considerably, with a range of 5. 0-128.2 pA and a mean value of 20.7+/-4.1 pA (n = 12 cells). The decay phase of miniature IPSCs was best fit by a single exponential, similar to evoked IPSCs. The mean time constant of decay was 6.4+/-0.6 ms, with a range of 0.2-20.1 ms. The mean 10-90% rise time was 1.9+/-0.2 ms, ranging from 0.2 to 6.3 ms. Evaluation of mIPSC kinetics revealed no evidence of dendritic filtering. Amplitude histograms of mIPSCs exhibited skewed distributions with several discernable peaks that, when fit with Gaussian curves, appeared to be spaced equidistantly, suggesting that mIPSC amplitudes varied quantally. The mean separation of Gaussian peaks ranged from 6.1 to 7.8 pA. The quantal distributions did not appear to be artifacts of noise. Exposure to saline containing low Ca(2+) and high Mg(2+) concentrations reduced the number of histogram peaks, but did not affect the quantal size. Mean mIPSC amplitude and quantal size varied with cell holding potential in a near-linear manner. Statistical evaluation of amplitude histograms verified the multimodality of mIPSC amplitude distributions and corroborated the equidistant spacing of peaks. Comparison of mIPSC values with published data from single GABA channel recordings suggests that the mean mIPSC conductance corresponds to the activation of 10-20 GABA(A) receptor channels, and that the release of a single inhibitory quantum opens 3-6 channels. Further comparison of mIPSCs with evoked inhibitory events suggests that a single interneuron may form, on average, 4-12 functional synapses with a pyramidal cell, and that 10-12 individual interneurons are engaged during recruitment of maximal population IPSCs. This suggests that inhibitory circuits are much more restricted in both the size of the unit events and effective number of connections when compared with excitatory inputs.

Development of glycinergic synaptic transmission to rat brain stem motoneurons.

Using an in vitro rat brain stem slice preparation, we examined the postnatal changes in glycinergic inhibitory postsynaptic currents (IPSCs) and passive membrane properties that underlie a developmental change in inhibitory postsynaptic potentials (IPSPs) recorded in hypoglossal motoneurons (HMs). Motoneurons were placed in three age groups: neonate (P0-3), intermediate (P5-8), and juvenile (P10-18). During the first two postnatal weeks, the decay time course of both unitary evoked IPSCs [mean decay time constant, taudecay = 17.0 +/- 1.6 (SE) ms in neonates and 5.5 +/- 0.4 ms in juveniles] and spontaneous miniature IPSCs (taudecay = 14.2 +/- 2.4 ms in neonates and 6.3 +/- 0.7 ms in juveniles) became faster. As glycine uptake does not influence IPSC time course at any postnatal age, this change most likely results from a developmental alteration in glycine receptor (GlyR) subunit composition. We found that expression of fetal (alpha2) GlyR subunit mRNA decreased, whereas expression of adult (alpha1) GlyR subunit mRNA increased postnatally. Single GlyR-channels recorded in outside-out patches excised from neonate motoneurons had longer mean burst durations than those from juveniles (18.3 vs. 11.1 ms). Concurrently, HM input resistance (RN) and membrane time constant (taum) decreased (RN from 153 +/- 12 MOmega to 63 +/- 7 MOmega and taum from 21.5 +/- 2.7 ms to 9.1 +/- 1.0 ms, neonates and juveniles, respectively), and the time course of unitary evoked IPSPs also became faster (taudecay = 22.4 +/- 1.8 and 7.7 +/- 0.9 ms, neonates vs. juveniles, respectively). Simulated synaptic currents were used to probe more closely the interaction between IPSC time course and taum, and these simulations demonstrated that IPSP duration was reduced as a consequence of postnatal changes in both the kinetics of the underlying GlyR channel and the membrane properties that transform the IPSC into a postsynaptic potential. Additionally, gramicidin perforated-patch recordings of glycine-evoked currents reveal a postnatal change in reversal potential, which is shifted from -37 to -73 mV during this same period. Glycinergic PSPs are therefore depolarizing and prolonged in neonate HMs and become faster and hyperpolarizing during the first two postnatal weeks.

