Unitary conductance changes at teleost Mauthner cell glycinergic synapses: a voltage-clamp and pharmacologic analysis

1. The magnitude and kinetics of inhibitory postsynaptic currents (IPSCs) evoked in the goldfish Mauthner (M-) cell by intracellular stimulation of identified presynaptic interneurons (unitary responses) and by activation of the recurrent collateral network were determined with single-and double electrode voltage-clamp techniques. 2. The peak magnitude of the inhibitory conductance changes were 5610 +/- 4800 nS (mean +/- SD; n = 13) for the collateral response, and 144 +/- 44 nS (n = 7) for the unitary IPSCs. These synaptic conductances, which are due to the opening of Cl- channels, were independent of the degree of Cl- -loading of the M-cell. 3. The peak amplitude of the collateral inhibitory postsynaptic potential (IPSP) was a constant fraction (0.52 +/- 0.06) of the driving force, which was determined from current-voltage plots for both types of IPSCs and ranged from 10 to 37 mV. These findings confirm indirect measurements from previous current-clamp studies and validate the normalization procedure used to previously calculate synaptic conductances from IPSP amplitudes, a method that therefore may be applicable to other central neurons. 4. At the resting membrane potential, the rise time of the unitary IPSCs was 0.34 +/- 0.07 ms (n = 18), whereas their decay was exponential, with a time constant of 5.7 +/- 1.1 ms (n = 16). 5. Iontophoretic and intramuscular applications of the glycine antagonist strychnine reduced or blocked M-cell inhibitory responses, without altering the excitability of the presynaptic neurons, or the driving force. 6. Amplitude fluctuations of unitary IPSPs recorded during partial blockade by strychnine were analyzed according to a binomial model of quantal transmitter release. In one experimental series, comparison of the binomial parameters before and after applying the antagonist indicated that only quantal size, q, was reduced, whereas n, the number of available release units, and p, the probability of release, were unaffected by strychnine. In a second series, the individual presynaptic cells were injected with horseradish peroxidase (HRP), and it was found that the correlation between n and the number of stained presynaptic boutons and, therefore, of active zones, was maintained in the presence of the drug. No evidence was found for silent synapses in these conditions. 7. The quantal conductance, gq, was estimated from the binomially derived quantal size, in millivolts, and the voltage-clamp measurements of the IPSP driving force and M-cell input conductance. gq averaged 21.5 nS in control conditions and 12.3 nS in the presence of strychnine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory interactions between spiny projection neurons in the rat striatum

The spiny projection neurons are by far the most numerous type of striatal neuron. In addition to being the principal projection neurons of the striatum, the spiny projection neurons also have an extensive network of local axon collaterals by which they make synaptic connections with other striatal projection neurons. However, up to now there has been no direct physiological evidence for functional inhibitory interactions between spiny projection neurons. Here we present new evidence that striatal projection neurons are interconnected by functional inhibitory synapses. To examine the physiological properties of unitary inhibitory postsynaptic potentials (IPSPs), dual intracellular recordings were made from pairs of spiny projection neurons in brain slices of adult rat striatum. Synaptic interactions were found in 9 of 45 pairs of neurons using averages of 200 traces that were triggered by a single presynaptic action potential. In all cases, synaptic interactions were unidirectional, and no bidirectional interactions were detected. Unitary IPSPs evoked by a single presynaptic action potential had a peak amplitude ranging from 157 to 319 microV in different connections (mean: 277 +/- 46 microV, n = 9). The percentage of failures of single action potentials to evoke a unitary IPSP was estimated and ranged from 9 to 63% (mean: 38 +/- 14%, n = 9). Unitary IPSPs were reversibly blocked by bicuculline (n = 4) and had a reversal potential of -62.4 +/- 0.7 mV (n = 5), consistent with GABA-mediated inhibition. The findings of the present study correlate very well with anatomical evidence for local synaptic connectivity between spiny projection neurons and suggest that lateral inhibition plays a significant role in the information processing operations of the striatum.     

