Active membrane properties of rat neostriatal neurons in an in vitro slice preparation

Summary The active membrane properties of rat neostriatal neurons have been studied in an in vitro slice preparation. All the neurons examined had resting membrane potentials of more than 50 mV and generated action potentials with amplitudes exceeding 70 mV. The morphological characteristics of the neurons identified by intracellular labeling with HRP indicated that they were medium spiny neurons. 1. Depolarizing current injection through the recording microelectrode generated slow depolarizing potentials and repetitive action potentials with frequencies ranging from less than 10 Hz to over 300 Hz. Adaptation of action potentials was observed when long duration depolarizing current was injected. 2. Depolarizing current injections revealed that the membrane of the striatal neuron had an anomalous rectification when the membrane potential was depolarized to the resting potential. A possible bases for the anomalous rectification might involve inactivation of K-conductance and slow inward Ca- and/or Na-currents. 3. Local electrical stimulation evoked depolarizing postsynaptic potentials (DPSPs) followed by long-lasting small depolarizations. In a double stimulation test, a potentiation of the test DPSP was observed at interstimulus time interval of up to 80 ms. Post-tetanic potentiation of DPSPs was also seen in these neurons. 4. Tests utilizing depolarizing current injection, intracellular Cl^– injection, and Cl-conductance blocking drugs indicated that the DPSPs were composed of EPSPs and overlapping IPSPs. 5. The nature of the longlasting small depolarization succeeding the DPSPs could not be conclusively determined. However, available data suggest that the slow inward Cacurrent may be responsible for this response. 6. In some neurons, antidromic responses were observed following local stimulation. Spike invasion into the somatic region was blocked by an injection of hyperpolarizing current to the neuron or by synaptic inputs evoked by conditioning local stimulation. These findings may explain the difficulties encountered by previous investigators in obtaining antidromic responses from neostriatal neurons in in vivo preparation.     

The role of calcium in the repetitive firing of neostriatal neurons

Summary The Ca⁺⁺-dependence of the repetitive firing of neostriatal neurons was studied in an in vitro slice preparation of the rat neostriatum. Neuronal firing was evoked by injecting depolarizing currents of 100–200 ms duration. In normal conditions, the mode of firing was tonic and showed very little adaptation. The frequency-current relation was linear over a wide range of frequencies. The repetitive firing was first enhanced and later suppressed by Co⁺⁺, Mn⁺⁺ and Cd⁺⁺. These effects on the repetitive firing by the Ca⁺⁺-channel blockers paralleled the suppression of the slow afterhyperpolarizing potential. The lowering (0.2 mM) of Ca⁺⁺ had similar effects. In the presence of TEA (up to 10 mM), the cell fired both Na⁺ and Ca⁺ action potentials. The results suggest that, as in other CNS neurons of the vertebrate, in neostriatal neurons the slow afterhyperpolarizing potential (AHP) is due to a Ca⁺⁺-activated K⁺-conductance, and that the AHP plays a crucial role in the repetitive firing of these neurons.     

Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro

Properties of the action potential and subthreshold response were studied in large layer V neurons in in vitro slices of cat sensorimotor cortex using intracellular recording and stimulation, application of agents that block active conductances, and a single-microelectrode voltage clamp (SEVC). A variety of measured parameters, including action-potential duration, afterpotentials, input resistance, rheobase, and membrane time constant, were similar to the same parameters reported for large neurons from this region of cortex in vivo. Action-potential amplitudes and resting potentials were greater in vitro. Most measured parameters were distributed unimodally, suggesting that these parameters are similar in all large layer V neurons irrespective of their axonal termination. The voltage response to subthreshold constant-current pulses exhibited both time and voltage dependence in the great majority of cells. Current pulses in either the hyperpolarizing or subthreshold depolarizing direction cause the membrane potential to attain an early peak and then decay (sag) to a steady level. On termination of the pulse, the membrane response transiently overshoots resting potential. Plots of current-voltage relations demonstrate inward rectification during polarization on either side of resting potential. Subthreshold inward rectification in the depolarizing direction is abolished by tetrodotoxin (TTX). The ionic currents responsible for subthreshold rectification and sag were examined using the SEVC. Steady inward rectification in the depolarizing direction is caused by a persistent, subthreshold sodium current (INaP) (54). Sag observed in response to a depolarizing current pulse is due to activation of a slow outward current, which superimposes on and partially counters the persistent sodium current. Both sag in response to hyperpolarizing current pulses and rectification in the hyperpolarizing direction are caused by a slow inward "sag current" that is activated by hyperpolarizing voltage steps. The sag current is unaltered by TTX, tetraethylammonium, (TEA), Co2+, Ba2+, or 4-aminopyridine. Fast-rising, short-duration action potentials can be elicited by an intracellular current pulse or by orthodromic or antidromic stimulation. Spikes are blocked by TTX. The form of the afterpotential following a directly evoked spike varies among cells with similar resting potentials. Biphasic afterhyperpolarizations (AHPs) with fast and slow components were most frequently seen. About 30% of the cells displayed a depolarizing afterpotential (DAP), which was often followed by an AHP. Other cells displayed a purely monophasic AHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca²⁺) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca²⁺, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.     

Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro

1. Primary afferent fiber-evoked synaptic responses and the mechanisms of spike and slow potential generation have been examined in adult rat substantia gelatinosa (SG) neurons in an in vitro transverse spinal cord slice preparation in which an attached dorsal root is retained. Intracellular recordings were made from SG neurons identified by morphological and electrophysiological criteria. Afferent fiber-evoked fast excitatory postsynaptic potentials (fast EPSPs) and slow EPSPs have been analyzed. 2. SG neurons had mean resting membrane potentials of -67.1 +/- 0.5 mV (mean +/- SE), mean input resistance of 257 +/- 17.7 (SE) M omega, and a mean time constant of 21.3 +/- 1.9 ms and exhibited spontaneous EPSPs. 3. Single low-intensity stimuli applied to the dorsal root using a suction electrode produced, in 70% of SG neurons, short-latency, presumed monosynaptic fast EPSPs which had a half decay time of 10-30 ms and an amplitude of 8-28 mV. The conduction velocity of afferent fibers evoking fast EPSPs was 2-7 m/s, corresponding to that of thinly myelinated A-delta-fibers. Dorsal root stimulation at higher intensities evoked, in 10% of SG neurons, long-latency and apparently monosynaptic EPSPs which had a time course and amplitude similar to that evoked by low-intensity stimulation. The conduction velocity of fibers evoking long-latency EPSPs was 0.4-2 m/s, suggesting that they constitute predominantly C-fibers. A-delta- and C-fiber-mediated fast EPSPs were detected in 20% of SG neurons examined. 4. Low-intensity stimuli produced slow EPSPs in 20% of SG neurons. Slow EPSPs were 3-15 mV in amplitude and of up to 2 min in duration. A-delta-fibers appeared to be responsible for the generation of slow EPSPs. Slow EPSPs were associated with an increase in membrane resistance and were decreased in amplitude with membrane hyperpolarization. 5. Action potentials in SG neurons had a mean amplitude of 76.3 +/- 1.1 mV and a mean duration of 1.0 +/- 0.07 ms. Na+ ions represent the main charge carrier during the rising phase of the action potential and Ca2+ ions contribute to the shoulder on the falling phase. 6. In 20% of SG neurons, subthreshold depolarizing pulses were followed by long-lasting slow-inactivating depolarizing potentials which were able to initiate spikes. The slow depolarizing potentials were blocked by TTX and enhanced by application of TEA and Ba2+, suggesting that Na+ and K+ are involved in this slow-inactivating potential.(ABSTRACT TRUNCATED AT 400 WORDS)     

An early outward conductance modulates the firing latency and frequency of neostriatal neurons of the rat brain

