Expanding neurochemical investigations with multi-modal recording: simultaneous fast-scan cyclic voltammetry, iontophoresis, and patch clamp measurements.

Multi-modal recording describes the simultaneous collection of information across distinct domains. Compared to isolated measurements, such studies can more easily determine relationships between varieties of phenomena. This is useful for neurochemical investigations which examine cellular activity in response to changes in the local chemical environment. In this study, we demonstrate a method to perform simultaneous patch clamp measurements with fast-scan cyclic voltammetry (FSCV) using optically isolated instrumentation. A model circuit simulating concurrent measurements was used to predict the electrical interference between instruments. No significant impact was anticipated between methods, and predictions were largely confirmed experimentally. One exception was due to capacitive coupling of the FSCV potential waveform into the patch clamp amplifier. However, capacitive transients measured in whole-cell current clamp recordings were well below the level of biological signals, which allowed the activity of cells to be easily determined. Next, the activity of medium spiny neurons (MSNs) was examined in the presence of an FSCV electrode to determine how the exogenous potential impacted nearby cells. The activities of both resting and active MSNs were unaffected by the FSCV waveform. Additionally, application of an iontophoretic current, used to locally deliver drugs and other neurochemicals, did not affect neighboring cells. Finally, MSN activity was monitored during iontophoretic delivery of glutamate, an excitatory neurotransmitter. Membrane depolarization and cell firing were observed concurrently with chemical changes around the cell resulting from delivery. In all, we show how combined electrophysiological and electrochemical measurements can relate information between domains and increase the power of neurochemical investigations.

Magnification in striate cortex and retinal ganglion cell layer of owl monkey: a quantitative comparison.

Magnification, the relative size of the neural representation of a portion of the visual field, decreases more rapidly with increasing visual field eccentricity in striate cortex than in the retinal ganglion cell layer of the owl monkey (Aotus trivirgatus); the proportion of the cells in striate cortex devoted to central vision is much larger than the comparable proportion of retinal ganglion cells. Magnification in striate cortex is a power function of magnification in the retinal ganglion cell layer. A formula for convergence (ganglion cells to cortical neurons) follows from this relationship.

Polarity of long-term synaptic gain change is related to postsynaptic spike firing at a cerebellar inhibitory synapse.

Long-term potentiation and depression (LTP and LTD) in excitatory synapses can coexist, the former being triggered by stimuli that produce strong postsynaptic excitation and the latter by stimuli that produce weaker postsynaptic excitation. It has not been determined whether these properties also apply to LTP and LTD in the inhibitory synapses between Purkinje neurons and the neurons of the deep cerebellar nuclei (DCN), a site that has been implicated in certain types of motor learning. DCN cells exhibit a prominent rebound depolarization (RD) and associated spike burst upon release from hyperpolarization. In these cells, LTP can be elicited by short, high-frequency trains of inhibitory postsynaptic potentials (IPSPs), which reliably evoke an RD. LTD is induced if the same protocol is applied with conditions where the amount of postsynaptic excitation is reduced. The polarity of the change in synaptic strength is correlated with the amount of RD-evoked spike firing during the induction protocol. Thus, some important computational principles that govern the induction of use-dependent change in excitatory synaptic efficacy also apply to inhibitory synapses.

Heterogeneous Ca2+ influx along the adult calyx of Held: a structural and computational study.

