Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl^–-mediated hyperpolarizations in respiratory neurons of the brainstem–spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of γ-aminobutyric acid (GABA), i.e. GABA_A, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO₂/HCO₃^–-free solutions, but shifted from about ?65 mV to values more positive than ?20 mV upon dialysis of the cells with 144 instead of 4 mm Cl^–. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABA_B receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K⁺ channel-mediated hyperpolarization (reversal potential ?88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABA_A and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABA_B receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.     

In vitro studies of the role of gamma-aminobutyric acid in inhibition in the lateral septum of the rat

Focal stimulation, stimulation of the fimbria, and stimulation of the medial septal area result in an inhibitory postsynaptic potential (IPSP) in lateral septal neurons. Increased stimulus intensity results in the appearance of a late hyperpolarizing potential (LHP). Treatment of the slice with bicuculline methiodide or picrotoxin results in blockade of the IPSP. When present, LHPs are enhanced in the presence of bicuculline or picrotoxin. Spontaneous and evoked IPSPs reverse near -70 mV, and LHPs reverse near -90 mV. Iontophoretic application of gamma-amino-butyric acid (GABA) results in hyperpolarizing, depolarizing, or biphasic potentials. Treatment with bicuculline or picrotoxin results in depression of biphasic GABA responses that appears selective for the depolarizing portion of the potential. At high concentrations of bicuculline, a portion of the hyperpolarizing GABA potential persists. The reversal potential of the depolarizing GABA potential is near -30 mV, and the reversal potential of monophasic hyperpolarizing GABA potential is near -70 mV. The bicuculline-resistant hyperpolarizing GABA response has a reversal potential near -90 mV. GABA activates three separate conductances on septal neurons, which are similar to those reported on hippocampal neurons. The resistance of the hyperpolarizing GABA potential to bicuculline appears to be due to the presence of a GABA-activated potassium conductance, which is similar to that activated by baclofen.     

Electrophysiological diversity of pyramidal-shaped neurons at the granule cell layer/hilus border of the rat dentate gyrus recorded in vitro

In the rat dentate gyrus, pyramidal-shaped cells located on the border of the granule cell layer and the hilus are one of the most common types of γ-aminobutyric acid (GABA)-immunoreactive neurons. This study describes their electrophysiological characteristics. Membrane properties, patterns of discharge, and synaptic responses were recorded intracellularly from these cells in hippocampal slices. Each cell was identified as pyramidal-shaped by injecting the marker Neurobiotin intracellularly (n =17). In several respects the membrane properties of the sampled cells were similar to “fast-spiking” cells (putative inhibitory interneurons) that have been described in other areas of the hippocampus. For example, input resistance was high (mean 91.3 megohms), the membrane time constant was short (mean 7.7 ms), and there was a large afterhyperpolarization following a single action potential (mean 10.5 mV at resting potential). However, the action potentials of most pyramidal-shaped cells were not as brief (mean 1.2 ms total duration) as those of most previously described fast-spiking cells. Many pyramidal-shaped neurons had strong spike frequency adaptation relative to other fast-spiking cells. Although these latter two characteristics were apparent in the majority of the sampled cells, there were exceptional pyramidal-shaped neurons with fast action potentials and weak adaptation, demonstrating the electrophysiological variability of pyramidal-shaped cells. Responses to outer molecular layer stimulation were composed primarily of excitatory postsynaptic potentials (EPSPs) rather than inhibitory postsynaptic potentials (IPSPs), and were usually small (EPSPs evoked at threshold were often less than 2 mV), and brief (less than 30 ms). There was variability, because in a few cells EPSPs evoked at threshold were much larger. However, regardless of EPSP amplitude, suprathreshold stimulation (up to 4 times the threshold stimulus strength) rarely evoked more than one action potential in any cell. The results suggest that stimulation of perforant path axons produces limited excitatory synaptic responses in pyramidal-shaped neurons. This may be one of the reasons why they are relatively resistant to prolonged perforant path stimulation. The pyramidal-shaped neurons located at the base of the granule cell layer have been associated historically with a basket plexus around granule cell somata, and have been called pyramidal “basket” cells. However, basket-like endings were rare and axon collaterals outside the granule cell layer were common. Many axon collaterals were as far from the granule cell layer as the outer molecular layer and the central hilus, and antidromic action potentials could be recorded in some cells in response to weak stimulation of these areas. Taken together with the electrophysiological variability, the results indicate that these cells are physiologically heterogeneous. © 1995 Wiley-Liss, Inc.     