Intracellular correlates of spatial memory acquisition in hippocampal slices: long-term disinhibition of CA1 pyramidal cells

Despite many advances in our understanding of synaptic models of memory such as long-term potentiation and depression, cellular mechanisms that correlate with and may underlie behavioral learning and memory have not yet been conclusively determined. We used multiple intracellular recordings to study learning-specific modifications of intrinsic membrane and synaptic responses of the CA1 pyramidal cells (PCs) in slices of the rat dorsal hippocampus prepared at different stages of the Morris water maze (WM) task acquisition. Schaffer collateral stimulation evoked complex postsynaptic potentials (PSP) consisting of the excitatory and inhibitory postsynaptic potentials (EPSP and IPSP, respectively). After rats had learned the WM task, our major learning-specific findings included reduction of the mean peak amplitude of the IPSPs, delays in the mean peak latencies of the EPSPs and IPSPs, and correlation of the depolarizing-shifted IPSP reversal potentials and reduced IPSP-evoked membrane conductance. In addition, detailed isochronal analyses revealed that amplitudes of both early and late IPSP phases were reduced in a subset of the CA1 PCs after WM training was completed. These reduced IPSPs were significantly correlated with decreased IPSP conductance and with depolarizing-shifted IPSP reversal potentials. Input-output relations and initial rising slopes of the EPSP phase did not indicate learning-related facilitation as compared with the swim and na?ve controls. Another subset of WM-trained CA1 PCs had enhanced amplitudes of action potentials but no learning-specific synaptic changes. There were no WM training-specific modifications of other intrinsic membrane properties. These data suggest that long-term disinhibition in a subset of CA1 PCs may facilitate cell discharges that represent and record the spatial location of a hidden platform in a Morris WM.     

2-Deoxyglucose-induced long-term potentiation of monosynaptic IPSPs in CA1 hippocampal neurons

In previous experiments on excitatory synaptic transmission in CA1, temporary (10-20 min) replacement of glucose with 10 mM 2-deoxyglucose (2-DG) consistently caused a marked and very sustained potentiation (2-DG LTP). To find out whether 2-DG has a similar effect on inhibitory synapses, we recorded pharmacologically isolated mononosynaptic inhibitory postsynaptic potentials (IPSPs; under current clamp) and inhibitory postsynaptic currents (IPSCs; under voltage clamp); 2-DG was applied both in the presence and the absence of antagonists of N-methyl-D-aspartate (NMDA). In spite of sharply varied results (some neurons showing large potentiation, lasting for >1 h, and many little or none), overall there was a significant and similar potentiation of IPSP conductance, both for the early (at approximately 30 ms) and later (at approximately 140 ms) components of IPSPs or IPSCs: by 35.1 +/- 10.25% (mean +/- SE; for n = 24, P = 0.0023) and 36.5 +/- 16.3% (for n = 19, P = 0.038), respectively. The similar potentiation of the early and late IPSP points to a presynaptic mechanism of LTP. Overall, the LTP was statistically significant only when 2-DG was applied in the absence of glutamate antagonists. Tetanic stimulations (in presence or absence of glutamate antagonists) only depressed IPSPs (by half). In conclusion, although smaller and more variable, 2-DG-induced LTP of inhibitory synapses appears to be broadly similar to the 2-DG-induced LTP of excitatory postsynaptic potentials previously observed in CA1.     

Mechanism for increased hippocampal synaptic strength following differential experience

Exposure to novel environments or behavioral training is associated with increased strength at hippocampal synapses. The present study employed quantal analysis techniques to examine the mechanism supporting changes in synaptic transmission that occur following differential behavioral experience. Measures of CA1 synaptic strength were obtained from hippocampal slices of rats exposed to novel environments or maintained in individual cages. The input/output (I/O) curve of extracellularly recorded population excitatory postsynaptic potentials (EPSPs) increased for animals exposed to enrichment. The amplitude of the synaptic response of the field potential was related to the fiber potential amplitude and the paired-pulse ratio, however, these measures were not altered by differential experience. Estimates of biophysical parameters of transmission were determined for intracellularly recorded unitary responses of CA1 pyramidal cells. Enrichment was associated with an increase in the mean unitary synaptic response, an increase in quantal size, and a trend for decreased input resistance and reduction in the stimulation threshold to elicit a unitary response. Paired-pulse facilitation, the percent of response failures, coefficient of variance, and estimates of quantal content were not altered by experience but correlated well with the mean unitary response amplitude. The results suggest that baseline synaptic strength is determined, to a large extent, by presynaptic release mechanisms. However, increased synaptic transmission following environmental enrichment is likely due to an increase in the number or efficacy of receptors at some synapses and the emergence of functional synaptic contacts between previously unconnected CA3 and CA1 cells.     