Pharmacological evidence for two types of postsynaptic glycinergic receptors on the Mauthner cell of 52-h-old zebrafish larvae.

The presence of homooligomeric and heterooligomeric glycine receptors (GlyRs) on the Mauthner (M) cell in the isolated medulla of 52-h-old zebrafish larvae was investigated by analysis of the effects of picrotoxin on glycine-gated channels and on glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Two functionally different GlyRs have been previously described on the M cell. The effects of picrotoxin on these two GlyRs were first analyzed by measuring the relative change in their total open probability (NP(o)) with picrotoxin concentration. Picrotoxin had no significant effect on the glycine channel with a single conductance level of 40-46 pS. In contrast, picrotoxin application decreased the NP(o) of the GlyR with multiple subconductance levels. On this GlyR, picrotoxin decreased in a concentration-dependent manner the occurrence of the 80- to 86-pS substate (median inhibiting concentration = 0.89 microM) and had no apparent effect on the 40- to 46-pS opening probability. Opening frequency and the mean open times of the 80- to 88-pS main conductance state were reduced in the presence of 10 microM picrotoxin, but their relative weight remained unchanged. These effects of picrotoxin were not voltage dependent. Picrotoxin also modified 40- to 46-pS kinetics. At 100 microM, picrotoxin evoked voltage-independent flickering during channel openings. Short and long mean open times were significantly decreased, whereas the relative proportion of long mean open times was increased. The medium closed time was decreased, whereas medium burst duration was increased. The burst frequency remained unchanged. Spontaneous glycinergic mIPSCs were recorded in the presence of 1 microM tetrodotoxin + 25 microM bicuculline (holding potential = -50 mV). Application of 10 microM picrotoxin did not change the frequency of the synaptic activity, whereas it decreased the amplitude of large mIPSCs. No effect was observed on the time to peak (0.8 ms) or the mean decay time constant (tau(d) = 7.7 ms). Increasing picrotoxin concentration to 100 microM resulted in a decrease of mIPSC frequency (35.6%), amplitude (39.8%), and tau(d) (from 7.7 to 5 ms). These results suggest that these two functionally different GlyRs correspond to alpha1 homooligomeric-like and alpha1/beta-heterooligomeric-like GlyRs, and that both are synaptically activated. Variation in the proportions of GlyR subtypes from one synapse to another could partly account for the broad amplitude distribution of mIPSCs recorded from the zebrafish M cell.

Modulation of GABAA receptor-mediated IPSCs by neuroactive steroids in a rat hypothalamo-hypophyseal coculture model.

1. We have used the whole-cell configuration of the patch-clamp technique to investigate the effects of neuroactive steroids on GABAA receptor-mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre- and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor-mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half-maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) reversibly potentiated IGABA in a dose-dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/- 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta-isomer of allopregnanolone, epipregnanolone (5 beta-pregnan-3 beta-ol-20-one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5-pregnen-3 alpha-ol-20-one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of -80 and -60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre- and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

Somatic voltage-gated potassium currents of rat hippocampal pyramidal cells in organotypic slice cultures.