Electrical properties and synaptic potentials of rabbit pancreatic neurons

Pancreatic ganglia receive innervation from a wide variety of extrinsic nerves and supply the predominant innervation to pancreatic acini, islets, and ducts. This study used intracellular recordings to investigate the electrical properties and synaptic potentials of rabbit pancreatic neurons. Neurons had a mean resting membrane potential of -54+/-0.4 mV and generated action potentials with a mean overshoot of 10+/-0.4 mV and a mean after-spike hyperpolarization (ASH) of 11+/-0.5 mV with duration of 210+/-19 ms. Action potentials exhibited a high threshold (-15+/-1 mV) for intracellular stimulation and a phasic firing pattern was observed in response to prolonged depolarizing currents. Stimulation of attached nerve bundles evoked multiple fast excitatory postsynaptic potentials (fEPSPs) which were abolished by hexamethonium in 75% of neurons, while a non-cholinergic fEPSP was observed in 25% of the neurons. Repetitive stimulation (3-30 Hz) evoked muscarinic slow EPSPs with a mean amplitude of 8+/-2 mV and duration of 5+/-1 s in a small subset (21%) of neurons. Exogenous muscarine evoked a mean slow depolarization of 10+/-1 mV amplitude in 22% of neurons tested. Following repetitive nerve stimulation non-cholinergic late, slow EPSPs with a mean amplitude of 4.3+/-0.4 mV were recorded in 32% of neurons. Nicotinic transmission was subject to inhibition mediated by presynaptic muscarinic receptors at low (0.5 Hz) stimulus frequencies in 80% of neurons. At higher frequencies (> or =1 Hz), either facilitation or depression of nicotinic transmission was observed depending on the ganglion studied. A population (9%) of neurons exhibited spontaneous, low-amplitude pacemaker-like potentials. Spontaneous fEPSPs and action potentials were also observed and these occasionally occurred in rhythmically timed bursts. Thus, distinct subpopulations of pancreatic neurons could be identified on the basis of both their intrinsic electrical properties and the receptors mediating and/or modulating synaptic transmission. These neurons function as critical sites of integration for synaptic input from extrinsic pancreatic nerves and thereby determine the postganglionic firing patterns presented to the pancreatic exocrine and endocrine secretory cells.     

Development of sensitivity to GABA and glycine in cultured cerebellar neurons

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine on developing cerebellar neurons cultured for 7-40 days were intracellularly investigated. All neurons tested dose-dependently responded to both GABA and glycine. In mature neurons (after 25 days in culture) these amino acids inhibited spontaneous spikes, decreased the membrane input resistance and induced either hyperpolarization or depolarization of membrane potential. The mean reversal potential was -47 mV for GABA and -43 mV for glycine. Immature neurons, 7-12 days in culture, which were not spontaneously firing, also behaved in a similar manner as the mature ones, though the membrane resistance was not so largely changed by GABA or glycine and the reversal potential was more positive (-39 mV for GABA, -37 mV for glycine). These reversal potentials were shifted toward 0 mV by lowering the external Cl- concentration in either mature or immature neurons. The effects of GABA and glycine on mature or immature neurons were more or less inhibited by all of picrotoxin, bicuculline and strychnine. The effective concentrations of these antagonists, however, were lower in general in immature neurons. In mature neurons, picrotoxin and bicuculline became more selective to GABA than glycine and strychnine became more selective to glycine than GABA. These results suggest that sensitivities to GABA and glycine differentiate into selective types in the course of maturing of cerebellar cultured neurons.     

Cerebellar macroneurons in microexplant cell culture. Postsynaptic amino acid pharmacology

Cerebellar neurons derived from 17- to 19-day-old fetal rats have been grown in a monolayer in microexplant cell culture, and intracellular recording coupled with iontophoresis of amino acid neurotransmitters has been employed to characterize their amino acid chemosensitivity. Although these cultures contain at least 3 different neuronal cell types, intracellular recordings were obtained from large neurons (diameter greater than 15 microns) with 1-5 dendritic shafts and fine dendritic arborizations and which could, on morphological grounds, be identified as Purkinje cells. All neurons with resting membrane potentials greater than 25 mV and with action potentials evoked by intracellular stimulation, responded to iontophoretically applied glutamate and GABA. There was essentially no chemosensitivity to glycine, beta-alanine or taurine. Aspartate application evoked only small responses at high iontophoretic currents. GABA reversibly increased membrane conductance and produced hyperpolarization at resting membrane potential with reversal potentials between -50 and -40 mV (5-10 mV more negative than resting membrane potential). Glutamate reversibly increased membrane conductance and produced depolarizing responses with extrapolated reversal potentials between 0 and -10 mV. Aspartate augmented glutamate responses at low iontophoretic currents which did not directly alter membrane potential or conductance. Thus Purkinje cells grown in the absence of parallel fiber and climbing fiber input develop autonomous neuropharmacologic specificity similar to that of Purkinje cells in vivo.     

