Regulation of Action Potential Size and Excitability in Substantia Nigra Compacta Neurons: Sensitivity to 4-Aminopyridine

S. Nedergaard

Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nedergaard, S.. Regulation of Action Potential Size and Excitability in Substantia Nigra Compacta Neurons: Sensitivity to 4-Aminopyridine. J. Neurophysiol. 82: 2903-2913, 1999. Slow, pacemaker-like firing is due to intrinsic membrane properties in substantia nigra compacta (SNc) neurons in vitro. How these properties interact with afferent synaptic inputs is not fully understood. In this study, intracellular recordings from SNc neurons in brain slices showed that spontaneous action potentials (APs) were attenuated when generated from lower than normal threshold. Such APs were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and could be related to non-N-methyl-D-aspartate (NMDA) receptor-mediated spontaneous excitatory postsynaptic potentials (EPSPs). The AP attenuation was reproduced by stimulus-evoked EPSPs and by current injections to the soma. APs evoked from holding potentials between -40 and -60 mV were reduced in width by Cd2+ (0.2 mM). Tetraethylammonium chloride (TEA, 10 mM) or 4-aminopyridine (4-AP, 5 mM) increased the AP width. However, at more negative holding potentials, Cd2+ and TEA were inefficacious, whereas 4-AP enlarged the AP, partly via induction of a Cd2+-sensitive component. A monophasic afterhyperpolarization (AHP), following attenuated APs, was little affected by either Cd2+ or TEA, but inhibited by 4-AP, which induced an additional, slow component, sensitive to Cd2+ or apamin (100 nM). The AP delay showed a discontinuous relation to the amplitude or slope of the injected current (delay shift), which was sensitive to low doses of 4-AP (0.05 mM). The initial time window before the delay shift was longer than the rise time of EPSPs. It is suggested that a 4-AP-sensitive current prevents or postpones discharge during slow depolarization's, but allows direct excitation by fast EPSPs. Fast excitation leads to AP attenuation, primarily due to strong activation of 4-AP-sensitive current. This seems to cause inhibition of the Ca2+ current during the AP and reduction of Ca2+-dependent K+ currents. Together, these properties are likely to influence the excitability and the local, somatodendritic effects of the AP, in a manner that discriminates between firing induced by the intrinsic pacemaker mechanism and fast synaptic potentials.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal responses to afferent inputs depend on nonlinear properties provided by voltage-dependent membrane conductances (Llinás 1988). In cells where the conductances interact to generate spontaneous spikes, the diversity of possible outcomes of a given stimulus could be particularly high. The mesencepalic dopaminergic (DA) neuron is an example of such a cell type. When recorded in vivo these neurons show a slow irregular firing or a burst firing pattern (Clark and Chiodo 1988; Grace and Bunney 1984a,b; Nissbrandt et al. 1994; Tepper et al. 1995). These firing patterns are believed to depend on afferent synaptic inputs, and evidence is accumulating that excitatory pathways, using glutamate as neurotransmitter (Smith et al. 1996), are involved in the control of firing in DA substantia nigra pars compacta (SNc) cells (Charlety et al. 1991; Chergui et al. 1994; Christoffersen and Meltzer 1995; Overton and Clark 1992). In the in vitro slice preparation, however, the spontaneous activity is characterized by a regular, pacemaker-like discharge, which has been attributed entirely to intrinsic membrane properties (Grace and Onn 1989; Harris et al. 1989; Nedergaard et al. 1993; Yung et al. 1991). This discrepancy is explained if the synaptic drive on the in vitro cells is low (due to the truncation of afferent fibers during slice preparation), and implies that the in vivo activity could represent the integration of both an intrinsic and an extrinsic influence on the discharge. Electrophysiological studies from DA neurons in vitro have shown that synaptic stimulation (Johnson and North 1992; Mereu et al. 1991) or exogenous application of glutamate agonists (Seutin et al. 1990; Wang and French 1993) elicits responses composed of both N-methyl-D-aspartate (NMDA)- and non-NMDA receptor-mediated events. However, it is not yet clear how, or to which extent, discrete excitatory inputs interfere with the background spontaneous activity and vice versa.