The calyx of Held is a morphologically complex nerve terminal containing hundreds to thousands of active zones. The calyx must support high rates of transient, sound-evoked vesicular release superimposed on a background of sustained release, due to the high spontaneous rates of some afferent fibers. One means of distributing vesicle release in space and time is to have heterogeneous release probabilities (Pr) at distinct active zones, which has been observed at several CNS synapses including the calyx of Held. Pr may be modulated by vesicle proximity to Ca2+ channels, by Ca2+ buffers, by changes in phosphorylation state of proteins involved in the release process, or by local variations in Ca2+ influx. In this study, we explore the idea that the complex geometry of the calyx also contributes to heterogeneous Pr by impeding equal propagation of action potentials through all calyx compartments. Given the difficulty of probing ion channel distribution and recording from adult calyces, we undertook a structural and modeling approach based on computerized reconstructions of calyces labeled in adult cats. We were thus able to manipulate placement of conductances and test their effects on Ca2+ concentration in all regions of the calyx following an evoked action potential in the calyceal axon. Our results indicate that with a non-uniform distribution of Na+ and K+ channels, action potentials do not propagate uniformly into the calyx, Ca2+ influx varies across different release sites, and latency for these events varies among calyx compartments. We suggest that the electrotonic structure of the calyx of Held, which our modeling efforts indicate is very sensitive to the axial resistivity of cytoplasm, may contribute to variations in release probability within the calyx.

Convergence of auditory-nerve fiber projections onto globular bushy cells.

Globular bushy cells are a key element of brainstem circuits that mediate the early stages of sound localization. Many of their physiological properties have been attributed to convergence of inputs from the auditory nerve, many of which are large with complex geometry, but the number of these terminals contacting individual cells has not been measured directly. Herein we report, using cats as the experimental model, that this number ranged greatly (9-69) across a population of 12 cells, but over one-half of the cells (seven of 12) received between 15 and 23 inputs. In addition, we provide the first measurements of cell body surface area, which also varies considerably within this population and is uncorrelated with convergence. For one cell, we were able to document axonal structure over a distance greater than 100 microm, between the soma and the location where the axon expanded to its characteristic large diameter. These data were combined with accumulated physiological information on vesicle release, receptor kinetics and voltage-gated ionic conductances, and incorporated into computational models for four cells that are representative of the structural variation within our sample population. This predictive model reveals that basic physiological features, such as precise first spike latencies and peristimulus time histogram shapes, including primary-like with notch and onset-L, can be generated in these cells without including inhibitory inputs. However, phase-locking is not significantly enhanced over auditory-nerve fibers. These combined anatomical and computational approaches reveal additional parameters, such as active zone density, nerve terminal size, numbers and sources of inhibitory inputs and their activity patterns, that must be determined and incorporated into next-generation models to understand the physiology of globular bushy cells.

Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus.

Intracellular recordings from the dorsal cochlear nucleus have identified cells with both simple and complex action potential waveforms. We investigated the hypothesis that cartwheel cells are a specific cell type that generates complex action potentials, based on their analogous anatomical, developmental, and biochemical similarities to cerebellar Purkinje cells, which are known to discharge complex action potentials. Intracellular recordings were made from a brain slice preparation of the guinea pig dorsal cochlear nucleus. A subpopulation of cells discharged a series of two or three action potentials riding on a slow depolarization as an all-or-none event; this discharge pattern is called a complex spike or burst. These cells also exhibited anodal break bursts, anomalous rectification, subthreshold inward rectification, and frequent inhibitory postsynaptic potentials (IPSPs). Seven complex-spiking cells were stained with intracellular dyes and subsequently identified as cartwheel neurons. In contrast, six identified simple-spiking cells recorded in concurrent experiments were pyramidal cells. The cartwheel cell bodies reside in the lower part of layer 1 and the upper part of layer 2 of the nucleus. The cells are characterized by spiny dendrites penetrating the molecular layer, a lack of basal dendritic processes, and an axonal plexus invading layers 2 and 3, and the inner regions of layer 1. The cartwheel cell axons made putative synaptic contacts at the light microscopic level with pyramidal cells and small cells, including stellate cells, granule cells, and other cartwheel cells in layers 1 and 2. The axonal plexus of individual cartwheel cells suggests that they can inhibit cells receiving input from either the same or adjacent parallel fibers and that this inhibition is distributed along the isofrequency contours of the nucleus.

Olfactory receptor neurons exist as distinct subclasses of immature and mature cells in primary culture.