1. The dominant voltage-gated K+ currents in the somatic membrane of CA3 pyramidal cells from hippocampal slice cultures were characterized using the cell-attached configuration of the patch-clamp recording method. The kinetics, the voltage dependence of activation and inactivation, and the pharmacological properties of the current were determined from ensemble averages of large numbers of episodes from multichannel patches. 2. Steady-state analysis revealed that this current was half-inactivated at the resting membrane potential (Vr), and fully inactivated when patches were held 40 mV positive to Vr. Inactivation was removed when patches were hyperpolarized by 50 mV from Vr. Inactivation was well described by the Boltzmann equation with a slope factor of 12.6 mV. Removal of inactivation of the peak outward current could be described by a time-dependent monoexponential function with a time constant of the order of 100 ms. In contrast, the time course of inactivation was very slow: a +40 mV depolarization relative to Vr of several seconds was required for complete inactivation of the total outward current. 3. When steady-state inactivation was removed by hyperpolarization, the outward current activated with a threshold 10 mV positive to Vr and was half-activated at a potential 57 mV positive to Vr. The conductance can be described in terms of a single Boltzmann equation with a slope factor of 13.5 mV. Activation and inactivation properties of the somatic conductance produce a small window current between +10 and +20 mV relative to Vr. 4. The outward current activated in a voltage-dependent manner in less than 10 ms with 500 ms depolarizing steps. A kinetic analysis of its decay revealed at least three components, with the following time constants: a fast (17 ms), a slowly (approximately 150 ms), and a very slowly inactivating component (in the range of seconds). 5. External application of 4-aminopyridine (4-AP) induced a dose-dependent block of the peak outward current with an IC50 of 28 microM. The inhibitory effect of 4-AP saturated at a concentration of 200 microM which blocked 80% of the total current. The slowly and very slowly inactivating components of the current were not observed with 20 mM tetraethylammonium (TEA) in the pipette solution. A fast transient ensemble current (mean decay time constant, 24 ms) persisted in the presence of extracellular TEA in 29% of the patches. 6. In summary, at least two distinct voltage-gated K+ currents were present at the somatic level of hippocampal pyramidal cells. The dominant one, which we named IK(AT), is sensitive to micromolar concentrations of 4-AP and millimolar concentrations of TEA, and contributes three kinetic components to the total outward current. The second is TEA insensitive, and contributes only a fast transient component of outward current probably corresponding to the classic A-type K+ current. Intracellular recordings in CA3 pyramidal cells showed that IK(AT) plays an important role in regulating the duration of the action potential.

Dopamine D1-like receptor-mediated presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones.

1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3.81 +/- 0.09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM). 2. Bath-applied dopamine (DA) reduced evoked EPSC amplitude by up to 54.2 +/- 2.3% with an IC50 of 19.9 microM in normal Krebs solution (2.5 mM Ca2+, 1.2 mM Mg2+) without effect on postsynaptic holding current. 3. DA (30 microM) reduced the mean frequency of spontaneous miniature EPSCs recorded in 0.5 microM tetrodotoxin without affecting their mean amplitude, rise time or decay time constant. This effect was diminished by 100 microM Cd2+. 4. The effect of DA on evoked EPSCs was mimicked by the D1-like receptor agonist, SKF 81297 (IC50 25.6 microM), but not by the D2-like receptor agonist R(-)-TNPA (30 microM) or (-)-quinpirole (30 microM), and was antagonized by the D1-like receptor antagonist R(+)-SCH 23390 (estimated dissociation constant KB = 1.7 microM) but not by the D2-like receptor antagonist S(-)-eticlopride (10 microM). 5. Forskolin (10 microM) reduced evoked EPSCs to approximately 60% of the control amplitude, and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D1-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

Inhibitory synaptic transmission in isolated patches of membrane from cultured rat spinal cord and medullary neurons.

1. To quantify the variability in the characteristics of inhibitory glycinergic and GABAergic currents at single synaptic connections between cultured rat embryonic spinal cord or medullary neurons, we have used patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in cell-attached patches. Experiments were performed with the patch pipette containing either a low-calcium internal saline to allow comparison with subsequent whole cell recordings or external saline with tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione, a solution that is more appropriate for bathing a nerve terminal. 2. The mIPSCs recorded from the synapses restricted to the cell-attached patches were characterized by their times to peak, amplitudes, and time constants of decay. The degree of variability in these characteristics was quantified with the use of the following model-independent parameters: the coefficient of variation, skewness, and kurtosis. The distribution of time to peak values has a mean value of 5.6 +/- 0.5 (SE) ms, has the lowest coefficient of variation (0.33 +/- 0.01), is fairly symmetrical, and has a Gaussian shape with respect to peakedness. On the other hand, both the amplitude and decay time constant distributions are highly skewed and more peaked than Gaussian distributions. The mean amplitude is -6.6 +/- 0.6 pA with a coefficient of variation of 0.60 +/- 0.05, whereas the mean decay time constant is 22.8 +/- 1.0 ms with a coefficient of variation of 0.81 +/- 0.03. 3. The amplitude distributions for spontaneous inhibitory currents recorded from cell-attached patches are best fitted by the sum of multiple Gaussians. The coefficient of variation for the first Gaussian peak fitted to the amplitude distributions is 0.290 +/- 0.028. 4. Decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.7 +/- 0.2 ms (n = 12), D2 = 11.2 +/- 0.7 ms (n = 11), D3 = 20.6 +/- 0.8 ms (n = 12), and D4 = 43.8 +/- 2.3 ms (n = 16). These mean values are essentially identical to those reported in the preceding paper for mIPSCs recorded in the whole cell configuration. 5. In eight neurons we were able to record mIPSCs both in cell-attached patches and in subsequent whole cell configurations. The properties of mIPSCs recorded from single synapses (i.e., times to peak, amplitude, and time constants of decay) show as much variability as those of mIPSCs recorded subsequently in the whole cell mode; that is, there are no statistically significant differences in the coefficients of variation, skewness, or kurtosis for the three different distributions.