Unpublished observations in this laboratory have indicated that pacemaker discharge involves a large variability in the size of individual action potentials (APs) in the cell soma. The reason for this variability is not obvious from the assumption that spike generation is at a fixed location and entirely related to an underlying slow oscillatory potential (Nedergaard et al. 1993). The present study was undertaken to describe more closely the spontaneous AP variability and to investigate the possible contribution from afferent synaptic inputs and intrinsic properties to the regulation of the AP.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Brain slice preparation and intracellular recording

Mesencephalic brain slices (coronal, 400 µm thick) were prepared from albino guinea pigs of either sex (250-350 g) as described previously (Nedergaard et al. 1993). The animals were deeply anesthetized with chloroform in an airtight container and killed by decapitation. The brain was removed, and a block of tissue containing the midbrain was isolated and used for slice preparation. Slices were transferred to a HEPES solution (see Drugs and solutions), bubbled with 95% O2-5% CO2, and stored at room temperature for at least 1 h before use. In the recording chamber the slice surface was at the interface between a humidified atmosphere of 95% O2-5% CO2 at 32-33°C and a standard perfusion medium (see below). Flow rate was 1.5 ml/min.

Intracellular recordings were made via glass microelectrodes, filled with 3 M potassium acetate (resistance 50-90 MOmega ), and connected to an AXOCLAMP 2A bridge amplifier (Axon Instruments, Foster City, CA). Signals were digitized and stored on videotape. At the end of each experiment the electrode was retracted a few micrometers from the cell and the extracellular potential recorded. Focal stimulation was performed by means of a bipolar, insulated platinum wire electrode, placed on the slice surface ventral to the recording site. Current (100-500 µs, variable strength) was delivered by a stimulus isolation unit (ISOLATOR 10, Axon Instruments).

Drugs and solutions

The HEPES storage solution contained (in mM) 120 NaCl, 2.0 KCl, 1.25 KH2PO4, 2.0 MgSO4, 2.0 CaCl2, 20 NaHCO3, 6.7 HEPES acid, 2.6 HEPES salt, and 10 glucose. The standard perfusion medium contained (in mM) 132 NaCl, 1.8 KCl, 1.25 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 20 NaHCO3, and 10 glucose. The following drugs were kept in stock solutions and dissolved in the perfusion medium to the final concentration immediately before use: bicuculline [10 µM, prepared in 10 mM stock (0.06 N HCl) from the free base], 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), D,L-2-amino-5-phosphonovaleric acid (APV, 50 µM), 4-aminopyridine (4-AP, 0.05-10 mM), CdCl2 (200 µM), tetraethylammonium chloride (TEA, 10 mM), and apamin (100 nM).

Data analyses

Signals were analyzed off-line on a PC computer, using SIGAVG software (CED, Cambridge, UK). AP thresholds were determined as the membrane potential at the point where the rate of depolarization started to increase above the baseline rate. The AP threshold and height were expressed in absolute voltage (extracellularly recorded voltage set to 0 mV). The threshold variation of spontaneous APs was calculated by subtracting the mean threshold from the most negative threshold, recorded in a period of 60-120 s. The AP duration was measured as the width at half-amplitude between threshold and peak. The rates of rise and fall were calculated as the average slope between 30 and 70% amplitude from threshold to peak. Spike afterhyperpolarization (AHP) measurement was made on visually identified subcomponents and the peak amplitude expressed in absolute voltage. The membrane input resistance (Rin) was calculated from the voltage deflection at the end of a 1-s negative current pulse of 0.1 nA. Unless stated otherwise, averaged data were expressed as means ± SE and Student's t-test used for statistical evaluation.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell identification

Intracellular recordings were made from SNc neurons, located lateral to the accessory optic tract. A total of 112 neurons were selected for investigation, based on an AP width of >1.5 ms at threshold, a slow, regular firing pattern, and a prominent sag during hyperpolarizing current pulses, which are distinguishing criteria for DA neurons in this preparation (Grace and Onn 1989; Yung et al. 1991). Neurons with AP amplitudes of <45 mV beyond threshold, or inability to fire repetitively in response to a 1-s long depolarizing current pulse, were not included. Most neurons (n = 95) fired in a slow (1.6 ± 0.9 Hz; mean ± SD), pacemaker-like rhythm during passive recording. The remaining 17 neurons were silent at rest.