The processes of neuronal differentiation and survival are key questions in neurobiology. The olfactory system possesses unique regenerative capacity, as its neurons are continually replaced throughout adulthood from a maintained population of precursor cells. Primary cultures of olfactory epithelium enriched in olfactory neurons would provide a useful model to study the processes of neurogenesis, differentiation and senescence. To determine whether immature olfactory neurons could be isolated in primary culture and to investigate the mechanisms underlying these processes, culture conditions which selectively favored the presence of immature olfactory neurons were optimized. Using low plating densities, a population of cells was identified which, by reverse transcription-polymerase chain reaction, demonstrated messages for olfactory neuronal markers, including Golf, olfactory cyclic nucleotide-gated channel and olfactory marker protein, as well as the p75 low-affinity nerve growth factor receptor. Immunocytochemical analysis showed that these putative immature olfactory neurons possessed immunoreactivity to G(olf), neuron-specific tubulin, neural cell adhesion molecule, synaptophysin and neurofilament. These neurons were defined as olfactory receptor neuron-1 cells. Under these conditions, a separate class of rarely occurring cells with different morphology demonstrated immunoreactivity to mature markers, such as adenylyl cyclase III and olfactory marker protein. Electrophysiologically, these cells displayed properties consistent with those of acutely dissociated olfactory receptor neurons. Another class of rarer cells which represented less than 2% of cells in culture demonstrated immunoreactivity to glial fibrillary acidic protein. These cultures can serve as a model for in vitro analysis of olfactory receptor neuronal development and maintenance, and provide a potential substrate for the development of cell lines.

A fast, high precision and inexpensive analog to digital board for PC-AT or compatible.

We describe a simple and inexpensive circuit for analog data acquisition with high speed and high resolution, for use in an IBM PC-AT or compatible. The circuit is suitable for a wide range of applications in electrophysiology. Features of the circuit include multichannel reading, programmable sampling rate, and direct memory access controlled dual buffering for continuous recording. A program written in the C programming language for control of data acquisition is also given.

Goldfish retinotectal transmission in vitro: component current sink-source pairs isolated by varying calcium and magnesium levels.

Field potentials and radial current source-densities (CSDs) evoked by optic tract stimulation were observed in goldfish tectum in vitro. The effects of different concentrations of calcium and magnesium were studied to improve understanding of component events in retinotectal transmission and interpretations of effects of pharmacological agents. Responses were of 3 forms: 'non-synaptic', 'subthreshold', and 'complex.' Subthreshold responses occurred when amplitudes were less than 40% of their maximal levels. They consisted of a sink-source pair with the sink in the superficial optic neuropil and the source 100-150 micron deeper. They were monophasic, rising in 1-2 ms and decaying as simple exponentials with time constants between 3.3 and 4.5 ms. Several other conditions which reduce amplitudes (besides low [Ca2+] and/or high [Mg2+]) produce responses of the subthreshold form. Complex responses, observed when amplitudes were 40-100% of maximal, were characterized by more rapid rise and decay and included, in the most extreme cases, a late, long, low-amplitude sink-source pair of opposite polarity. We propose that the time course of decay of subthreshold responses is determined by the passive cable properties of bi- or multistratified neurons with one dendritic arbor in the optic neuropil and a second arbor 100-250 micron deeper. Complex responses probably include recurrent inhibition to depolarized dendrites of the optic neuropil, latent by 4 ms to the monosynaptic excitation. Pharmacological assessments of retinotectal transmission may be made more precise by using low [Ca2+]/high [Mg2+] media to isolate monosynaptic activity.

Electrophysiological analysis of synaptic distribution in CA1 of rat hippocampus after chronic ethanol exposure.