Summary An in vitro slice preparation was used to obtain intracellular recordings of neostriatal neurons. Indirect evidence for the presence of an early outward conductance in neostriatal neurons is presented. With near threshold stimulation neostriatal neurons fired very late during the pulse. The long firing latency was associated with a slow (ramp-like) depolarization. In the presence of TTX the slow depolarization was lost and outward-going rectification dominated the subthreshold response. This finding demonstrated that both, outward and inwardgoing conductances play a role during the ramp-like depolarization. Outward-going rectification during depolarizing responses could be further augmented if the depolarizing stimulus was preceded by a conditioning hyperpolarization. A conditioning hyperpolarization prolonged the firing latency and slowed the firing frequency. A conditioning depolarization had opposite effects. After TTX treatment, the response showed a hyperpolarizing sag when depolarizing stimulation was preceded by conditioning hyperpolarization. 4-AP (0.5–2.5 mM) blocked the effects of the conditioning hyperpolarization on the firing latency and on the voltage trajectory. 4-AP also disclosed a slow depolarization which could produce neuronal firing very early during the pulse. This depolarization was TTX-sensitive and Co⁺⁺-insensitive. In contrast to 4-AP, TEA (20 mM) did not produce a reduction in the firing latency but disclosed a membrane oscillatory behavior most probably produced by the interplay of these opposing conductances: the slow inward (probably Na⁺) and the transient outward (probably K⁺). Repetitive firing during 4-AP treatment was of the phasictonic type with an initial burst riding on the initial Co⁺⁺-insensitive slow depolarization and a somehow irregular train of spikes during the remainder of the stimulation. Action potentials during 4-AP treatment were followed by an afterdepolarization which dominated the initial part of the interspike interval.     

Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents.

1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones.

1. Depolarization-induced voltage and conductance changes were studied in frog montoneurones in isolated, perfused spinal cord slices. Two types of afterhyperpolarization are observed following action potentials in normal Ringer, a fast afterhyperpolarization lastin 5-10 msec and a slow afterhyperpolarization lasting 60-200 msec. Both afterhyperpolarizations are mediated by an increased K+ conductance. 2. The slow afterhyperpolarization and conductance increase underlying it are selectively and reversibly inhibited by perfusion with solutions containing low [Ca2+] (less than or equal to 0-2 nM) or the Ca2+ antagonists Mn2+ (1mM) or Co2+ (5 mM), and are enhanced by perfusion with high [Ca2+]. 3. Addition of 2-5 mM tetraethylammonium ion (TEA+) to the perfusing solution prolongs the falling phase of the action potential and abolishes the fast afterhyperpolarization, but does not inhibit the slow afterhyperolarization. 4. When the voltage-dependent Na+ current is blocked by perfusion with TTX (10-5 M), intracellularly applied depolarizing current steps evoke fast and slow hyperpolarizations with kinetics and pharmacological sensitivities similar to those of the fast and slow afterhyperpolarizations, respectively. The fast hyperpolarization is maximally activated by brief, intense depolarizations, the slow hyperpolarization by prolonged, less intense depolarizations. 5. These pharmacological and kinetic data demonstrate that in frog motoneurones the repolarization-fast afterhyperpolarization sequence and the slow afterhyperpolarization are produced by different K+ conductance systems. The fast K+ conductance activates rapidly on depolarization, decays rapidly on repolarization, and is TEA+ sensitive, while the slow K+ conducatance activates and decays more slowly and is Ca2+-dependent. 6. Motoneurones perfused with TEA+ and TEA often show a slow, regenerative depolarizing response to applied depolarizing currents. These regenerative depolarizations are probably produced by an influx of Ca2+, because they persist in isotonic CaCl2 and are blocked by Mn2+ or low [Ca2+]. The Ca2+-dependence of the slow afterhyperpolarization and the increase in slow afterhyperpolarization magnitude observed following the slow Ca2+ potentials suggest that a depolarization-evoked Ca2+ influx activates the K+ conductance underlying the slow afterhyperpolarization. 7. Motoneurones in which the slow Ca2+ and K+ conductance systems have been enhanced by high [Ca2+] or blocked by Mn2+ show altered discharge patterns in response to intracellularly applied depolarizing current steps. Perfusion with twice normal [Ca2+] (4 mM) causes montoneurones to discharge more slowly at all current intensities, and reduces the slope of the 'steady-state' frequency-current relationship. Mn2+-perfused motoneurones exhibit fairly normal high-frequency discharge at the onset of the current step, but unlike normal motoneurones, do not discharge at frequencies below 60/sec...     