Posttetanic potentiation and presynaptically induced long-term potentiation at the mossy fiber synapse in rat hippocampus.

A form of long-term potentiation (LTP) is induced at the mossy fiber (MF) synapse in the hippocampus by high-frequency presynaptic stimulation (HFS). It is generally accepted that induction of this form of LTP (MF LTP) does not depend on postsynaptic Ca2+ current gated by N-methyl-D-aspartate receptors, but it has remained controversial whether induction depends on postsynaptic depolarization and voltage-gated entry of Ca2+. There are also contradictory data on the time course of both LTP and post-tetanic potentiation (PTP), a shorter duration form of potentiation observed at MF synapses immediately following HFS. It has been proposed that some of these differences in results may have arisen because of difficulties in isolating monosynaptic responses to MF input. In the present study, whole cell recording was used to observe excitatory postsynaptic currents (EPSCs) elicited in CA3 pyramidal cells by input from MFs. Postsynaptic cells were dialyzed with 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and F- to inhibit postsynaptic mechanisms that required Ca2+, cells were under voltage clamp during HFS, and conditions were selected to minimize the likelihood of polysynaptic contamination. Under these conditions, HFS nevertheless induced robust LTP (mean magnitude, 62%). The possibility that EPSCs were contaminated by polysynaptic components was investigated by exposing the slices to a suppressing medium (one that partially blocked neurotransmission). EPSC waveforms did not change shape during suppression, indicating that contamination was absent. The LTP observed always was accompanied by prominent PTP that lasted through the first 5 to 15 min following HFS (mean decay time constant, 3.2 min). Induction of this LTP was not cooperative; there was no relationship between the size of responses and the magnitude of the LTP induced. LTP magnitude also was unrelated to the extent to which postsynaptic cells depolarized during HFS. These results show that high rates of presynaptic MF activity elicit robust LTP whether or not there is accompanying postsynaptic depolarization or increase in the concentration of postsynaptic Ca2+. High-frequency MF activity also results in a PTP that is unusually large and long.

Effects of the GABA uptake inhibitor tiagabine on inhibitory synaptic potentials in rat hippocampal slice cultures.

1. The effects of the gamma-aminobutyric acid (GABA) uptake blocker tiagabine on inhibitory synaptic potentials (IPSPs) were examined with microelectrode and whole-cell recording from CA3 pyramidal cells in rat hippocampal slice cultures. 2. Tiagabine (10-25 microM) greatly prolonged the duration of monosynaptic IPSPs elicited in the presence of excitatory amino acid antagonists but had no effect on their amplitude. Part of the prolonged time course resulted from a GABAB receptor-mediated component that was not detectable under control conditions. 3. The mean decay time constant of the underlying GABAA receptor-mediated synaptic current was increased from 16 to 250 ms. Spontaneous miniature IPSPs recorded with whole-cell clamp were unaffected by tiagabine. Pentobarbital sodium, in contrast, increased the decay time constant of both evoked and spontaneous GABAA-mediated currents. 4. Tiagabine (25 microM) inhibited spontaneous and evoked epileptiform bursting induced by increasing the extracellular potassium concentration to 8 mM. 5. We conclude that GABA uptake plays a significant role in determining the time course of evoked IPSPs and also limits the likelihood that GABAB receptors are activated.

Pulsatile release of acetylcholine by nerve terminals (synaptosomes) isolated from Torpedo electric organ.