Ethanol enhances synaptically evoked GABAA receptor-mediated responses in cerebral cortical neurons in rat brain slices.

Previous intracellular electrophysiological studies on rat hippocampal brain slices have shown very little effect of acute ethanol application on synaptically evoked GABAA receptor-mediated responses recorded in CA1 pyramidal neurons. The present study was designed to compare the effects of ethanol on pyramidal neurons in the hippocampus and cerebral cortex. Using conventional intracellular microelectrodes (60-80 M omega) to impale cortical neurons in brain slices, 80 mM ethanol application did not affect the membrane input impedance nor evoked EPSPs, but significantly affected the resting membrane potential (usually a 2-5 mV hyperpolarization). When stimulus-evoked GABAA-mediated IPSCs were studied using whole-cell recordings from cortical neurons voltage-clamped at depolarizing potentials, monophasic IPSCs were evoked that were blocked by bicuculline, increased by pentobarbital, and enhanced by ethanol superfusion in a dose dependent manner over the range of 20-160 mM. Hippocampal IPSCs recorded under identical conditions were not enhanced by ethanol. Parallel studies of GABA-stimulated 36Cl- flux measurements in microsacs prepared from hippocampal, cerebral cortical and cerebellar tissue demonstrated that ethanol significantly enhanced (30-50%) 36Cl- flux in microsacs derived from the cerebral cortex and cerebellum, but not in microsacs prepared from the hippocampus. These results demonstrate that there are clear brain region-dependent differences in the way that GABAA receptor function is altered by acute ethanol, and that these differences are apparent not only as an enhancement of responses to exogenous GABA, but also as a facilitation of the responses to endogenous GABA released from inhibitory nerve terminals during synaptic activation.     

An intracellular analysis of the entopeduncular inputs on the centrum medianum-parafascicular nuclear complex in cats

Intracellular recording from the CM-PF neurons was performed by stimulation of the EN, the caudate nucleus (Cd), the cerebellar nuclei (CN) and the motor cortex in the cat under Nembutal anesthesia. Twenty-seven neurons in the CM-PF nuclear complex and two neurons near the habenular nucleus received monosynaptic inhibitory postsynaptic potentials (IPSPs; latency of 1.0–4.0 ms, mean 2.3 ms) by EN stimulation. Cd stimulation evoked excitatory postsynaptic potentials (EPSPs) followed by long hyperpolarizations in most of the CM-PF neurons and produced antidromic activation in 7 neurons. Six neurons received EPSPs (latencies of 4–7 ms) by cortical stimulation. CN stimulation affected only two neurons in the present study. Intracellular HRP staining revealed that some CM-PF neurons have polygonal or spindle-shaped somata with fine, long and sparsely spinous dendrites.     

Participation of low-threshold calcium spikes in excitatory synaptic transmission in guinea pig medial frontal cortex

We studied the activation of low-threshold calcium spikes (LTS) by excitatory postsynaptic potentials in pyramidal neurons from guinea pig medial frontal cortex with intracellular recording. We used extracellular bicuculline and phaclofen and intracellular QX-314 to block inhibitory synaptic potentials and sodium currents. Postsynaptic potentials were evoked by stimulation of layer I. We found that large (> 10-15 mV) excitatory synaptic potentials evoked from membrane potentials more negative than -75 mV were able to trigger LTS. The activation of LTS resulted in an increase of the rising slope or amplitude of the synaptic potentials depending on the size of the excitatory postsynaptic potential (EPSP). We used 100 microM NiCl2 to confirm the presence of LTS as part of the EPSPs. The N-methyl-D-aspartate (NMDA) and non-NMDA components of the excitatory synaptic potentials were isolated using (+/-)2-amino-5-phosphonovaleric acid (APV; 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM); both components could, independently, trigger an LTS. With recordings made with K+ acetate-filled electrodes, we show that the activation of LTS was critical to allow excitatory synaptic potentials to reach the threshold of action potential firing; also, this amplification of synaptic responses produced the firing of more than a single action potential by the postsynaptic cell. These results demonstrate that in cortical pyramidal neurons the activation of low-threshold calcium spikes results in the amplification of synaptic responses.     