Synaptic and non-synaptic mechanisms underlying low calcium bursts in the in vitro hippocampal slice

Summary 1. The epileptiform activity generated by lowering extracellular [Ca⁺⁺] was studied in the CA1 subfield of rat hippocampal slices maintained in vitro at 32° C. Extracellular and intracellular recordings were performed with NaCl and KCl filled microelectrodes. 2. Synaptic potentials evoked by stimulation of the stratum radiatum and alveus were blocked upon perfusion with artificial cerebrospinal fluid (ACSF) containing 0.2 mM Ca⁺⁺, 4 mM Mg⁺⁺. Blockade of synaptic potentials was accompanied by the appearance of synchronous field bursts which either occurred spontaneously or could be induced by stimulation of the alveus. 3. Both spontaneous and stimulus-induced low Ca⁺⁺ bursts recorded extracellularly in stratum pyramidale consisted of a negative potential shift with superimposed population spikes. This extracellular event was closely associated with intracellularly recorded action potentials rising from a prolonged depolarization shift. Steady hyperpolarization of the cell membrane potential decreased the amplitude of the depolarizing shift suggesting that synaptic conductance were not involved in the genesis of the low Ca⁺⁺ burst. 4. Spontaneous depolarizing inhibitory potentials recorded in normal ACSF with KCl filled microelectrodes were reduced in size in low Ca⁺⁺ ACSF. However, small amplitude potentials could still be observed at a time when low Ca⁺⁺ bursts were generated by hippocampal CA1 pyramidal neurons. 5. Bicuculline methiodide, an antagonist of -aminobutyric acid (GABA), was capable of modifying the frequency of occurrence and the shape of synchronous field bursts. The effects evoked by bicuculline methiodide were, however, not observed when 81–100% of NaCl was replaced with Na-Methylsulphate. Hence, it was concluded that in low Ca⁺⁺ ACSF even though large release of transmitter such as those following electrical activation of stratum radiatum or alveus cannot be observed, small spontaneous release of the inhibitory transmitter GABA seems to persist. 6. Substitution of NaCl with Na-Methylsulphate also caused changes in the synchronous field bursts which were different from those observed following application of bicuculline methiodide. These findings suggest that in low Ca⁺⁺ ACSF, in addition to residual GABAergic Cl^- mechanisms, non-synaptic Cl^- conductances might play a role in controlling the excitability of hippocampal neurons.Supported by grants from the MRC of Canada (MA-8109) and Sick Children Foundation to MA     

Blockade of SK-type Ca2+-activated K+ channels uncovers a Ca2+-dependent slow afterdepolarization in nigral dopamine neurons

Sharp electrode current-clamp recording techniques were used to characterize the response of nigral dopamine (DA)-containing neurons in rat brain slices to injected current pulses applied in the presence of TTX (2 microM) and under conditions in which apamin-sensitive Ca2+-activated K+ channels were blocked. Addition of apamin (100-300 nM) to perfusion solutions containing TTX blocked the pacemaker oscillation in membrane voltage evoked by depolarizing current pulses and revealed an afterdepolarization (ADP) that appeared as a shoulder on the falling phase of the voltage response. ADP were preceded by a ramp-shaped slow depolarization and followed by an apamin-insensitive hyperpolarizing afterpotential (HAP). Although ADPs were observed in all apamin-treated cells, the duration of the response varied considerably between individual neurons and was strongly potentiated by the addition of TEA (2-3 mM). In the presence of TTX, TEA, and apamin, optimal stimulus parameters (0.1 nA, 200-ms duration at -55 to -68 mV) evoked ADP ranging from 80 to 1,020 ms in duration (355.3 +/- 56.5 ms, n = 16). Both the ramp-shaped slow depolarization and the ensuing ADP were markedly voltage dependent but appeared to be mediated by separate conductance mechanisms. Thus, although bath application of nifedipine (10-30 microM) or low Ca2+, high Mg2+ Ringer blocked the ADP without affecting the ramp potential, equimolar substitution of Co2+ for Ca2+ blocked both components of the voltage response. Nominal Ca2+ Ringer containing Co2+ also blocked the HAP evoked between -55 and -68 mV. We conclude that the ADP elicited in DA neurons after blockade of apamin-sensitive Ca2+-activated K+ channels is mediated by a voltage-dependent, L-type Ca2+ channel and represents a transient form of the regenerative plateau oscillation in membrane potential previously shown to underlie apamin-induced bursting activity. These data provide further support for the notion that modulation of apamin-sensitive Ca2+-activated K+ channels in DA neurons exerts a permissive effect on the conductances that are involved in the expression of phasic activity.     

