l-Aspartic acid potentiates ‘slow’ inward current in cultured spinal cord neurons

Intracellular recordings (current-and two electrode voltage-clamp) were made from mouse spinal cord neurons grown dissociated in tissue culture. Neurons were bathed in elevated concentrations of calcium (Ca) and sometimes tetraethylammonium (TEA). Brief depolarizing current injections activated graded ‘after-depolarizations’ which summated to trigger prolonged all-or-none action potentials. Under voltage-clamp both of these active potentials were manifest as ‘slow’ inward current. Net inward current was observed in some neurons during 0.5–1.0 s depolarizing command steps. However, in the majority of cases the inward current was seen as large inward current tails (outward current relaxations) upon repolarization of the membrane potential to holding values. Cadmium (Cd) blocked this slow inward current, ‘after-depolarizations’ and prolonged action potentials. Applications of l-aspartic acid increased the magnitude of net inward current evoked by command steps and potentiated and prolonged inward current tails. This potentiation and prolongation of voltage-dependent inward current likely accounts for the prolonged action potentials or ‘bursting’ characteristic of responses to l-aspartic acid and related amino acids such as N-methyl-d-aspartic acid.     

In vitro electrophysiology of rat subicular bursting neurons

Intracellular recordings were used to study the electrophysiological properties of rat subicular neurons in a brain slice preparation in vitro. Cells were classified as bursting neurons (n = 102) based on the firing pattern induced by depolarizing current pulses. The bursting response recorded at resting membrane potential (−66.1 ± 6.2 mV, mean ± SD n = 94) was made up of a cluster of fast action potentials riding on a slow depolarization and was followed by an afterhyperpolarization. Tonic firing occurred at a membrane potential of approximately −55 mV. A burst also occurred upon termination of a hyperpolarizing current pulse. Tetrodotoxin (TTX, 1 μM) blocked the burst and decreased or abolished the underlying slow depolarization. These effects were not induced by the concomitant application of the Ca²⁺ channel blockers Co²⁺ (2 mM) and Cd²⁺ (1 mM). Subicular bursting neurons displayed voltage- and time-dependent inward rectifications of the membrane during depolarizing and hyperpolarizing current pulses. The inward rectification in the depolarizing direction was abolished by TTX, while that in the hyperpolarizing direction was blocked by extracellular Cs⁺ (3 mM), but not modified by Ba²⁺ (0.5–1 mM), TTX, or Co²⁺ and Cd²⁺. Tetraethylammonium (10 mM)-sensitive, outward rectification became apparent in the presence of TTX. These results suggest that neurons in the rat subiculum can display voltage-dependent bursts of action potentials as well as membrane rectification in the depolarizing and hyperpolarizing directions. These results also indicate that activation of a voltage-gated Na⁺ conductance may be instrumental in the initiation of bursting activity. Hippocampus 7:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Low-threshold calcium spikes in hypothalamic neurons recorded near the paraventricular nucleus in vitro

From guinea-pig hypothalamic slices, intracellular studies demonstrate the existence of neurons responding to depolarizing current pulses by bursts of fast spikes riding on slow depolarizing potentials, when activated at the resting potential or from hyperpolarized levels (44 cells). Slow depolarizing potentials have a mean amplitude of 17.6 mV and a mean duration of 65.2 msec. They are also produced at the termination of hyperpolarizing current pulses. The ionic basis for these slow potentials have been investigated. Fast spikes constituting the burst discharge are blocked by TTX but the slow component is unaffected, being blocked by Co++ and enhanced by Ba++. Taken together, these results show that the slow depolarizing potentials are generated by a low-threshold Ca++ conductance which is de-inactivated by membrane hyperpolarization. When the neurons are spontaneously active, they exhibit bursts arising from slow depolarizing potentials reminiscent of those evoked by direct stimulation. They also show longer episodes of repetitive firing. Twelve neurons were intracellularly stained and were found in the periphery of the paraventricular nucleus (PVN), in close proximity to the groups of neurophysin-positive neuroendocrine neurons present in the lateral part of this nucleus. Injected neurons have the morphology of reticular cells, judging by their few multipolar, rectilinear and sparsely branched dendrites. Some of their processes are directed towards PVN. Because of their intrinsic electrophysiological properties and their possible relationships with PVN, the population of cells described in the present study may play a role in functions relating to the PVN.     