Characteristics of spontaneous APs

Spontaneous AP properties were analyzed in 49 neurons. The majority of APs had similar threshold and shape and were followed by a biphasic AHP, with a fast (fAHP) and a slow (sAHP) component. In most cells, however, some of the APs did not conform to the normal characteristics. These (referred to as variable APs) were generated at low and variable thresholds (ranging from a few millivolts to >15 mV negative to the average threshold), reached less positive overshoot potentials, and had shorter duration than the average AP (Fig. 1, A and B). The AHP following the variable APs had an early peak and fast decay with no distinct sAHP (Fig. 1A). Individual APs showed a gradual decrease in overshoot and half-width in proportion to their threshold (Fig. 1C). Statistical comparison (see Table 1) between normal and variable APs [with a large (>8 mV) difference in threshold] showed a significant difference in both overshoot and half-width. The reduced width of the variable APs was reflected in a faster rate of fall and a reduced ratio of rate of rise to rate of fall. Their mean rate of rise was slightly higher than the control APs, but this difference was not statistically significant. The fAHP following the variable APs reached a similar peak potential as control APs.



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Fig. 1. Normal and variable action potentials (APs) in a substantia nigra compacta (SNc) neuron. A: record of spontaneous discharge. One AP (b) is generated from a hyperpolarized membrane potential at a short interval from the preceding normal AP (a). B: the APs marked in A shown enlarged. The differentiated records are shown in inset. Note similar maximum rate of rise, but faster rate of fall of b compared with a. C: plots from the same cell of overshoot and half-width vs. threshold of all APs generated in 120 s (n = 115). Bicuculline 10 µM was present.


                              
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Table 1. Characteristics of spontaneous APs

Addition of the GABAA receptor blocker bicuculline (10 µM) had no detectable effect on the discharge properties. The glutamate receptor antagonist CNQX (10 µM) applied alone, or together with APV (50 µM), caused a marked reduction of the AP variability (Fig. 2A). The threshold variation of spontaneous APs (see METHODS) in 46 cells in the absence of CNQX ranged between 1.0 and 16.7 mV (mean, 7.7 ± 0.5 mV). This value was reduced from 8.9 ± 1.3 mV to 2.7 ± 0.5 mV in seven cells exposed to CNQX (Fig. 2B). Application of APV alone (n = 6) had little and inconsistent effects on the variation in AP threshold and size (Fig. 2B).



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Fig. 2. Effects of glutamate receptor antagonists on AP characteristics. A: samples of spontaneous APs from a control period (bicuculline 10 µM present) and during coapplication of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and D,L-2-amino-5-phosphonovaleric acid (APV; 50 µM). Note reduction in threshold variation and regularization of the AP shape. B: the maximal negative deviation from the mean threshold measured during steady-state firing (60-120 s), before (control) and during application of either 50 µM APV or 10 µM CNQX. Each pair of values represents one experiment.

Spontaneous excitatory postsynaptic potentials (EPSPs)

These observations indicate that the AP shape is influenced by spontaneous EPSPs. To verify the possible existence of such potentials, voltage records sampled over several minutes were examined in each cell. Isolated transient depolarization's, with a rising slope fast enough to compare with the initial phase of full spikes, were found in 46 of the 49 cells (Fig. 3). These putative EPSPs occurred at irregular frequencies (ranging from <1 to >10 per min), had a time-to-peak between 1.0 and 3.0 ms, and varied in height in individual cells and between cells from 2 to 20 mV. They persisted during hyperpolarization below firing threshold (n = 20). The transients were not found in records of similar length in the presence of 10 µM CNQX (n = 7). A quantitative comparison between the threshold variation of APs and the transient depolarization's was not attempted, due to the large variability in frequency and size.