A sealed cranial window system for simultaneous recording of blood flow, and electrical and optical signals in the rat barrel cortex

The dorsal commissural nucleus (DCN) in the lumbosacral spinal cord (L6–S1) receives primary afferent fibers from both pelvic and pudendal nerves in rats. However, the physiological and pharmacological properties of synaptic responses of the DCN neurons to stimulation of those nerves remain unclear. We have developed a longitudinal spinal cord (L6–S1) slice preparation from mature rats that retained both nerves attached. Blind whole-cell recordings were made from the DCN neurons in this preparation. In most neurons, mono- and/or poly-synaptic fast excitatory postsynaptic potentials (EPSPs) were evoked by electrical stimulation of either the pelvic or pudendal nerve. These EPSPs were mediated by activation of Aβ/Aδ and/or C fibers (conduction velocities, 0.5–17.3 m/s), and were abolished by CNQX. Fast EPSPs elicited by either pelvic or pudendal nerve stimulation were occasionally accompanied by bicuculline- and strychnine-sensitive IPSPs. In one-third of the neurons tested, mono- and/or poly-synaptic EPSPs were elicited by the stimulation of both the pelvic and pudendal nerves, indicating convergence of the visceral and somatic primary afferent inputs from the pelvic region onto the DCN neurons. The preparation is applicable to study the mechanism of the integration of the visceral and somatic inputs in the spinal cord.     

Dependence of Retinogeniculate Transmission on Membrane Voltage in the Cat

The mammalian lateral geniculate nucleus seems organized to gate or control the gain of retinogeniculate transmission, the result of which is then relayed to the visual cortex. We have performed in vivo intracellular studies of retinogeniculate transmission along these retino-geniculo-cortical pathways in cats by recording the retinally evoked excitatory postsynaptic potential (EPSP) in geniculate neurons. In cats, these pathways are organized into two parallel and functionally distinct channels, the X and Y pathways. We found that nearly all geniculate X cells display a fairly conventional voltage dependency for their retinally evoked EPSPs, because the amplitudes of these EPSPs decrease fairly linearly with membrane depolarization as the EPSP reversal potential is approached. Rare X cells and all Y cells, however, show an unconventional response: over a wide range of membrane potentials, their EPSP amplitudes increase with membrane depolarization. This increase does not result from alterations in neuronal input resistance and instead seems due to changes in synaptic conductance. The underlying cause of this voltage dependency remains to be determined. None the less, it does afford an interesting means by which retinogeniculate transmission can be gated, since non-retinal inputs (e.g. corticogeniculate axons) that can control a relay Y cell's membrane potential can also modulate the cell's EPSP amplitude.     

Intrinsic subthreshold oscillations extend the influence of inhibitory synaptic inputs on cortical pyramidal neurons

Fast inhibitory synaptic inputs, which cause conductance changes that typically last for 10–100 ms, participate in the generation and maintenance of cortical rhythms. We show here that these fast events can have influences that outlast the duration of the synaptic potentials by interacting with subthreshold membrane potential oscillations. Inhibitory postsynaptic potentials (IPSPs) in cortical neurons in vitro shifted the oscillatory phase for several seconds. The phase shift caused by two IPSPs or two current pulses summed non‐linearly. Cholinergic neuromodulation increased the power of the oscillations and decreased the magnitude of the phase shifts. These results show that the intrinsic conductances of cortical pyramidal neurons can carry information about inhibitory inputs and can extend the integration window for synaptic input.     

Postnatal development of neuronal connections in cat visual cortex studied by intracellular recording in slice preparation

Postnatal development of neuronal connections in cat visual cortex (area 17) was studied in slice preparations obtained from kittens aged 1–18 weeks after birth and adult cats by recording intracellularly excitatory (EPSP) and inhibitory postsynaptic potentials (IPSP) evoked in cortical cells by stimulation of white matter. The EPSPs were already present in all cells at 1 week of age. Their efficiency assessed by their maximum rate of rise was low initially and increased progressively with age. In contrast, the IPSPs were absent in half of the cells at 1 week and almost all of the cells came to demonstrate inhibition by 9 weeks except for a few layer II-III cells. At all ages about three-quarters of the IPSPs had GABA_A-mediated early and GABA_B-mediated late components with different time course, reversal potential and sensitivity to GABA antagonists, while the remaining IPSPs had only the early component. The efficiency of both IPSPs assessed by the associated conductance increase showed an increase of more than twice from 1 to 5 weeks, reaching the same level as adults. The time course of the development of inhibition demonstrated in this study paralleled the time course of the development of selective visual responsiveness in cortical cells, suggesting that the postnatal maturation of inhibitory connections is a basis of maturation of visual responsiveness.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglia neurons