Electrical membrane properties of rat subthalamic neurons in an in vitro slice preparation

The electrical membrane properties of subthalamic (STH) neurons and their response characteristics to stimulation of the internal capsule (IC) were studied in an in vitro slice preparation. Most STH neurons recorded exhibited spontaneous repetitive firing. The input resistance of STH neurons was 146 +/- 48 M omega and showed both an anomalous and a delayed rectification when the membrane was hyperpolarized or depolarized by current injections. In neurons with the membrane potential less negative than 65 mV, depolarizing current pulses generated repetitive firing with the maximum frequency of up to 500 Hz. Two types of tetrodotoxin (TTX)-resistant cobalt-sensitive potentials, slow depolarizing potential and slow action potential, were observed in STH neurons. The slow depolarizing potential had a long duration (over 500 ms in some cases) and was able to trigger repetitive firing. The slow action potential had a duration of about 30 ms and triggered a burst of firing. The slow action potential was seen only when the neurons were hyperpolarized to more negative than 65 mV by a current injection. Electrical stimulation of IC evoked monosynaptic inhibitory postsynaptic potentials (IPSPs) in most of the neurons examined. The polarity of IPSPs was reversed in the depolarizing direction by intracellular injection of Cl-. Bath application of bicuculline markedly suppressed IPSPs and unmasked monosynaptic excitatory postsynaptic potentials (EPSPs). The EPSP was able to trigger a slow depolarization with repetitive firing or a slow action potential with burst of firing when the neuron was hyperpolarized by a continuous current injection. The results demonstrated that STH neurons in an in vitro preparation have spontaneous discharges, high input resistance, capability to generate high-frequency firing, and Ca potentials. The pattern of responses of STH neurons to synaptic inputs is dependent on their membrane potentials.     

The effects of QX-314 on medullary respiratory neurones

The synaptic and current-evoked responses of respiratory neurones located in the nucleus of the tractus solitarius, the para- and retroambigual regions and the nucleus ambiguus, were examined after voltage-dependent sodium currents were blocked by intracellular application of the quaternary lidocaine derivative QX-314. (1) QX-314 abolished orthodromically and antidromically evoked action potential discharge. Only antidromic action potentials recovered during negative DC current injection. (2) QX-314 did not alter the amplitude or duration of small and short excitatory and inhibitory postsynaptic potentials evoked by vagus or superior laryngeal nerve stimulation. Larger and longer waves of spontaneous membrane depolarizations, however, were slightly diminished. (3) The repetitive discharge evoked by depolarizing current pulses was blocked by QX-314. Positive current pulses produced less membrane depolarization than under control and often evoked only a single action potential at the beginning of the pulse, indicating that QX-314 interferes with the processes responsible for repetitive firing. (4) When fast spike discharges were completely blocked, positive current pulses occasionally evoked depolarizing 'spikes' and potentials which were followed by a hyperpolarization. We conclude that a noninactivating sodium inward current and calcium currents contribute to the electroresponsiveness of respiratory neurones.     

Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation

The electrical membrane properties of rat substantia nigra pars compacta (SNC) neurons were studied in an in vitro slice preparation. Some of the recorded neurons were intracellularly labeled with HRP and were found to have morphological characteristics resembling the presumed SNC dopaminergic neurons, as reported by others. The input resistance of SNC neurons at resting membrane potential ranged between 70 and 250 M omega. The membrane resistance showed strong anomalous rectification when the membrane was hyperpolarized by current injection. The anomalous rectification was decreased by the addition of tetraethylammonium bromide (TEA) to the bathing Ringer solution. Injection of depolarizing current or termination of hyperpolarizing current induced slow depolarizing potentials. Their amplitude was dependent on the membrane potential and the current intensity. In neurons treated with tetrodotoxin (TTX) and TEA, slow action potentials were triggered from the slow depolarizing potentials. Both the slow depolarizing potential and slow action potential were TTX resistant and abolished by superfusion of Ca2+-free medium. Long duration hyperpolarizations were observed following the injection of depolarizing current pulses. The hyperpolarization was abolished by the superfusion of Ca2+-free medium or decreased by addition of TEA to the Ringer solution indicating an involvement of a Ca2+-dependent K+-conductance in generation of the hyperpolarization. The long duration hyperpolarization was also observed following action potentials. The spike after hyperpolarization consisted of an initial short duration fast component and a long lasting component. The amplitude of both components seems to be reduced but not abolished by TEA (up to 10 mM). When hyperpolarizing current pulses were applied to neurons that were held either continuously depolarized or were superfused with Ca2+-free medium, the pattern of the membrane potential after the offset of current pulses consisted of an initial fast and a later slow ramp-shaped phase. The latter was associated with a membrane conductance increase and interpreted to be due to an early K+ current. This early K+ current was relatively resistant to TEA. Injections of strong depolarizing currents triggered action potentials with multiple inflections on their rising phase. The amplitudes of action potentials changed abruptly during current application. These data indicate that SNC neurons have multiple generation sites for action potential.     