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Fig. 3. Putative spontaneous excitatory postsynaptic potentials (EPSPs). A: selected voltage traces obtained during slow discharge (left) and during hyperpolarization below the threshold for regular firing (right). Fast, subthreshold depolarization's (a and d) and full APs (b and c; truncated) are shown enlarged in B. Bicuculline (10 µM) and APV (50 µM) were present throughout. The subthreshold depolarization's were not seen after subsequent application of 10 µM CNQX (not illustrated).

Comparison of EPSP-evoked and current-pulse-evoked APs

In the presence of 10 µM bicuculline, EPSPs were produced by focal stimulation. Discharge induced by individual EPSPs consisted of a single AP, generated at or near the EPSP peak. The EPSP rise time (from beginning of depolarization to peak) was on average 2.8 ± 0.2 ms in 22 neurons held at membrane potentials between -60 and -70 mV (mean: -62 ± 1 mV; bicuculline 10 µM present). The EPSP-evoked APs were attenuated and had fast decaying AHPs, both when the membrane potential was kept stable by hyperpolarizing holding current (Fig. 4A) and during free firing. In the latter case, an unattenuated AP with normal AHP was evoked in a few trials, where the stimulation coincided with a potential close to the normal AP threshold. The degree of attenuation (reduced overshoot and half-width) varied with the prestimulus voltage similar to the variable APs. At -60 mV, or below, the apparent firing threshold was typically 5-10 mV negative to normal. In the presence of 50 µM APV the EPSP rise time was unaltered, although the amplitude was sometimes slightly reduced (n = 6). The evoked APs failed, together with a complete block of the EPSP, after application of 10 µM CNQX (Fig. 4A; n = 8).



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Fig. 4. Comparison of EPSP- and current pulse-evoked APs. A, left: spontaneous AP (s) and AP generated by evoked EPSP (e) from a hyperpolarized potential (stimulus artifact denoted by star) in the presence of bicuculline (10 µM) and APV (50 µM). Record after addition of 10 µM CNQX is included. A, right: APs evoked by current pulses of 1-10 ms duration. Note similar threshold and shape of EPSP- and current-evoked APs. B: responses in the same cell to longer depolarizing current pulses (50-200 ms), adjusted to the minimal strength that gave an AP. The APs are aligned to the right. Note decreased overshoot and duration with decreasing latency from the pulse onset, and the lower threshold of the early AP on the 50-ms pulse.

In the same experiments depolarizing current pulses were adjusted to mimic EPSPs. Single APs, evoked by short (1-10 ms) pulses, resembled EPSP-induced APs, when compared in the same cell, at similar membrane potential (Fig. 4A). Weaker pulses, which failed to fire the cell within ~10 ms, needed to be sustained for much longer periods (>= 80 ms) to evoke an AP. These long-delay APs were normal-sized and were generated near the threshold of spontaneous APs (Fig. 4B).

Effects of ion channel blockers on the AP and AHP

Ion channel blockers were used to examine the possible contribution of intrinsic conductances to the AP variability. To minimize the interference from spontaneous synaptic activity, these experiments were done in the presence of CNQX (10 µM), APV (50 µM), and bicuculline (10 µM). Injection of constant current (positive or negative, depending on the resting state of the cell), combined with brief depolarizing current pulses (2-4 ms), were used to generate APs from membrane potentials between -40 and -75 mV.

The Ca2+ channel blocker Cd2+ (200 µM) significantly reduced the half-width of APs generated at -40 mV (n = 5). This effect was associated with an increased rate of fall and disappearance of the shoulder on the falling phase of the AP (Fig. 5Aa). Hyperpolarization between -40 and -60 mV led to a progressive decrease in the effect of Cd2+. From -60 mV there was no detectable effect on the AP duration (Fig. 5, Aa and Ba). The AP height was not altered significantly by Cd2+ at any potential, although a slight increase in the mean overshoot was noted at holding potentials negative to -60 mV (Fig. 5Ba).