Synaptic and intrinsic control of membrane excitability of neostriatal neurons. II. An in vitro analysis

1. The effects of intrinsic membrane properties on the spontaneous and synaptically evoked activity of neostriatal neurons were studied in an in vitro slice preparation with the use of intracellular recordings. The recorded neurons did not show spontaneous action potentials at rest; depolarizing current pulses triggered a tonic firing pattern. 2. Subthreshold spontaneous depolarizing potentials (SDPs) were observed in 52% of the recorded neurons. The amplitude of these potentials at rest ranged between 2 and 15 mV, and their duration between 4 and 100 ms. The frequency and the amplitude of the SDPs were functions of the membrane potential: membrane depolarization by constant positive current increased the frequency of the SDPs and reduced their amplitude; hyperpolarization of the membrane decreased their frequency and increased their amplitude. Often, at membrane potentials more negative than -90 mV, SDPs were completely suppressed. 3. SDPs were blocked by low calcium-cobalt containing solutions. In the presence of tetrodotoxin (TTX, 1-3 microM), SDPs were completely abolished in 50% of the tested neurons; in the remaining neurons, small (1-4 mV) TTX-resistant SDPs were observed. In most of the neurons, bicuculline (BIC, 10-100 microM) and low concentrations of tetanus toxin (5-10 micrograms/ml) did not clearly affect the SDPs. Higher concentrations of tetanus toxin (100 micrograms/ml) blocked the SDPs as well as the synaptic potentials evoked by intrastriatal stimulation. 4. At resting membrane potential, intrastriatal stimulation produced a fast depolarizing postsynaptic potential (EPSP) that was reduced by BIC (10-100 microM). The relationship between EPSP amplitude and membrane potential was studied either by utilizing K(+)-chloride electrodes or by the use of cesium-chloride electrodes. In both these cases, the reversal potential for the EPSPs was between 0 and -14 mV. In cesium-loaded neurons, the decrease of the EPSP, usually observed at negative membrane potentials (below -85 mV), was clearly reduced. Internal cesium prolonged the duration of the SDPs and the EPSPs evoked by intrastriatal stimulation. 5. The relationship between spontaneous and evoked synaptic activity and membrane potential was studied in the presence of different external potassium blockers. 4-Aminopyridine (4AP, 0.1-1 mM) increased the EPSP amplitude and the frequency of the SDPs, but did not decrease membrane rectification and the shunt of the EPSPs present at negative membrane potentials. On the contrary, rectification of the membrane and the shunt of the EPSPs below -85 mV were clearly reduced by tetraethylammonium (TEA, 10-20 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple types of Na+ currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia

Small (<25 μm in diameter) neurons of the dorsal root ganglion (DRG) express multiple voltage-gated Na⁺ channel subtypes, two of which being resistant to tetrodotoxin (TTX). Each subtype mediates Na⁺ current with distinct kinetic property. However, it is not known how each type of Na⁺ channel contributes to the generation of action potentials in small DRG neurons. Therefore, we investigated the correlation between Na⁺ currents in voltage-clamp recordings and corresponding action potentials in current-clamp recordings, using wild-type (WT) and Na_V1.8 knock-out (KO) mice, to clarify the action potential electrogenesis in small DRG neurons. We classified Na⁺ currents in small DRG neurons into three categories on the basis of TTX sensitivity and kinetic properties, i.e., TTX-sensitive (TTX-S)/fast Na⁺ current, TTX-resistant (TTX-R)/slow Na⁺ current, and TTX-R/persistent Na⁺ current. Our concurrent voltage- and current-clamp recordings from the same neuron revealed that the action potentials in WT small DRG neurons were mainly dependent on TTX-R/slow Na⁺ current mediated by Na_V1.8. It was surprising that a large portion of TTX-S/fast Na⁺ current was switched off in WT small DRG neurons due to a hyperpolarizing shift of the steady-state inactivation (h _∞), whereas in KO small DRG neurons which are devoid of TTX-R/slow Na⁺ current, the action potentials were generated by TTX-S/fast Na⁺ current possibly through a compensatory shift of h _∞ in the positive direction. We also confirmed that TTX-R/persistent Na⁺ current mediated by Na_V1.9 actually regulates subthreshold excitability in small DRG neurons. In addition, we demon strated that TTX-R/persistent Na⁺ current can carry an action potential when the amplitude of this current was abnormally increased. Thus, our results indicate that the action potentials in small DRG neurons are generated and regulated with a combination of multiple mechanisms that may give rise to unique functional properties of small DRG neurons.     