Membrane properties and adrenergic responses in locus coeruleus neurons of young rats

Intracellular recordings were made from locus coeruleus neurons in slices taken from rats 8-26 d of age. Neurons from these animals exhibited spontaneous action potentials, which were superimposed on slow (0.3-3 Hz) rhythmic depolarizations. The frequency of these potentials was closely related to the age of the animals from which the slice was taken, the slowest frequencies being observed in tissues from the youngest animals. In adult animals, such rhythmic activity was only rarely observed under normal recording conditions. The rhythmic depolarizations had a slow rate of rise and fall, were 3-15 mV in amplitude, were not affected by tetrodotoxin, and were abolished in solutions that contained elevated magnesium content. When the membrane potential was hyperpolarized by passing current through the recording electrode, the depolarizing rhythmic activity persisted even at very negative potentials (-120 mV). These depolarizations appear to be generated by the inward movement of calcium ions, probably in dendritic regions of the neuron. Superfusion of phenylephrine caused membrane depolarizations, increased the frequency of action potentials and of the slow, rhythmic depolarizations in about 80% of the cells from young rats, whereas it had no effect or a depressant action on cells from adults. Noradrenaline hyperpolarized the cells through an alpha 2-adrenoceptor and abolished the slow depolarizations. In cells from young rats, the hyperpolarization produced by noradrenaline reached a maximum and then declined, such that there was a "sag" in the membrane potential toward the resting potential following the peak of the hyperpolarization. Following the washout of noradrenaline, the membrane potential repolarized before moving toward the resting level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of neurokinin receptors modulates K and Cl channel activity in cultured astrocytes from rat cortex

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.     

Corticosterone Reduces Synaptic Inhibition in Rat Hippocampal and Neocortical Neurons in vitro

We examined the effect of corticosterone (10(-7) to 10(-5) M) on membrane properties and postsynaptic potentials, by means of intracellular recordings from neocortical and hippocampal CA1 pyramidal neurons of the intact adult rat in vitro. Corticosterone reduced both the early and the late components of the orthodromically-evoked inhibitory postsynaptic potential in both structures. The glucocorticoid receptor antagonist RU 38486 (10(-6) M) prevented this effect in the hippocampus. In hippocampal, but not in neocortical pyramidal neurons, corticosterone reduced a depolarizing membrane transient evoked by a depolarizing current step and increased the threshold for eliciting action potentials evoked by depolarizing current pulses. Corticosterone did not detectably alter the afterhyperpolarization following repetitive neuronal discharges evoked by current injection, in either the neocortex or in the hippocampus. Excitatory postsynaptic potentials, action potentials, membrane potential and membrane input resistance were also unchanged. The decrease in synaptic inhibition together with the reduction of electrical excitability in the hippocampus, would imply a modulation of response characteristics in pyramidal neurons such that repeated synaptic inputs become more efficient and low frequency input is blunted.     

Nociceptive integration in the rat spinal cord: role of non-linear membrane properties of deep dorsal horn neurons

Deep dorsal horn neurons (DHNs) involved in nociception can relay long-lasting inputs and generate prolonged afterdischarges believed to enhance the transfer of nociceptive responses to the brain. We addressed the role of neuronal membrane properties in shaping these responses, by recording lamina V DHNs in a slice preparation of the rat cervical spinal cord. of 256 neurons, 102 produced accelerating discharges in response to depolarizing current pulses, whereas the other neurons showed spike frequency adaptation. Two mechanisms mediated the firing acceleration: a slow inactivation of a K⁺ current expressed upon activation of the neuron from hyperpolarized holding potentials, and the expression of a regenerative plateau potential activating around resting membrane potential. The increase in firing frequency was much stronger when sustained by the plateau potential (71 DHNs, 28%). A few neurons produced adaptation and both types of acceleration, in different membrane potential domains, showing that the firing pattern of a deep DHN is not a rigid characteristic. Plateau potentials could be elicited by stimulation of nociceptive primary afferent fibres. The bistability associated with plateau potentials permitted afterdischarges. Because plateau potentials had slow activation kinetics and were voltage-dependent, the neurons had non-linear input–output relationships in both the amplitude and time domains. Nociceptive primary afferent stimulation elicited intense and prolonged responses in plateau-generating DHNs, while brief bursts of spikes were evoked otherwise. These results indicate that in a population of deep DHNs, intense firing and prolonged afterdischarges in response to nociceptive stimulation depend on non-linear intrinsic membrane properties.     