This study investigated the long-lasting effects of chronic ethanol consumption on the distribution of Schaffer collateral-commissural (SCH/COM) afferents within stratum radiatum of rat hippocampal CA1. Experimental animals were fed an ethanol-containing liquid diet for 20 weeks but were withdrawn from the special diet for at least 8 weeks prior to acute electrophysiological recordings. Field potential laminar analyses were performed by stepping the recording electrode in 25 microns increments through CA1 and sampling evoked potentials at each point. One-dimensional current-source density (CSD) was calculated from the field potential laminar profiles to enhance spatial resolution of current sources and sinks. Stimulation of the SCH/COM afferents elicits short-latency, negative field potentials throughout the synaptic terminal zone (stratum radiatum). CSD analysis in normal animals revealed that the synaptic currents generated in stratum radiatum concentrate into bimodal yet overlapping components, peaking 71.3 microns and 228.3 microns from the pyramidal cell layer. Chronic ethanol treatment produced: (1) a 13.2% shrinkage of the overall extent of current sinks in stratum radiatum; (2) a 37.4% reduction in the spatial extent of the sink proximal to the cell layer; and (3) an increase in the amplitude of the more distal sink. We tentatively propose the proximal and distal sinks to reflect a separation of the COM and SCH afferents, respectively. Chronic ethanol thus appeared to have selectively produced persistent damage to the COM-CA1 pathway.

Effects of deafferentation on the electrophysiology of ventral cochlear nucleus neurons.

When cochlear pathology impairs the afferent innervation of the ventral cochlear nucleus (VCN), electrical responses of the auditory brainstem are altered and changes in cell and synaptic morphology are observed. However, the impact of deafferentation on the electrical properties of cells in the VCN is unknown. We examined the electrical properties of single neurons in the anterior and posterior VCN following bilateral cochlear removal in young rats. In control animals, two populations of cells were distinguished: those with a linear subthreshold current-voltage relationship and repetitive firing of action potentials with regular interspike intervals (type I), and those with rectifying subthreshold current-voltage relationships and phasic firing of 1-3 action potentials (type II). Measures of action potential shape further distinguished these two groups. Two weeks following cochlear removal, both electrical response patterns were still seen. Type I cells showed a higher input resistance. Deafferented single-spiking type II cells were slightly more depolarized, had smaller action potentials, smaller afterhyperpolarizations and shorter membrane time constants, whereas multiple-spiking type II cells were apparently unaffected. These changes in the electrical properties of VCN neurons following cochlear injury may adversely affect central processing of sounds presented acoustically or electrically by prostheses.

Properties of cochlear nucleus neurons in primary culture.

Dissociated primary cell cultures were derived from the cochlear nuclei (CN) of postnatal rats using standard techniques. Cultured cells differentiated morphologically, but their dendritic profiles were generally less specialized than those of CN cells in vivo. Physiologically, cultured cells could be divided into three classes: tonic, phasic and non-spiking cells, which differed in many of their fundamental biophysical properties. The percentage of cultured cells that spiked repetitively increased over time to a maximum of 85% at 6 days. However, the percentage of cells that produced action potentials decreased with time in culture, from 91% during the first 8 days to less than 40% after 9 days. CN cells were successfully cultured in both serum-supplemented and serum-free (Neurobasal) media. More neurons survived at low plating densities in Neurobasal than in medium containing serum, although neuronal survival was similar at higher densities. Few neurons raised in the serum-free medium were spontaneously active; other response properties were similar to those of cells grown in the presence of serum. Although differentiation of CN cells in culture did not completely mirror the in vivo developmental pattern, these experiments demonstrate that primary culture represents a viable method for the in vitro study of CN neurons.

Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro.

Intracellular recordings were made from neurons of the guinea pig dorsal cochlear nucleus in an in vitro brain slice preparation. The membrane properties of the cells were studied, and the membrane potentials were manipulated by current injection to determine how intrinsic conductances might alter the cell discharge patterns. Eleven cells were marked with Lucifer yellow. Ten of these cells were identified as the large pyramidal cells of layer 2 of this nucleus, and 1 cell was identified as a "vertical" cell in layer 3. Two kinds of action potentials were observed: simple spikes and complex spikes. This report discusses only cells with simple spikes. Simple spiking cells (60/72 recorded cells; all stained cells were simple spiking cells) discharged in a regular fashion with depolarization, and had linear frequency-current relationships up to 2 nA with a mean slope of 116 Hz/nA. The discharge rate was approximately constant throughout the current pulse. Responses of simple spiking cells to depolarizing current steps superimposed on a steady-state membrane hyperpolarization were studied. When the membrane has been held hyperpolarized, small current pulses produce a long-latency regular train of action potentials. Larger current pulses superimposed on membrane hyperpolarization can produce a short-latency action potential followed by a long silent interval (i.e., a long first interspike interval), and finally a regular train of spikes. It is concluded that the membrane conductances of DCN pyramidal cells are capable of generating at least 3 discharge patterns (regular firing, long first spike latency, and long first interspike interval) depending on the state of the membrane potential prior to a depolarizing current step. These responses are similar to the "chopper," "buildup," and "pauser" discharge patterns reported for these cells in vivo in response to tone bursts. The modulation of the intrinsic membrane conductances by membrane polarization and the possible contribution of these conductances to the generation of DCN discharge patterns provide new insights into the mechanisms underlying the responses of DCN cells to acoustic stimuli.