Dendritic origin of late events in optical recordings from salamander olfactory bulb.

1. Optical recordings of membrane-potential changes were used to characterize the origin and properties of the electrical signals from the dendritic level in slices of the salamander olfactory bulb. 2. The optical events were correlated with field-potential waves recorded simultaneously. Both responses exhibited patterns similar to those found in other species. 3. Orthodromic stimulation evoked a compound action potential in the olfactory nerve fibers, followed by two additional principal waves (N1 and N2). These field-potential waves reflected excitatory postsynaptic potentials at the primary mitral/tufted and granule cell dendrites, respectively. 4. Extrinsic optical signals from horizontal slices stained with the pyrazo-oxonal dye RH-155 showed a characteristic sequence of depolarizing and hyperpolarizing events. All of the signals exhibited a wavelength dependence expected for this dye and were abolished in the presence of high K+ in the bath. 5. According to their time courses, depolarizing responses under normal recording conditions were divided into two components, fast and slow. Orthodromic stimuli evoked a fast presynaptic response that represents synchronous compound action potentials from olfactory nerve fibers. At subglomerular levels, additional fast responses could often be recorded at the peri/subglomerular level and in the mitral/tufted somata region. These postsynaptic responses partially coincided with the rising phase of a different depolarizing signal, a slow component characterized by its prolonged time course. 6. With orthodromic stimulation, this slow signal attained its largest amplitude in the zone between the glomeruli and the superficial part of the external plexiform layer (EPL). Antidromic stimuli evoked a signal with some similarities to the one evoked orthodromically, but originating in deeper EPL regions. 7. Slow components were characterized by their Ca dependence. Low Ca2+ medium, or calcium channel blockers, suppressed this optical component, whether evoked orthodromically, antidromically, or by direct stimulation. In addition, Ba2+ (2.5-3.6 mM) in the bath did not abolish these responses, suggesting that they do not reflect a glial depolarization in response to elevated extracellular K+ concentration ([K+]o). 8. Locally applied stimuli next to the glomerular layer elicited these signals in 5-10 microM tetrodotoxin (TTX) or in low extracellular Na+ concentration ([Na+]o) medium, but antidromic or orthodromic stimuli failed to evoke the response under these conditions. The sizes of the responses to local stimuli remained constant, but an increase in their duration was observed in either TTX or low [Na+]o. 9. gamma-Aminobutyric acid (GABA) and baclofen reduced the size of the slow components in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for initiation of calcium spikes in C-cells of the rabbit nodose ganglion

In Na⁺-free solution or in tetrodotoxin (TTX, 10^–6 M) solution, direct electrical stimulation of nodose ganglion C-cells of the rabbit elicited regenerative action potentials. The amplitude of the action potentials generated in these neurons is dependent on the external Ca²⁺ concentration ([Ca²⁺]₀). These action potentials were characterized by reduced amplitude, prolonged duration, and graded responses to changes of the stimulation intensity. Either removal of [Ca²⁺]₀ or application of organic Ca²⁺-blocking agents, diltiazem (10^–5 M) or verapamil (10^–5 M), abolished these action potentials. The conduction of action potentials along the axon was blocked in Na⁺-free solution or by application of TTX. The, present results provide evidence for the initiation of Ca²⁺-dependent action potentials in the soma membrane of mammalian nodose ganglia, and suggest that the Ca²⁺ ion plays an important role in the development of action potentials in the C-cell of rabbit nodose ganglia.     

Effects of adrenaline on the action potential of sympathetic ganglion cells in bullfrogs.