Intracellular injection of apamin reduces a slow potassium current mediating afterhyperpolarizations and IPSPs in neocortical neurons of cats

Electrophysiologic effects of intracellularly injected apamin, a Ca2+-dependent K+ channel blocker, were investigated in neurons of the motor cortex of awake cats. Single-electrode voltage clamp techniques were used to measure changes in membrane currents including those that were synaptically activated. All changes occurred within 2-4 min after pressure injection of apamin with partial recovery observed within 8-15 min. Apamin selectively abolished an outward current that mediated a slow afterhyperpolarization (AHP) following intracellular depolarizing current pulses and action potentials without influencing the time course of the action potentials or an associated fast AHP component. In addition apamin increased the number and frequency of spike discharges evoked by the depolarizing current pulses and produced a small increase in the rate of background firing activity. The baseline resting potential and input resistance were essentially unchanged by apamin. Apamin also diminished a late, slowly decaying component of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) elicited by stimulation of the ventrolateral thalamus or the pyramidal tract. The apamin-induced changes were concomitant with a decrease of the decay time constant of both IPSPs and IPSCs and a positive shift in their reversal potential. The results suggest that the late, slowly decaying component of these inhibitory postsynaptic responses is generated by an apamin-sensitive Ca2+-dependent K+ conductance which is also responsible for the slow AHP.     

Inactivation of calcium channels in rat pituitary intermediate lobe cells

The voltage-dependent inactivation of Ca currents was explored in dissociated intermediate lobe (IL) cells from the rat pituitary. On the basis of current-voltage relations two main inward currents could be identified in this cell, a transient current, (I-t), and a sustained current, (I-s). Inactivation was explored either by changing the holding potential and testing the change in the inward currents during a brief test pulse, or, by depolarizing the membrane and following the decay of the evoked inward current. Three current decay rates were identified, each with a characteristic dependence on membrane potential. The fastest decay rate (tau 1), was attributed to the inactivation of the I-t current and had a value of 57 ms at -40 mV, decreasing to 10 ms at -10 mV (extrapolated value of 6 ms at 0 mV). The other two decay rates, tau 2 and tau 3, decreased monotonically with depolarization of the membrane potential and reflected the inactivation of the I-s current with values of 1.8 and 20 s at 0 mV. I-s inactivation and reactivation was found to occur even in the normal resting potential range of this cell. These properties of the calcium channels can explain the voltage-dependent inactivation of secretion that has been observed previously in this and other secretory cells. In addition, they suggest that calcium currents, and hence secretion, may be modulated by external factors that cause small, but sustained, changes in the resting potential of the IL cell.     

Characterization of the neurons of the mouse hypogastric ganglion: morphology and electrophysiology

The somata of mouse hypogastric ganglion cells injected with Lucifer yellow were ovoid in shape, lacked dendritic processes but gave rise to a single axonal process. Antidromic activation demonstrated that some of the cells contained in this ganglion innervated the vas deferens. The passive and active membrane properties of the ganglion cells were determined in current clamp experiments. Cells fired tetrodotoxin-sensitive action potentials in response to intracellularly-applied depolarizing current. In voltage clamp evidence was obtained for both a persistent inward calcium current at potentials between -30 and -40 mV and a transient calcium current evoked by step depolarizations to around -20 mV. In current clamp, however, cells did not fire calcium action potentials in the presence of tetrodotoxin. Three potassium currents, IM (blocked by 1 mM barium and by 30 microM bethanechol), IA (blocked by 2 mM 4-aminopyridine) and IK(Ca) fast (blocked by 100 microM cadmium and by 5 mM tetraethylammonium) were characterized in these neurons. In addition, IK(Ca) slow was observed in a small proportion of cells. Fast, all-or-nothing, excitatory synaptic potentials were recorded in response to single stimuli applied to the afferent fibres running to the ganglion. In most cells the excitatory synaptic potentials were suprathreshold for action potential initiation and were markedly reduced or abolished by 100 microM mecamylamine, 1 mM hexamethonium and following desensitization to 100 microM nicotine. Excitatory synaptic potentials arose from stimulation of a single presynaptic nerve process and are typical of strong synaptic inputs.     