Inter-bouton variability of synaptic strength correlates with heterogeneity of presynaptic Ca(2+) signals

The elevation of presynaptic calcium concentration is a crucial step in excitation-secretion coupling. However, the amplitudes of action-potential-induced presynaptic calcium transients can display high variability among different terminals. The aim of this study was to clarify whether, at individual boutons, synaptic strength correlates with the average amplitude of presynaptic calcium transients. Low-density collicular cultures were loaded with the calcium indicator Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1. Action potentials were blocked with tetrodotoxin. Presynaptic terminals were identified with FM4-64, a use-dependent vesicle marker. Presynaptic calcium influx was elicited by a focal electrical stimulation of single boutons. Whole cell patch-clamp and calcium imaging techniques were used to record GABAergic evoked inhibitory postsynaptic currents (eIPSCs) and presynaptic fluorescence changes in the stimulated terminal. To make the eIPSCs from different boutons comparable, they were normalized to the mean value of miniature IPSCs (mIPSCs) of the postsynaptic cell. Records from 47 boutons showed that eIPSCs varied between 0.5 and 3.0 and presynaptic calcium transients varied between 0.1 and 1.3. However, there was a strong correlation between the mean amplitudes of eIPSCs and presynaptic calcium responses. The eIPSC-[Ca(2+)](pre) relationship allows to use the amplitudes of presynaptic calcium transients as an indicator of release efficacy and, in a set of contacts made by one axon, to predict the relative impact of individual terminals.     

Use-dependent depression of IPSPs in rat hippocampal pyramidal cells in vitro

We have used intracellular recording techniques to study the use-dependence of evoked inhibitory postsynaptic potentials (IPSPs) in rat CA1 hippocampal pyramidal cells. We determined reversal potentials and conductance changes associated with IPSPs and responses to directly applied gamma-aminobutyric acid (GABA). The IPSP depression could be seen after a single conditioning stimulus. This depression appeared to be due primarily to a 50% decrease in IPSP conductance (gIPSP). Trains of stimulating pulses (50 pulses at 5 or 10 Hz) produced more pronounced effects than a single conditioning pulse. Suprathreshold repetitive stimulation of stratum radiatum (SR) produced epileptiform burst firing and greater depression of IPSPs than did alvear (ALV) or subthreshold SR stimulation. During suprathreshold SR stimulation the IPSP was nearly abolished and the membrane potential could become less negative than the resting potential. A masking effect of facilitated depolarizing potentials on IPSPs was unlikely since IPSPs accompanied by little or no depolarizing potential were also depressed by SR trains. The 75% reduction in IPSP conductance found after repetitive stimulation confirmed that an overlapping conductance was not responsible for the depression of the IPSP. The GABA-induced conductance increase was not depressed by identical trains. Trains of stimulation induced depolarizing shifts in equilibrium potentials for the IPSP (EIPSP) and GABA (EGABA) of approximately 10 mV. These shifts were always greater after SR trains than after ALV trains. Simultaneous recordings of membrane potential and extracellular potassium concentration ([K+]o) with K+-sensitive microelectrodes revealed a direct correlation between the two during a stimulus train. Membrane potential depolarized as much as 18 mV from the peak of the IPSP and [K+]o could increase to a maximum of 10 mM during some trains. A depressant effect (of approximately 50%) of K+ on IPSPs was demonstrated by brief pressure ejection of K+ near the soma. We conclude that repetitive stimulation depresses gIPSP and shifts EIPSP in the depolarizing direction. Whereas gIPSP began to decline after a single conditioning pulse, the additional depression of IPSPs produced by stimulus trains was due in large part to shifts in EIPSP. Depression of gIPSP was not due to desensitization or block of ionic conductances, since gGABA was not reduced. The EIPSP may change as a result of increases in [K+]o.     

Synaptic inhibition of cat phrenic motoneurons by internal intercostal nerve stimulation.