The effects of catecholamines (adrenaline, noradrenaline and isoproterenol) on ionic conductance changes during the generation of action potentials of bullfrog sympathetic and spinal ganglion cells were studied with intracellular microelectrodes. In sympathetic ganglion cells, adrenaline (3X10(-5)-1X10(-3)M) reversibly decreased the peak amplitude and positive after-potential of action potentials, and prolonged the duration of spike potentials without changes in the resting membrane potential and conductance in the Ringer solution. The maximum rates of rise and fall of spike potentials were also decreased. The action of noradrenaline was similar to that of adrenaline, but isoproterenol did not show any effects. Adrenaline (3X10(-5)-3X10(-4)M) markedly depressed the peak amplitude and maximum rate of rise of both TEA-potential and Ca-potential produced either in TEA solution containing TTX or in the isotonic CaCl2 solution. Similar actions were observed with noradrenaline but not isoproterenol. In spinal ganglion cells, catecholamines did not show any effects of the action potentials in Ringer and TEA solutions. It was concluded that adrenaline inhibited the increases in Ca2+, K+ and Na+ conductances during the generation of action potentials of sympathetic ganglion cells.     

The pathway for the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia

Intracellular and sucrose gap recording techniques were used to examine synaptically evoked potentials and the response of neurons in bullfrog paravertebral sympathetic ganglia to muscarinic agonists. These neurons were defined as either B or C cells on the basis of the conduction velocity of antidromically evoked action potentials. Following stimulation of preganglionic C-fibers in the rostral portion of the VIIIth spinal nerve, a fast nicotinic excitatory postsynaptic potential (EPSP) and a slow atropine-sensitive inhibitory postsynaptic potential (IPSP) could be recorded intracellularly in C cells of the IXth and Xth paravertebral ganglia treated with 70 microM d-tubocurarine chloride (dTC). Under these conditions, local iontophoretic application of acetylcholine (ACh) could produce a slow hyperpolarization of C cell membrane potential. ACh hyperpolarizations or slow IPSPs were not detected in ganglionic B cells. Stimulation of the preganglionic B-fibers in the sympathetic chain produced a fast nicotinic EPSP and a slow muscarinic EPSP in ganglionic B cells. A small population of C cells also received cholinergic B-fiber innervation from the sympathetic chain and exhibited a slow IPSP upon tetanic stimulation of this pathway. When curarized ganglia were examined by means of sucrose gap recording, superfusion of the muscarinic agonist, methacholine (MCh), produced an initial hyperpolarization (MChH) followed by a depolarization (MChD). Both responses were blocked by atropine and therefore presumably reflect the activation of muscarinic receptors involved in the generation of the slow IPSP and the slow EPSP, respectively. Although synaptic transmission was blocked by Ringer solution containing 4 mM Co2+, neither this solution nor 10 microM tetrodotoxin reduced the amplitude of the MChH. The MChH was slightly reduced by Ringer solution containing 0.1 mM Ca2+, however, the response could be restored by the addition of 6 mM Mg2+. These results indicate that the MChH in curarized bullfrog sympathetic ganglia results from a direct muscarinic action on ganglionic cells. This suggests that the slow IPSP is mediated by ACh released from cholinergic preganglionic fibers that make synaptic contact with ganglionic C cells.     

Jittery trains induced by synaptic-like currents in cerebellar inhibitory interneurons.

Cerebellar inhibitory interneurons respond to parallel fiber input with a characteristic train of action potentials. Here we show that the characteristics of these trains reflect the intrinsic properties of the interneurons. In in vitro cerebellar slices, the response of these neurons to synaptic-like current resembles their in vivo response to parallel fiber input-a train of action potentials characterized by a gradual increase in interspike interval and spike amplitude. A large variability in spike timing, or jitter, was observed, the last action potential emerging from a slow depolarizing wave that lasted beyond the synaptic current and was prevented by either TTX or membrane hyperpolarization. While response duration was weakly dependent on current intensity, the variability of the overall duration was closely related to the variability of the timing of the last action potential. Blocking the Ca(2+) currents or partial blockade of the delayed rectifier (TEA 2 mM) decreased the excitability, leading to a decrease in the duration and variability of the response and increasing its dependence on stimulus intensity. Increased duration and variability was observed in the presence of Cs(+) ions (5 mM) that blocked an h-like current. We conclude that a persistent Na(+) current governs the duration of the response, whereas the synaptic current and the spiking mechanism shape its pattern. The large variability between trials is due to the stochastic nature of the persistent Na(+) current. Thus unless precise timing is achieved by a network of interconnected neurons, these results vote against temporal coding as a player in the cerebellar computational processing.     