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Fig. 5. Voltage-dependent effects of ion channel blockers on the AP. A: APs generated from -40 mV (top traces) and from -65 mV holding potential following a brief (3-4 ms) current pulse (bottom traces). Records from 3 different cells are shown before (Con.) and during application of either 200 µM Cd2+ (a), 10 mM tetraethylammonium (TEA; b), or 5 mM 4-aminopyridine (4-AP; c), or combined as indicated. Ba-Bc: plots of AP overshoot and half-width vs. holding potential in control periods () and during application of blockers (open circle ); Cd2+ (200 µM, n = 4), TEA (10 mM, n = 5), 4-AP (5 mM, n = 5). Error bars = SE.

In separate experiments, TEA (10 mM) caused a broadening of the AP at -40 mV (half-width increased by >100%; n = 7), and induced a plateau-like delay during the repolarizing phase (Fig. 5Ab). The AP height was not consistently affected. The TEA effect decreased gradually with holding potentials between -40 and -60 mV, and became undetectable negative to -60 mV (Fig. 5, Ab and Bb, n = 7). Addition of 200 µM Cd2+ in the presence of TEA blocked the plateau induced from -40 mV, and reduced the AP half-width to less than control (Fig. 5Ab). Coapplication of 200 µM Cd2+ and 10 mM TEA had no effect on the half-width of APs generated from holding potentials negative to -60 mV (Fig. 5Ab; n = 5).

The response to 5 mM 4-AP was tested in 11 neurons. Here, a lowering of the spontaneous firing threshold (3-5 mV) was noted in four of seven active neurons after exposure to 4-AP. Compared to control APs, evoked from equal potentials, the half-width increased in all neurons exposed to 4-AP. This effect involved a decreased rate of fall, starting from the beginning of the repolarization, near the AP peak, and without a distinct plateau phase as observed with TEA (Fig. 5Ac). The effect of 4-AP persisted during hyperpolarization below -60 mV (Fig. 5, Ac and Bc). The relative increase in half-width was between 70 and 90% at all membrane potentials (n = 5). The AP height was largely unaltered by 4-AP at the more depolarized potentials. However, from -60 mV the height increased significantly in response to 4-AP, an effect that became larger with further hyperpolarization. Thus the strong voltage dependency of the AP overshoot, seen under control conditions, was clearly diminished in the presence of 4-AP (Fig. 5Bc). In three experiments where 4-AP was already present, addition of Cd2+ caused a reduction of the AP width. The latter effect of Cd2+ persisted at hyperpolarized holding potentials (Fig. 5Ac).

Figure 6 shows the effects of ion channel blockers on the AHP of individual spikes. The biphasic AHP generated around -40 mV was highly sensitive to 200 µM Cd2+, which reduced the amplitude of the early peak (fAHP) and blocked the sAHP component (Fig. 6Aa). In 10 mM TEA alone, the fAHP decreased in amplitude, whereas the sAHP persisted (Fig. 6Ab). On the other hand, 4-AP (5 mM) had little and variable effects on this AHP; of 10 cells examined, the fAHP did not change to any detectable degree (n = 4, example in Fig. 6Ac), or showed a small increase or decrease (by 2-4 mV, n = 6). The variation in the differences was not statistically significant when tested by the Wilcoxon test for pair differences (2alpha  > 0.05). The sAHP was not consistently altered by 4-AP. When evoked from -60 mV, or below, the monophasic AHP, evoked in normal medium, showed little sensitivity to Cd2+ alone (Fig. 6Aa; n = 5). Similarly, TEA did not alter the overall shape of this AHP (Fig. 6Ab) and had inconsistent and nonsignificant effects on the amplitude (unaltered in 4 of 7 cells; decreased by 2-3 mV in 3 cells; Wilcoxon test: 2alpha  > 0.05). Coapplication of TEA and Cd2+ had no further effect (Fig. 6Ab; n = 5). In contrast, 4-AP (5 mM) induced two distinct changes in this AHP. First, the peak amplitude was markedly reduced in all cells tested (range: 5-18 mV; n = 11). The effect was not complete, as an early AHP was still discernible in most cells held between -60 and -70 mV (Fig. 6Ac). Second, an additional, late component developed in the presence of 4-AP, which was reflected in a delayed decay phase and prolongation of the AHP (10 of 11 cells). The entire AHP complex, in the presence of 4-AP, was inhibited by subsequent application of 200 µM Cd2+ (Fig. 6Ac; n = 4). The 4-AP-induced, late AHP component was blocked by 100 nM apamin (Fig. 6B; n = 4), and the early peak was markedly reduced in 3 of 3 cells by 10 mM TEA, added in the presence of 4-AP (Fig. 6B).