Intracellular recordings from 65 phrenic motoneurons (PMNs) in the C5 segment and recordings of C5 phrenic nerve activity were made in 27 pentobarbitone-anesthetized, paralyzed, and artificially ventilated adult cats. Inhibition of phrenic nerve activity and PMN membrane potential hyperpolarization (48/55 PMNs tested) was seen after stimulation of the internal intercostal nerve (IIN) at a mean latency to onset of 10.3 +/- 2.7 ms. Reversal of IIN-evoked hyperpolarization (n = 14) by injection of negative current or diffusion of chloride ions occurred in six cases, and the hyperpolarization was reduced in seven others. Stimulation of the IIN thus activates chloride-dependent inhibitory synaptic inputs to most PMNs. The inhibitory phrenic nerve response to IIN stimulation was reduced by ipsilateral transection of the lateral white matter at the C3 level and was converted to an excitatory response by complete ipsilateral cord hemisection at the same level. After complete ipsilateral hemisection of the spinal cord at C3 level, stimulation of the IIN evoked both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) in PMNs (n = 10). It was concluded that IIN stimulation can evoke both excitatory and inhibitory responses in PMNs using purely spinal circuitry, but that excitatory responses are normally suppressed by a descending pathway in intact animals. Fifteen PMNs were tested for possible presynaptic convergence of inputs in these reflex pathways, using test and conditioning stimuli. Significant enhancement (>20%) of IPSPs were seen in seven of eight IIN-evoked responses using pericruciate sensorimotor cortex (SMC) conditioning stimuli, but only one of five IIN-evoked responses were enhanced by superior laryngeal nerve (SLN) conditioning stimuli. The IIN-evoked IPSP was enhanced in one of two motoneurons by stimulation of the contralateral phrenic nerve. It was concluded that presynaptic interneurons were shared by the IIN and SMC pathways, but uncommonly by other pathways. These results indicate that PMNs receive inhibitory synaptic inputs from ascending thoracocervical pathways and from spinal interneurons. These inhibitory reflex pathways activated by afferent inputs from the chest wall may play a significant role in the control of PMN discharge, in parallel with disfacilitation following reduced activity in bulbospinal neurons projecting to PMNs.     

Conductance changes underlying a late synaptic hyperpolarization in hippocampal CA3 neurons

1. Single-electrode current- and voltage-clamp techniques were employed to study properties of the conductance underlying an orthodromically evoked late synaptic hyperpolarization or late inhibitory postsynaptic potential (IPSP) in CA3 pyramidal neurons in the rat hippocampal slice preparation. 2. Late IPSPs could occur without preceding excitatory postsynaptic potentials at the resting membrane potential and were graded according to the strength of the orthodromic stimulus. The membrane hyperpolarization associated with the late IPSP peaked within 140-200 ms after orthodromic stimulation of mossy fiber afferents. The late IPSP returned to base line with a half-decay time of approximately 200 ms. 3. As determined from constant-amplitude hyperpolarizing-current pulses, the membrane conductance increase during the late IPSP, and the time course of its decay, were similar whether measurements were made near the resting membrane potential or when the cell was hyperpolarized by approximately 35 mV. 4. When 1 mM cesium was added to the extracellular medium to reduce inward rectification, late IPSPs could be examined over a range of membrane potentials from -60 to -140 mV. For any given neuron, the late IPSP amplitude-membrane potential relationship was linear over the same range of membrane potentials for which the slope input resistance was constant. The late IPSP reversed symmetrically near -95 mV. 5. Intracellular injection of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid or extracellular application of forskolin, procedures known to reduce or block certain calcium-dependent potassium conductances in CA3 neurons, had no significant effect on the late IPSP. 6. Single-electrode voltage-clamp techniques were used to analyze the time course and voltage sensitivity of the current underlying the late IPSP. This current [the late inhibitory postsynaptic current (IPSC)] began as early as 25 ms after orthodromic stimulation and reached a peak 120-150 ms following stimulation. 7. The late IPSC decayed with a single exponential time course (tau = 185 ms). 8. A clear reversal of the late IPSC at approximately -99 mV was observed in a physiological concentration of extracellular potassium (3.5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of Ia-inhibitory synaptic input to cat spinal motoneurons evoked by vibration of antagonist muscles.