Responses to parallel fiber stimulation in the guinea pig dorsal cochlear nucleus in vitro.

1. Parallel fibers of the guinea pig dorsal cochlear nucleus (DCN) were electrically stimulated at the pial surface of the nucleus in a brain-slice preparation. Extracellular field potentials produced by the parallel fibers and postsynaptic cells, and the response of single units were identified and characterized. Responses were compared with those reported for stimulation of parallel fibers in the cerebellum and to those seen with electrical stimulation of the auditory nerve. 2. Stimulation of the DCN parallel fibers generates a consistent set of extracellular field potentials. In layer 1 of the DCN, a short-latency triphasic wave (P1(1)-N1(1)-P2(1)) is followed by a slower negative wave (N2(1)). The onset phase of the N2(1) often exhibits a small positive notch (P2a1). In layer 2, an initial triphasic wave (P1(2)-N1(2)-P2(2)) is followed by a short-latency negative wave (N2(2)) and a slower positive wave (P3(2)). The N1(2) is approximately coincident with the N1(1), whereas the P3(2) is coincident with N2(1). The falling phase of the P3(2) is sometimes interrupted by a brief negative deflection (N3(2)). These field potentials are similar, but not identical to those reported for parallel fiber stimulation in the cerebellum in vivo (15). These responses differ substantially from those produced in the DCN by electrical stimulation of the auditory nerve (50). 3. Low-calcium solutions and pharmacologic manipulations were used to separate pre- and postsynaptic response components in the field potential records. When the slice is bathed in a low-calcium solution the P2a1, N2(1), N2(2), P3(2), and the brief late deflections are abolished. However, the P1(1)-N1(1)-P2(1) and P1(2)-N1(2)-P2(2) remain unaffected. A similar separation of pre- and postsynaptic components can be achieved with 100 microM adenosine or 0.5 mM kynurenic acid. It is concluded that the P1(1)-M1(1)-P2(1) wave is the compound action potential of the unmyelinated parallel fibers, whereas the longer-latency field potential components are generated postsynaptically. 4. The conduction velocity of the parallel fiber volley was measured to be 0.30 m/s at the pial surface, in a line approximately parallel to the strial axis of the nucleus. Mapping experiments reveal that the spread of the P1(1)-N1(1)-P2(1) is greatest along the strial axis, and more limited in the orthogonal direction. 5. Single units were recorded in layer 2. At a distance of 500-700 microns from the stimulating electrode, the latencies of single-unit discharges fall between 2.5 and 4 ms, at the time of the N2(2).(ABSTRACT TRUNCATED AT 400 WORDS)

Outward currents in isolated ventral cochlear nucleus neurons.