Substance P augments a persistent slow inward calcium-sensitive current in voltage-clamped spinal dorsal horn neurons of the rat

Rat spinal dorsal horn neurons in slice preparations perfused with Ringer solution containing 0.5-1 microM TTX and/or 10-20 mM tetraethylammonium at 29 degrees C, were studied by using a single microelectrode voltage-clamp technique. Slow persistent inward currents were recorded during depolarizing voltage commands to membrane potentials positive to about -40 mV. The inward current was depressed by removing external Ca, or by adding 0.1-0.2 mM Cd, 5 mM Co or 0.1 mM verapamil, and was increased by adding Ba or Bay-K 8644. Substance P (SP) augmented a persistent slow inward Ca-sensitive current in a dose-dependent manner. It is suggested that this effect may be instrumental in generating the SP-evoked slow depolarization, increase in membrane excitability, and the 'bursting' behavior in the immature rat dorsal horn neurons. In addition, in some neurons SP reduced the M-like current, which effect may contribute to, but not explain, generation of the SP-induced slow depolarization.     

Photoresponses of an extraocular photoreceptor associated with a decrease in membrane conductance in an opisthobranch mollusc

The photoresponse of an extraocular photoreceptor, the photoresponsive neuron (A-P-1) in the abdominal ganglion of Onchidium verruculatum, was studied by using a voltage-clamp with two micropipettes and a monochromatic light. When the A-P-1 was voltage-clamped at resting membrane potential levels, light induced a slowly developing inward current which peaked at about 20 s. A decrease in membrane conductance accompanied this light-induced current which corresponded to the depolarizing photoreceptor potential in the unclamped A-P-1. The relationship between the peak of the current response and light intensity could be predicted by using the modified Michaelis-Menten equation. The spectral sensitivity for the photoresponse had a peak at 490 nm. The steady-state light-induced current was a non-linear function of the membrane potential. The current-voltage relationship for the instantaneous light-induced current was almost linear. In normal (10 mM K+) saline, the polarity of the instantaneous light current reversed from inward to outward at about -67 mV, and doubling the external K+ from 10 to 20 mM shifted the reversal potential to about-50 mV, similar to that predicted by a K+-electrode. These results suggest that the light-induced current or the depolarizing receptor potential of A-P-1 is due to the light suppression of a voltage- and time-dependent K+ current.     

Contrasting properties of K+ conductances induced by baclofen and gamma-aminobutyric acid in slices of the guinea pig hippocampus

Properties of membrane K+ conductances induced by baclofen and gamma-aminobutyric acid (GABA) in the hippocampus were investigated by using guinea-pig brain slices. Baclofen hyperpolarized the membrane and decreased the input resistance of pyramidal cells through the activation of a membrane K+ conductance. GABA caused a biphasic response in pyramidal cells, consisting of hyperpolarizing and depolarizing components. Combined application of picrotoxin and bicuculline eliminated the major part of the depolarizing component of the biphasic response and produced a relatively pure hyperpolarizing response which was also mediated by an increase in K+ conductance. The K+ conductance change induced by baclofen showed prominent inward rectification. However, the K+ conductance induced by GABA did not show an obvious rectifying property. The K+ conductance activated by baclofen was strongly antagonized by a low concentration (5 x 10(-6) M) of 4-aminopyridine (4-AP). In contrast, the K+ conductance activated by GABA was insensitive to 4-AP even at a high concentration of 10(-3) M. The slow inhibitory postsynaptic potential (slow i.p.s.p.) evoked by stimulation of the mossy fibres was totally suppressed by a low concentration of baclofen (5 x 10(-6) M). Whereas GABA (10(-3) M) decreased the amplitude of the slow i.p.s.p., the reduction of the amplitude was proportional to the decrease in the amplitude of the electrotonic potentials produced by constant inward current injections. These results suggest that the hyperpolarizations induced by GABA and baclofen may be generated by K+ conductances of different kinetic and pharmacologic properties.     