1. Steady-state inhibitory postsynaptic potentials (IPSPs) were evoked in tibialis anterior and extensor digitorum longus motoneurons of the cat by using tendon vibration to activate Ia-afferent fibers from the antagonist medial gastrocnemius muscle. 2. The effective synaptic currents (IN) underlying the steady-state IPSPs were measured by the use of a modified voltage-clamp technique. The amplitudes of the effective synaptic currents (1.62 +/- 0.66 nA, mean +/- SD; n = 20) extended over a fivefold range (0.5-2.7 nA) but were not correlated with the intrinsic properties of the motoneurons or with putative motor unit type. 3. We calculated the synaptic conductance (GS) underlying the steady-state Ia IPSPs from measurements of motoneuron input conductance during the activation of the Ia synaptic input. As was expected from Ohm's law, the Ia-inhibitory GS and IN were correlated (r = 0.49; P less than 0.05). Like IN, GS (175 +/- 202 nS, mean +/- SD; n = 20) was not correlated with the intrinsic properties of the motoneurons. 4. As has been reported previously for transient Ia IPSPs, the amplitudes of the steady-state IPSPs were correlated with motoneuron input resistance (r = 0.74; P less than 0.001) and homonymous Ia excitatory postsynaptic synaptic potential (EPSP) amplitude (r = 0.72; P less than 0.001). 5. The amplitudes of the steady-state Ia IPSPs and the homonymous Ia EPSPs were plotted on logarithmic axes. The slope (0.59) was significantly less than 1, which indicates that the gradient of Ia inhibition across the motoneuron pool is less steep than that of Ia excitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy and short-term plasticity at GABAergic synapses between Purkinje and cerebellar nuclei neurons

Although the entire output of the cerebellar cortex is conveyed to the deep cerebellar nuclei neurons (DCNs) via the GABAergic synapses established by Purkinje cells (PCs), very little is known about the strength and dynamic properties of PC-DCN connections. Here we show that activation of PC-DCN unitary connections induced large conductance changes (11.7 nS) in DCNs recorded in whole cell patch configuration in acute slices, suggesting that activity of single PCs might significantly affect the output of its target neurons. Based on the large unitary quantal content (18) inferred from calculations of PC-DCN quantal size (0.65 nS) and the near absence of failures in synaptic transmission during control conditions, we conclude that PC-DCN connections are highly multi-sited. The analysis of dynamic properties of PC-DCN synapses demonstrated remarkable paired pulse depression (PPD), maximal at short intervals (paired pulse ratio of 0.15 at 7-ms interval). We provide evidence that PPD is presynaptic in origin and release-independent. In addition, multiple pulse stimulation revealed that PC-DCN synapses exhibited larger sensitivity to dynamic than to steady signals. We postulate that the, otherwise paradoxical, combination of marked short-term depression with strong multi-sited connections is optimal to transfer dynamic information at unitary level by performing spatial average of release probability across the numerous release sites. This feature could enable these synapses to encode presynaptic time-varying signals of single PCs as moment-to-moment changes in synaptic strength, a capacity well suited to the postulated role of cerebellum in control of temporal aspects of motor or cognitive behaviors.     

Monosynaptic inhibition of pallidal neurons by axon collaterals of caudato-nigral fibers

Summary The effects produced in the entopeduncular nucleus (ENT) by stimulation of the substantia nigra (SN) were studied in cats anesthetized with pentobarbital. SN stimulation evoked positive field potentials localized in ENT and long latency IPSPs in ENT neurons. The IPSP seemed to be produced monosynaptically since 1. it was evoked by single shocks to SN and had a constant shape and latency, 2. there was no temporal facilitation of the second IPSP when two shocks were applied to SN at various intervals, and 3. an IPSP of similar shape, but shorter latency, was evoked from the diencephalic region between SN and ENT. Linear regression analysis of the latency vs. conduction distance for these two IPSPs, mediated by the same fibers, indicated a synaptic delay of less than 0.7 msec and a conduction velocity for these fibers of about 1 m/sec.Stimulation of the caudate nucleus evoked IPSPs with shapes similar to those evoked from SN; and there was a strong interaction between SN and caudate-evoked IPSPs, suggesting that they were mediated by the same fibers. Acute experiments on cats in which the caudate nucleus had been destroyed 3 to 7 weeks earlier showed that SN stimulation did not evoke positive field potentials in ENT or IPSPs in ENT cells. It was concluded that SN stimulation antidromically activated caudato-nigral fibers that monosynaptically inhibited ENT cells via axon collaterals.     