1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Unitary, quantal and miniature gaba-activated synaptic chloride currents in cultured neurons from the rat superior colliculus

The aim of this study was to identify the conductance change induced by one quantum of γ-aminobutyric acid from axonal release sites on cultured superior colliculus neurons. Unitary (single cell-activated) inhibitory postsynaptic currents and spontaneous synaptic activity were recorded with patch clamp techniques in the whole cell configuration while superfusing the entire neuron with normal saline. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and in reduced[CA 2+] o/[Mg 2+] o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 μm. Smaller neurons were chosen for recording to render a standard deviation of the “instrumental” noise of less than 1.5pA at a holding voltage of −80 mV. After two weeks in vitro, the percentage of synaptically connected tectal neurons exceeded 50%. At holding voltages of −80 mV (Cl − equilibrium potential −12mV) minimal amplitudes of unitary inhibitory postsynaptic currents were as low as 7–10 pA, while maximal amplitudes exceeded 500 pA. The mean time to peak and time constant of decay were 3.0 and 34.4ms, respectively ( n = 31). Fluctuating unitary inhibitory postsynaptic currents were deemed to be compound postsynaptic responses. Multiple Gaussian equations could be fitted to the amplitude histograms of unitary postsynaptic currents. This procedure rendered a quantal size between 5.0 and 10.9 pA (mean 7.1 pA; S.D. 1.78 pA) in five neurons from mature cultures. The amplitudes of statistically determined quantal inhibitory postsynaptic currents were slightly smaller than the independent estimate from somatic miniature inhibitory postsynaptic currents. The latter had a mean amplitude of 9.1 pA (S.D. 3.3 pA, n = 23), a mean time to peak of 1.65 ms ( n = 9), and a mean time constant of decay of 16.2 ms ( n = 9). Single channel recording from outside-out patches showed three to four main conductance states ranging from 9 to 22 pS. Single channel closures at the 21–24 pS level were occasionally observed during relaxation of miniature currents. The small size of whole cell quantal inhibitory postsynaptic currents and somatic miniature currents indicates that one GABA quantum opened only 5–15 single Cl − channels.

Characterization of membrane currents in dissociated adult rat pineal cells.

1. Membrane currents, particularly the outward components, were studied in pineal cells acutely dissociated from adult rats using the whole-cell variant of the patch-clamp technique. 2. In current clamp, outward constant current elicited a transient graded depolarizing response. A sustained membrane rectification developed within 20 ms; this phenomenon was reduced in cells internally dialysed with 120 mM-CsCl. 3. Study of the membrane current revealed the existence of a transient and a delayed outward current. These currents were virtually eliminated when the cell was internally dialysed with CsCl. 4. The delayed outward current, isolated from a holding potential of -50 mV, activated at potentials near -20 mV, reached a steady-state current amplitude within 60 ms and had little or no decay during steps up to 400 ms in duration. This component was reduced by 80% or more with the addition of 5 mM-TEA. 5. From -100 mV, the transient outward current reached a peak within 15 ms and decayed with a single-exponential time course. The mean decay time constant was 66 +/- 10 ms (at -33 mV) and it showed little voltage sensitivity. This current, which activated at potentials positive to -60 mV and displayed half-inactivation at -76 +/- 8 mV, was reduced by 50% with the addition of 5 mM-4-AP (4-amino-pyridine). 6. In the presence of external Ca2+, the current-voltage relationship for the delayed current did not display a region of negative-slope conductance (N-shape). Increasing the intracellular ionized Ca2+ concentration by varying the Ca-EGTA buffer ratio did not alter the dependence of the current on the membrane potential. 7. Block of outward currents with internal Cs+ revealed a small (less than 90 pA) inward Ca2+ current when the external Ca2+ concentration was increased to 10 mM. From a holding potential of -50 mV, it had a threshold at -30 mV and peaked at +5 mV. Evidence for an inward Na+ current was not obtained. 8. We conclude that acutely dissociated pineal cells display two distinct K+ currents: (i) a slowly activating, sustained current similar to the delayed rectifier (IK); and (ii) a transient A-current (IA). At normal Ca2+ concentrations, no macroscopic Ca2+-activated outward current was observed.