Slow EPSPs evoked in the neurons of the rabbit isolated superior cervical ganglion were studied using intracellular microelectrodes. Two types of EPSPs occurring in different neurons were found. The type I slow EPSPs showed an increase during hyperpolarization of the membrane and a decrease during its depolarization. Input resistance of the neurons during the response either decreased or remained unchanged. The type II slow EPSPs were increased by depolarization and decreased by hyperpolarization with the reversal potential -78.9 +/- 3.6 mV. Depolarization evoked by acetylcholine or carbocholine was followed by an increase in the input resistance in 53% of neurons with reversal potential -83.2 +/- 6.7 mV. It is concluded that in the first group of the neurons the nature of the slow EPSP is similar to that of ordinary EPSP. The main component underlying the ionic mechanism of slow EPSP in the other group of the neurons is a decrease in potassium conductance of the membrane.     

Recruitment of inhibition by enhanced activation of synaptic NMDA responses in the rat cerebral cortex

Intracellular recordings of layer V neurons from rat neocortical slices were obtained to examine the effects of reducing extracellular magnesium on inhibition. Magnesium-free solutions induced interictal and ictal-like events in cortical neurons. Changes in synaptic events underlying epileptogenesis were studied when extracellular calcium was raised (from 2 to 3–7 mM) since this delayed seizure activity. With increasing time of exposure of cells to magnesium-free solutions, there was a significant increase in the size and duration of both the depolarizing and slow synaptic hyperpolarizing responses, but the fast synaptic hyperpolarizationsignificantly declined in amplitude. When cells were recorded with cesium acetate-filled microelectrodes slow hyperpolarizing responses were blocked, but depolarization of cells to 0 mV allowed an isolated fast hyperpolarizing response to be recorded following synaptic stimulation. The amplitude of this response was unchanged after exposure to magnesium-free solutions. Synaptic responses of cells initially bathed in an (NMDA) antagonist (CPP) were unchanged by subsequent exposure to magnesium-free solutions. CPP exposure by itself caused in depolarization duration, increase in fast hyperpolarizing amplitude, and decrease in slow hyperpolarization amplitude and duration. When the fast hyperpolarization was viewed in isolation (cesium recording electrodes) at 0 mV, the amplitude of this event was unchanged by exposure to CPP. Given these results stimulus-response characteristics of neocortical neurons were reassessed under control conditions. With higher intensity stimuli larger depolarizing and slow hyperpolarizing responses were evoked, but the fast hyperpolarization showed a decremental response. These effects were reversed when CPP was added. When NMDA activity was enhanced by exposure to magnesium-free solutions or electrical stimulation, the amplitude of excitatory events and slow hyperpolarizations increased, but fast inhibitory responses showed limited capacity for incremental recruitment. This suggests fast inhibition is saturated (maximal) at submaximal levels of excitation, and can be overcome by increasing levels of excitation. Such a process is active under physiological conditions, altering the efficacy of inhibition.     

An intracellular recording study of stimulus-specific response properties in cat area 17

Stimulus-specific response properties, such as direction or orientation selectivity, were studied intracellularly in cells recorded from area 17 of the cat. In all 5 direction selective complex cells and one orientation selective simple cell successfully studied, visually evoked excitatory postsynaptic potentials (EPSPs) were tuned to the preferred direction or orientation. Visually evoked inhibitory postsynaptic potentials (IPSPs) were also tuned to the preferred direction/orientation of stimulus. IPSPs evoked by the non-preferred stimulus when present were smaller than those evoked by the preferred stimulus. IPSPs were undetected in two of the 5 cells tested. These results suggest that directionally/orientationally tuned EPSPs make a major contribution to stimulus specificity in visual cortical neurons but IPSPs evoked by a stimulus with null-direction/orientation may sharpen the stimulus specificity.     