Passive electrical membrane properties of rat neostriatal neurons in an in vitro slice preparation

The passive electrical membrane properties of rat neostriatal neurons were studied in in vitro slice preparations. The data are only from neurons having stable resting membrane potentials of more than 50 mV and able to generate action potentials of amplitudes greater than 70 mV evoked by local or intracellular stimulation. All neurons measured for current-voltage relationship (n = 52) showed non-linearity of the input resistance in the hyperpolarizing direction. The mean input resistance at the resting membrane potential was 16.6 M omega. Depolarizing postsynaptic potentials evoked by local stimulation were decreased both in their amplitude and half-decay time by inward current injections exceeding more than 1 nA due to the strong membrane rectification at these levels of hyperpolarization. The mean membrane time constant (tau 0) was 5.3 ms, as measured from the semilogarithmic plots of transmembrane potential shift produced by small hyperpolarizing current pulses. In some neurons, the equalizing term (tau 1) could be determined as well and had a mean value of 1.0 ms. Measurement of (tau 0) using the strength-latency relation showed a similar value (5.0 ms) to that measured from the voltage transients. Intracellular labeling of the recorded neurons with horseradish peroxidase suggested that the recordings were obtained from medium spiny neurons.     

An aminopyridine-sensitive, early outward current recorded in vivo in neurons of the precruciate cortex of cats using single-electrode voltage-clamp techniques

Studies were performed in cortical neurons to determine if voltage- and time-dependent membrane currents could be recognized and characterized in the dynamic, in vivo state. Intracellular measurements made in neurons of the precruciate cortex of awake cats with single-electrode voltage-clamp (SEVC) techniques disclosed an early outward current to depolarizing command steps in 124 of 137 cells studies. The voltage-dependent properties of the early outward current closely resembled those of A-currents studied in vitro in verterbrate and invertebrate neurons. The current was activated rapidly at onset latencies of less than two ms, fell to flat plateau levels within 60–120 ms during sustained depolarization, and was reduced or eliminated in 22 of 23 cells following intracellular administration of 3- or 4-aminopyridine. The magnitude of outward current in response to depolarizing commands was increased by preceding steady hyperpolarization and reduced by preceding steady depolarization. (The steady potentials were of 9.8 s duration and ±40 mV apart from the holding potentials.) Since return to the holding potentials occurred 80 ms before the onset of the command steps, the changes in membrane properties that were induced lasted beyond cessation of the steady polarizing stimuli themselves. Spiking did not prevent recognition of the early outward current as judged from its appearance before and after intracellular application of QX-314 to reduce spike activity. Apart from fast inward currents associated with spike potentials, the early outward current was the most conspicuous and characteristic membrane current noted in these recordings. An additional current component that was noted but not characterized in these studies was a slow, depolarization-induced inward current that could be reduced by intracellular injection of QX-314.     

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.     

Synaptic control of excitability in isolated dendrites of hippocampal neurons

The apical dendrites of CA1 pyramidal cells were isolated from their cell bodies by making cuts through proximal stratum radiatum of transverse hippocampal slices from the guinea pig. This lesion separated the distal apical dendritic elements from the somata, basal dendrites, and 50 to 100 microns of the proximal apical dendritic tree. Orthodromic stimuli in stratum radiatum evoked excitatory synaptic responses in isolated dendrites, but no phasic inhibitory components could be detected. In spite of this surgically produced disinhibition, orthodromic stimuli did not elicit burst activity at the resting membrane potential. However, isolated dendrites and intact dendrites could generate multiple slow spike activity when directly stimulated with depolarizing current pulses. When isolated dendrites were depolarized by DC current, excitatory postsynaptic potentials could evoke subthreshold intrinsic slow depolarizations, or repetitive slow spikes, similar to responses elicited by depolarizing current pulses alone. After exposure to bicuculline (5 microns), both intact and isolated dendrites generated bursts of activity following synaptic activation. A possible mechanism for this action of bicuculline is blockade of a residual GABA-mediated inhibition which was not expressed as a postsynaptic hyperpolarization in isolated dendrites. This bicuculline-sensitive event was capable of depressing dendritic excitability in the absence of the recurrent inhibitory synaptic input and was very effective in controlling burst activity. Our results indicate that the dendritic electrical behavior is dependent on a complex interaction between synaptic and voltage-sensitive events.