Cortically evoked pre-and postsynaptic inhibition of impulse transmission to the dorsal spinocerebellar tract

Summary Synaptic actions evoked from primary afferents and the sensorimotor cortex in neurones of the dorsal spinocerebellar tract were investigated: 1. Stimulation of the anterior lobe of the cerebellum produced a small IPSP in only one but not in the other six neurones examined. 2. IPSPs were induced not only from group I fibres (in 41% of group I neurones) but also from cutaneous and/or high threshold muscle afferents (in 37%). 3. Stimulation of the contralateral sensorimotor cortex evoked IPSPs in 80% of group I neurones. The IPSP had a latency of 10–15 msec and lasted for 40–100 msec. EPSPs were evoked from the cortex in a small number of neurones. 4. Effects from the cortex were compared with those from primary afferents in individual neurones. The cortical IPSPs were induced independently of whether the neurone received monosynaptic EPSP from extensor or flexor group I fibres. The cortical IPSPs (or EPSPs) occurred more frequently in neurones which exhibited polysynaptic IPSPs (or EPSPs) from primary afferents. 5. The few FRA neurones encountered were all excited from the cortex.Excitability measurements of primary afferent terminals in or near Clarke's column showed that a terminal depolarization is evoked from the cortex in group Ib but not in Ia afferents.The relative importance of post-and presynaptic inhibition of transmission to the DSCT is discussed.     

Spatial synaptic distribution of recurrent and group Ia inhibitory systems in cat spinal motoneurones

1. The reversal potentials of several types of inhibitory post-synaptic potentials (IPSPs) have been studied in cat spinal motoneurones with and without modification of intracellular chloride ion (Cl(-)) concentration. Single barrel intracellular micropipette electrodes have been used.2. When studied with potassium citrate filled micropipettes, the reversal potential for IPSPs evoked by stimulation of antagonist group Ia afferents is the same as that for recurrent IPSPs evoked by antidromic stimulation of motoneurone axon collaterals, confirming earlier observations (Araki, Ito & Oscarsson, 1961; Coombs, Eccles & Fatt, 1955).3. Studied with potassium chloride filled micropipettes. the reversal potential for the group Ia IPSP was found to be different from that for the recurrent IPSP when the amount of Cl(-) diffusing or iontophoretically injected into the motoneurone was small. The amount of difference in reversal potential varied from cell to cell but when present the group Ia IPSP reversed to a depolarizing potential more readily than the recurrent IPSP in all cases.4. Interaction between recurrent IPSPs and monosynaptic excitatory post-synaptic potentials (EPSPs) was also studied and the amount of non-linearity of potential summation was measured.5. The results are consistent with the hypothesis that the terminations of Renshaw cells responsible for the recurrent IPSP are located largely on the proximal dendrites of motoneurones, while the terminations of the interneurones generating the group Ia IPSP appear to be closer to or on the cell somata.     

Synaptic potentials evoked in spiny neurons in rat neostriatal grafts by cortical and thalamic stimulation.