Neurons of the ventral cochlear nucleus (VCN) perform diverse information processing tasks on incoming activity from the auditory nerve. We have investigated the cellular basis for functional diversity in VCN cells by characterizing the outward membrane conductances of acutely isolated cells using whole-cell, tight-seal, current- and voltage-clamp techniques. The electrical responses of isolated cells fall into two broad categories. Type 1 cells respond to small depolarizations with a regular train of action potentials. Under voltage clamp, these cells exhibit a noninactivating outward current for voltage steps positive to -35 mV. Analysis of tail currents reveals two exponentially decaying components with slightly different voltage dependence. These currents reverse at -73 mV, near the potassium equilibrium potential of -84 mV, and are blocked by tetraethylammonium (TEA). The major outward current in Type I cells thus appears to be mediated by potassium channels. In contrast to Type I cells, Type II cells respond to small depolarizations with only one to three short-latency action potentials and exhibit strong rectification around -70 mV. Under voltage clamp, these cells exhibit a noninactivating outward current with a threshold near -70 mV. Analysis of tail currents reveals two components with different voltage sensitivity and kinetics. A low-threshold current with slow kinetics is partly activated at rest. This current reverses at -77 mV and is blocked by 4-aminopyridine (4-AP) but is only partly affected by TEA. The other component is a high-threshold current activated by steps positive to -35 mV. This current is blocked by TEA, but not by 4-AP. A simple model based on the voltage dependence and kinetics of the slow low-threshold outward current in Type II cells was developed. The model produces current- and voltage-clamp responses that resemble those recorded experimentally. Our results indicate that the two major classes of acoustic response properties of VCN neurons are in part attributable to the types of outward (potassium) conductances present in these cells. The low-threshold conductance in the Type II (bushy) cells probably plays a role in the preservation of information about the acoustic stimulus phase from the auditory nerve to central auditory nuclei involved in low-frequency sound localization.

Transient potassium currents regulate the discharge patterns of dorsal cochlear nucleus pyramidal cells.

Pyramidal cells in the dorsal cochlear nucleus (DCN) show three distinct temporal discharge patterns in response to sound: "pauser," "buildup," and "chopper." Similar discharge patterns are seen in vitro and depend on the voltage from which the cell is depolarized. It has been proposed that an inactivating A-type K+ current (IKI) might play a critical role in generating the three different patterns. In this study we examined the characteristics of transient currents in DCN pyramidal cells to evaluate this hypothesis. Morphologically identified pyramidal cells in rat brain slices (P11-P17) exhibited the three voltage-dependent discharge patterns. Two inactivating currents were present in outside-out patches from pyramidal cells: a rapidly inactivating (IKIF, tau approximately 11 msec) current insensitive to block by tetraethylammonium (TEA) and variably blocked by 4-aminopyridine (4-AP) with half-inactivation near -85 mV, and a slowly inactivating TEA- and 4-AP-sensitive current (IKIS, tau approximately 145 msec) with half-inactivation near -35 mV. Recovery from inactivation at 34 degrees C was described by a single exponential with a time constant of 10-30 msec, similar to the rate at which first spike latency increases with the duration of a hyperpolarizing prepulse. Acutely isolated cells also possessed a rapidly activating (<1 msec at 22 degrees C) transient current that activated near -45 mV and showed half-inactivation near -80 mV. A model demonstrated that the deinactivation of IKIF was correlated with the discharge patterns. Overall, the properties of the fast inactivating K+ current were consistent with their proposed role in shaping the discharge pattern of DCN pyramidal cells.

Voltage-gated Ca2+ conductances in acutely isolated guinea pig dorsal cochlear nucleus neurons.

Although it is known that voltage-gated Ca2+ conductances (VGCCs) contribute to the responses of dorsal cochlear nucleus (DCN) neurons, little is known about the properties of VGCCs in the DCN. In this study, the whole cell voltage-clamp technique was used to examine the pharmacology and voltage dependence of VGCCs in unidentified DCN neurons acutely isolated from guinea pig brain stem. The majority of cells responded to depolarization with sustained inward currents that were enhanced when Ca2+ was replaced by Ba2+, were blocked partially by Ni2+ (100 microM), and were blocked almost completely by Cd2+ (50 microM). Experiments using nifedipine (10 microM), omegaAga IVA (100 nM) and omegaCTX GVIA (500 nM) demonstrated that a variety of VGCC subtypes contributed to the Ba2+ current in most cells, including the L, N, and P/Q types and antagonist-insensitive R type. Although a large depolarization from rest was required to activate VGCCs in DCN neurons, VGCC activation was rapid at depolarized levels, having time constants <1 ms at 22 degrees C. No fast low-threshold inactivation was observed, and a slow high-threshold inactivation was observed at voltages more positive than -20 mV, indicating that Ba2+ currents were carried by high-voltage activated VGCCs. The VGCC subtypes contributing to the overall Ba2+ current had similar voltage-dependent properties, with the exception of the antagonist-insensitive R-type component, which had a slower activation and a more pronounced inactivation than the other components. These data suggest that a variety of VGCCs is present in DCN neurons, and these conductances generate a rapid Ca2+ influx in response to depolarizing stimuli.