Interaction between longitudinal and circular muscle in intestine of cat

1. Slow waves recorded from isolated longitudinal muscle averaged 13 mV and had slow rate of rise (0.04 V/sec) whereas when recorded from intact segments the amplitude averaged 27 mV and the rate of rise was more rapid (0.09 V/sec), often with a notch between the initial peak and the plateau. Membrane potentials of longitudinal muscle were similar in isolated and intact preparations (- 66 mV). Resting potentials of circular muscle averaged - 67 mV.

2. Small bundles of circular muscle tested in the double sucrose gap produced activity, either spontaneously or in response to stimulation, which fell into three categories: fast spikes (50-200 msec duration), slow spikes (1-5 sec duration), and small graded responses. The duration of fast spikes could be increased severalfold by the addition of TEA; the graded responses were converted to full-sized spikes by TEA.

3. Treatment of circular muscle with Ca-free Krebs solution eliminated spikes, and in intact preparations reduced the amplitude and rate of rise of slow waves and eliminated the notch on slow waves.

4. Current—voltage curves of longitudinal muscle show delayed rectification in the depolarizing quadrant; similar curves of circular muscle show anomalous rectification, i.e. a region where a very small current causes a large voltage change.

5. Non-polarized electrotonic coupling between longitudinal and circular layers indicates low-resistance pathways. Apparent space constants of longitudinal muscle are greater when attached to circular muscle than when isolated.

6. It is concluded that small slow potentials originate rhythmically in longitudinal muscle, that these spread passively to circular muscle where a regenerative amplification occurs which depends on Ca conductance and the amplified slow waves spread back to the longitudinal layer. In the intact intestine pacemaking is, therefore, separate from propagation and the circular muscle provides the bulk of depolarizing current for propagation.

     

Membrane properties and synaptic responses of the guinea pig nucleus accumbens neurons in vitro

1. The membrane properties and synaptic responses of guinea pig nucleus accumbens neurons in vitro were studied with intracellular recording methods. 2. The population of neurons could be divided into groups of low (20-60 M omega, average 46.5 M omega) and high (60-180 M omega, average 96.5 M omega) input resistance. The resting membrane potential in both groups was approximately -70 mV. 3. Other membrane properties were quite similar in both groups. Inward rectification occurred at potentials more negative than -80 mV; this was blocked by Cs+ (2 mM). Membrane potential oscillations were observed at potentials between -65 and -55 mV; these were blocked by tetrodotoxin (TTX, 0.5 microM). Outward rectification occurred at potentials less negative than -45 mV; this was depressed by tetraethylammonium (TEA, 10 mM). 4. Action potentials elicited by small depolarizing current pulses (2-5 ms, 0.3-0.5 nA) were approximately 95 mV in amplitude and 1.0 ms in duration. The afterhyperpolarization following each action potential was less than 30 ms in duration, and no accommodation of action-potential discharge was seen at frequencies up to 40 Hz. The action potentials were reversibly blocked by TTX (0.3 microM). In addition, TTX-insensitive, Ca2+-dependent spikes were evoked by passing larger and more prolonged current pulses (greater than 40 ms, greater than 0.5 nA) across the membrane. 5. Focal electrical stimulation of the slice surface with low intensity (1 ms, less than 10 V) elicited excitatory postsynaptic potentials (EPSPs) in neurons of both high- and low-resistance groups. The reversal potential (+10.2 mV) for the EPSPs was close to the reversal potential (+7.7 mV) of the responses to glutamate applied in the superfusing solution. The N-methyl-D-aspartic acid (NMDA) receptor antagonists, D-alpha-aminoadipic acid (1 mM) and DL-2-amino-5-phosphonovaleric acid (DL-APV, 250 microM), reversibly depressed the EPSP; the glutamate uptake inhibitor, L-aspartic acid-beta-hydroxamate (50 microM), or removal of Mg2+ from the superfusate, augmented the EPSP. 6. When the intensity of the focal stimulus was increased (1 ms, greater than or equal to 10 V), a second larger depolarizing response (duration, 800 ms to 2 s) could be evoked in addition to the smoothly graded EPSP. This was seen only in cells of the high-resistance group (90-130 M omega).(ABSTRACT TRUNCATED AT 400 WORDS)