1. Fetal rat striatal primordia were implanted into the neostriatum of adult rats 2 days after kainic acid lesion. Two to 6 mo after transplantation, in vivo intracellular recording and staining were performed to study the responses of spiny neurons in the grafts to the cortical and thalamic stimuli. The physiological characteristics and synaptic responses of 27 cells recorded in the grafts were compared with a sample of 23 neurons recorded from the surrounding host neostriatum in the same animals. Nineteen of the graft neurons and 19 of the host neurons were identified as spiny neurons by intracellular staining with biocytin. The responses of the remaining neurons were the same as those of identified spiny cells. 2. The spontaneous synaptically driven membrane potential shifts and long-lasting responses to afferent stimulation that are characteristic of neostriatal cells in normal animals were greatly reduced or absent in graft neurons. Presumably this reflects the reduction in synaptic input to the grafts and the lack of convergence of inputs from diverse sources. 3. Short-latency synaptic responses to cortical and thalamic stimulation were present and could consist of either excitatory postsynaptic potentials (EPSPs) or inhibitory postsynaptic potentials (IPSPs). The IPSPs were accompanied by a membrane conductance increase, and their reversal potentials could be altered by injection of chloride ions. Several minutes after impaling the cell, the IPSPs gradually disappeared, and the same stimuli could then evoke EPSPs. The disappearance of the IPSPs was independent of the presence of chloride in the electrodes. Most of the EPSP responses appeared to be monosynaptic but occurred at longer latencies than those seen in host neurons of the same type. 4. In cells not exhibiting IPSPs, or after the IPSP responses disappeared, cortical or thalamic stimulation could evoke slow depolarizing potentials and bursts of action potentials. These could not be evoked by current injection. They could be prevented or delayed by an exaggerated action potential after hyperpolarization that developed in neurons maintained in a depolarized state for several seconds, but could not be prevented by passage of hyperpolarizing current from the recording electrode. 5. The input resistance of graft spiny neurons was higher than that of the host cells, and time constants were longer. Both of these properties appeared to be due to the absence of the strong inward rectification that is usually present at resting membrane potentials in neostriatal neurons.     

Regulation of the timing and pattern of action potential generation in rat subthalamic neurons in vitro by GABA-A IPSPs

The regulation of activity in the subthalamic nucleus (STN) by GABAergic inhibition from the reciprocally connected globus pallidus (GP) plays an important role in normal movement and disorders of movement. To determine the precise manner in which GABAergic synaptic input, acting at A-type receptors, influences the firing of STN neurons, we recorded the response of STN neurons to GABA-A inhibitory postsynaptic potentials (IPSPs) that were evoked by supramaximal electrical stimulation of the internal capsule using the perforated-patch technique in slices at 37 degrees C. The mean equilibrium potential of the GABA-A IPSP (EGABA-A IPSP) was -79.4 +/- 7.0 mV. Single IPSPs disrupted the spontaneous oscillation that underlies rhythmic single-spike firing in STN neurons. As the magnitude of IPSPs increased, the effectiveness of prolonging the interspike interval was related more strongly to the phase of the oscillation at which the IPSP was evoked. Thus the largest IPSPs tended to reset the oscillatory cycle, whereas the smallest IPSPs tended to produce relatively phase-independent delays in firing. Multiple IPSPs were evoked at various frequencies and over different periods and their impact was studied on STN neurons held at different levels of polarization. Multiple IPSPs reduced and/or prevented action potential generation and/or produced sufficient hyperpolarization to activate a rebound depolarization, which generated a single spike or restored rhythmic spiking and/or generated a burst of activity. The pattern of IPSPs and the level of polarization of STN neurons were critical in determining the nature of the response. The duration of bursts varied from 20 ms to several hundred milliseconds, depending on the intrinsic rebound properties of the postsynaptic neuron. These data demonstrate that inhibitory input from the GP can produce a range of firing patterns in STN neurons, depending on the number and frequencies of IPSPs and the membrane properties and voltage of the postsynaptic neuron.     

Pre- and postsynaptic actions of baclofen: blockade of the late synaptically-evoked hyperpolarization of CA1 hippocampal neurones