Repetitive inhibitory postsynaptic potentials evoked by 4-aminopyridine in hippocampal neurons in vitro

The effects of topical application of microdrops containing 4-aminopyridine (4-AP) on properties of CA₁ neurons were examined in the hippocampal slice preparation. 4-AP triggered repetitive large (4–10 mV) hyperpolarizing potentials (HPs) having a short rise time and slow (3–4 s) decay. There was a marked decrease in input resistance during the HPs. The HPs are likely to be caused by an increase in potassium conductance; their reversal potential was 15–20 mV negative to rest, the reversal potential shifted in the depolarizing direction when the slice was bathed in high potassium medium, and it was the same with KCl or potassium acetate recording electrodes. The HPs were not generated by release of neurotransmitter substances from terminals of extrinsic afferents since they were present in slices taken from deafferented hippocampus but they were blocked by tetrodotoxin (TTX) or Cd and Mn, indicating that they are synaptic potentials of local origin. HPs were still present when Ca-dependent K currents were blocked by acetylcholine and noradrenaline. Three of 56 cells recorded in the hippocampus could be classified as interneurons. They emitted high frequency trains of action potentials in response to 4-AP, at a rate corresponding to the HPs recorded in all other neurons. It is suggested that 4-AP excites a specific type of interneuron which in turn generates large K-mediated inhibitory postsynaptic potentials in the pyramidal neurons of CA₁ region of the hippocampus.     

Effects of ethanol on CA1 and CA3 pyramidal cells in the hippocampal slice preparation: an intracellular study

Superfusion of ethanol (10-350 mM) sometimes caused weak hyperpolarization, but more often elicited weak depolarization or biphasic depolarizing, hyperpolarizing responses in CA1 and CA3 pyramidal neurons of the hippocampal slice. The occasional polarizations were sometimes accompanied by, but not always correlated with, small increases or decreases in input resistance. However, many cells in both areas showed no detectable change in membrane potential (36% of cells) or input resistance (57% of cells), even at very high ethanol concentrations (86-200 mM). Spontaneous spiking, when present, was occasionally accelerated or decelerated, although in CA3 a biphasic speeding-slowing sequence was often seen. The afterhyperpolarizations following bursts of action potentials evoked by current (CA1) or occurring spontaneously (CA3) were most often either slightly reduced in amplitude (CA3) or not affected (CA1) by ethanol superfusion. In contrast, synaptic potentials evoked by stimulation of the hilar mossy fiber pathway (for CA3) or the stratum radiatum (for CA1) were more sensitive to ethanol: excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) were most often reduced in amplitude in both CA1 and CA2, even at low ethanol concentrations (10-50 mM). The action on IPSPs may be exerted presynaptically, because responses to locally applied GABA were little affected. These results suggest that hippocampal evoked synaptic activity may be more sensitive than postsynaptic membrane properties to physiologically relevant ethanol concentrations.     

Bicuculline- and Phaclofen-Resistant Hyperpolarizations Evoked by Glutamate Applications to Stratum Lacunosum-Moleculare in CA1 Pyramidal Cells of the Rat Hippocampus In Vitro

In the hippocampus, different types of interneurons may mediate distinct gamma-aminobutyric acid (GABA) responses, i.e. the early and late inhibitory postsynaptic potentials (IPSPs). To verify this hypothesis, intracellular recordings were obtained from CA1 pyramidal cells (n=63) in rat hippocampal slices. Glutamate (1 mM) was locally ejected in stratum lacunosum-moleculare to activate interneurons in this region. Glutamate-evoked hyperpolarizing responses were characterized in pyramidal cells and compared to the early IPSP and the late IPSP elicited by stratum radiatum electrical stimulation. Several characteristics were similar for the glutamate-evoked IPSPs and late IPSPs: their amplitude was small (-3.4 versus -4.9 mV, respectively), each was associated with a small conductance increase (5.0 versus 9.3 nS, respectively), their peak latency was slow (124.4 versus 129.8 ms, respectively) and in the majority of cells, each displayed little response reversal. However, the equilibrium potential of the glutamate IPSP (-76.5 mV) was similar to that of the early IPSP (-73.8 mV). Perfusion with a low Ca2+ (0.5 mM)/high Mg2+ (8 mM) medium or with tetrodotoxin (1 microM), which blocked synaptic transmission, also reduced the glutamate IPSP. Therefore the glutamate IPSP may be mediated indirectly by inhibitory interneurons. The GABAA antagonist bicuculline (10 microM), or picrotoxin (10-20 microM), blocked the early IPSP, but not the glutamate IPSP. The GABAB antagonist phaclofen (1 mM) attenuated the late IPSP, but did not affect the glutamate IPSP. The results of these experiments suggest that glutamate stimulation of interneurons in stratum lacunosum-moleculare evokes a slow IPSP different from the GABA-mediated early and late IPSPs in CA1 pyramidal cells of the hippocampus.