Glycine-evoked currents in acutely dissociated neurons of the guinea pig ventral cochlear nucleus.

1. Glycine was applied to acutely dissociated neurons of the guinea pig ventral cochlear nucleus (VCN) with the use of iontophoresis. With approximately equal chloride concentrations in the extra- and intracellular solutions (i.e., chloride equilibrium potential = 0 mV), cells held at -60 mV responded with inward currents that were 1-10 nA in amplitude, had rise times of approximately 50 ms, and decayed to half of the peak amplitude in 50-600 ms. More than 95% of cells with diameters > 12 microns responded to glycine. Response amplitude and area increased with increasing duration of the iontophoretic pulse. Response amplitude saturated at pulse durations of 60-80 ms, whereas response area did not exhibit saturation for pulse durations of 10-100 ms. 2. The glycine antagonist strychnine was added to the extracellular solution at concentrations of 0.5-500 nM to evaluate its effect on glycine-evoked responses. Strychnine produced a 50% reduction in the response at a concentration of 12 nM and the dose-response function had a limiting slope (Hill coefficient) of 1.4. 3. Changes in glycine-evoked currents as a function of cell membrane potential were examined in the presence of tetrodotoxin, tetraethylammonium chloride, and 4-aminopyridine, which block sodium and potassium conductances activated by depolarization. Both the amplitude and the decay of glycine-evoked currents displayed a voltage dependence. Under conditions where the glycine currents reversed at -35 mV, the amplitudes of responses evoked at membrane potentials of 0 mV were 2.3 times larger than those of responses evoked at -70 mV. The decay time constant at 0 mV was 1.49 times longer than that at -70 mV. 4. Acutely dissociated neurons of the VCN previously have been classified on the basis of the absence (type I) or presence (type II) of a low-threshold outward current. Type I cells fire repetitively in response to current pulses, whereas type II cells fire transiently. Glycine-evoked responses were compared in cells identified electrophysiologically as type I or type II on the basis of previously established criteria under voltage clamp. The average amplitudes of responses recorded at a membrane potential of -70 mV were 1.1 and 1.3 nA for type I and type II cells, respectively. The rise time of the glycine current for the two groups of cells was similar (52 ms for type I and 57 ms for type II), but the decay of currents to half-maximum amplitude following the offset of the iontophoretic pulse was longer in type II cells (340 ms) than in type I cells (173 ms). No differences between the two groups were noted with regard to the outward rectification of peak currents or the voltage dependence of current decay. 5. The reversal potential of glycine-evoked responses was determined in extracellular solutions with varying chloride concentrations. The change in the glycine reversal potential (54 mV) for a 10-fold change in chloride concentration was similar to the change in the chloride equilibrium potential (58 mV) over the same range of extracellular chloride concentrations. A similar result was obtained by maintaining the extracellular chloride concentration constant and varying the chloride concentration in the intracellular solution. Glycine-evoked responses were not affected by changes in the potassium or sodium equilibrium potentials. The glycine receptors are therefore principally permeable to chloride. 6. In the VCN, glycine-mediated currents are readily evoked from the majority of larger neurons, indicating an abundance of glycine receptors on the somata and proximal processes of these neurons. The properties of glycine receptors in VCN and other areas of the nervous system are generally similar. The voltage dependence of glycine-evoked currents implies that the inhibitory effectiveness of glycine receptors in VCN increases nonlinearly with depolarization.