Summary Using intracellular recording techniques, the effects of -p-chlorophenyl-GABA (baclofen) on passive membrane properties and postsynaptic potentials of CA1 pyramidal neurones were investigated. In experiments where only the hyperpolarizing action of baclofen was precluded by conventional current clamp techniques, 20 M (±) baclofen blocked the early GABA-mediated IPSP and also a late hyperpolarization which, since it could be evoked by orthodromic stimulation subthreshold for spike firing, would not be expected to be produced by a Ca²⁺-activated increase in potassium conductance (AHP), but to be a transmitter-mediated event. In addition the conductance increase associated with this late IPSP evoked by subthreshold stimulation and also that associated with the AHP produced by spike activation were abolished. Baclofen also appeared to increase the duration of EPSPs, an event possibly related to loss of IPSPs. The hyperpolarization produced by baclofen was associated with an increased conductance of the resting membrane, an event possibly associated with an elevated potassium flux. To preclude this postsynaptic effect as a cause of reduced synaptic responses, tetraethylammonium chloride (TEA), a compound which decreases conductance and depolarizes the membrane of CA1 pyramidal neurones by a reduction of a leak or resting potassium conductance (gK), was added to the bathing medium. A comparison of the effect of TEA on the hyperpolarizations with that of baclofen was undertaken since TEA also interferes with the increased gK evoked by Ca²⁺ inflow during spike activation. Whereas TEA reduced only an early phase of the postspike hyperpolarization possibly related to the AHP, baclofen abolished the remaining late IPSP. While loss of the AHP or IPSPs individually did not provoke additional spike activity, the abolition of both components promoted extra action potentials in response to synaptic excitation. Baclofen also increased the reduced conductance evoked by TEA towards control levels and caused membrane hyperpolarization. Thus baclofen is considered to evoke its postsynaptic effects through an increased membrane potassium conductance which TEA may also affect to reduce membrane conductance. The resultant uncontrolled hyperpolarization (even in the presence of TEA) occurring in inhibitory interneurones might contribute to the disinhibition recorded in this study.     

Estimation of single channel conductance underlying synaptic transmission between pyramidal cells in the visual cortex

Axon collaterals originating from pyramidal cells are one of the most abundant presynaptic elements in the neocortical circuits. To understand a quantitative aspect of synaptic transmission between pyramidal cells, we attempted to estimate single channel conductance by applying non-stationary noise analysis to unitary excitatory postsynaptic currents. Simultaneous recordings were carried out in two pyramidal cells of superficial layers in visual cortical slices. Unitary postsynaptic currents, which were evoked by action potentials of presynaptic cells impaled with conventional sharp electrodes, were recorded from postsynaptic cells with whole-cell patch clamp techniques. Estimated single channel conductance was 12.8 3.8(S.D.) pS for kittens and 10.4 +/- 1.5 pS for rats. Dividing these values by the conductance for unitary postsynaptic currents, we calculated the number of non-N-methyl-D-aspartate receptor channels activated during the postsynaptic currents. The obtained estimates were 52 (kittens) and 41 (rats). To further estimate the number of channels involved in each quantal event, we analysed amplitude histograms of miniature and spike-evoked excitatory postsynaptic currents. The derived number of estimates from these two kinds of histograms agreed quite well; about 20 channels were required for individual quantal events. Assuming open probability of non-N-methyl-D-aspartate receptor channels to be 0.7, our results suggest that the number of channels available for synaptic transmission between individual pyramidal cells would be 74 (kittens) and 59 (rats). We propose that at pyramidal-pyramidal synapses, the number of open channels is several times smaller than that previously reported for the synapses between geniculo-cortical afferent and layer IV spiny stellate cells.     

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 gamma-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 [Ca2+]o/[Mg2+]o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 microns. Smaller neurons were chosen for recording to render a standard deviation of the "instrumental" noise of less than 1.5 pA 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 -12 mV) 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 34.4 ms, 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.     

GABA(A)-dependent chloride influx modulates reversal potential of GABA(B)-mediated IPSPs in hippocampal pyramidal cells

Changes in intracellular chloride concentration, mediated by chloride influx through GABA(A) receptor-gated channels, may modulate GABA(B) receptor-mediated inhibitory postsynaptic potentials (GABA(B) IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABA(A) receptor-mediated IPSPs (GABA(A) IPSPs) on the properties of GABA(B) IPSPs. At relatively positive membrane potentials (near -55 mV), mossy fiber--evoked GABA(B) IPSPs were reduced (compared with their magnitude at -60 mV) when preceded by GABA(A) receptor--mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABA(B) IPSP. The mossy fiber--evoked GABA(B) IPSP showed a positive shift in reversal potential (from -99 to -93 mV) when it was preceded by a GABA(A) IPSP evoked at cell membrane potential of -55 mV as compared with -60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABA(B) IPSP and a concurrent shift of GABA(B) IPSP reversal potential from -98 to -90 mV. We conclude that in hippocampal pyramidal cells, in which "resting" membrane potential is near action potential threshold, chloride influx via GABA(A) IPSPs shifts the reversal potential of subsequent GABA(B) receptor--mediated postsynaptic responses in a positive direction and reduces their magnitude.