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Fig. 6. Voltage-dependent effects of ion channel blockers on the spike AHP. A: AHPs following APs elicited from -40 mV (top traces) and by brief current pulses from -65 mV (bottom traces). Superimposed sweeps are taken before (Con.) and during application of blockers as indicated. Note the Cd2+-sensitive, slow component, generated in the presence of 4-AP. Records (a-c) are from 3 different experiments. B: AHPs (at -58 mV holding Vm) recorded in control medium (Con.), with 5 mM 4-AP present (4-AP), and after subsequent addition of either 100 nM apamin (4-AP + Apamin, top record), or, in another neuron, 10 mM TEA (4-AP + TEA, bottom record). Note block of the slow component in the presence of apamin, and reduction of the fast component by TEA.

Application of TEA alone caused a consistent increase in the Rin (on average by 13%, n = 7). Neither 4-AP (n = 11) nor Cd2+ (n = 5) had detectable effects on the Rin.

4-AP effect on action potential delay and threshold

As indicated above (Fig. 4), the AP attenuation seems to rely on the depolarizing current being strong enough to give a short delay. The possibility that the delay itself is influenced by 4-AP-sensitive processes was next examined. AP delays, monitored from the onset of positive current steps from a holding potential between -60 and -70 mV, were inversely related to the current intensity. However, with high intensity, the delay shifted abruptly from an average of 82 ± 8 ms to 7 ± 1 ms (n = 18; mean holding Vm = -65 ± 1 mV; Fig. 7A). Logarithmic plots of delay versus current intensity showed an almost linear relationship on either side of the delay shift; the slope on the left being consistently smaller than on the right side of the shift (Fig. 7C, top graph). In the presence of 4-AP (0.05-10 mM, n = 15), AP delays were generally reduced (Fig. 7A) and accompanied by a lowered firing threshold (Fig. 7B). These effects were dose dependent and were already marked at the lowest 4-AP concentration used (0.05 mM; Fig. 7B). Furthermore, with doses between 0.05 and 0.5 mM, the length of the delay shift decreased dramatically, and the delay-current relationship approximated a uniform slope (Fig. 7C). A quantitative estimate of the delay shift at different 4-AP concentrations was obtained in six cells (calculations made from linear regression lines; see Fig. 7 legend). On average, the control value was reduced by 60% in the presence of 0.05 mM, and by 93% in 0.5 mM 4-AP (Fig. 7D, holding Vm: -65 ± 1 mV, n = 6). A further increase in dose gave a small additional effect (99% reduction obtained in 10 mM 4-AP). For comparison, the dose dependency of the 4-AP effect on the AP half-width was examined in the same cells at similar holding potentials. Here, the broadening of the AP became detectable at 0.2 mM 4-AP and increased progressively with the concentration (Fig. 7D). The response showed no sign of saturation at high doses (415% increase in half-width in 10 mM 4-AP, n = 6).



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Fig. 7. Effects of 4-AP on the AP delay. A: responses to current steps of incrementing intensity from a holding potential of -60 mV in a control period (left), and after application of 5 mM 4-AP (right). Only the 1st AP in each sweep is shown. Note the abrupt shortening of AP delay (delay shift) in the control records at high current intensity, and the reduction of delays in the presence of 4-AP. B: APs evoked by a 0.1-nA current step in control medium (Con.; threshold of -41 mV indicated by broken line) and during application of 0.05, 0.5, and 5 mM 4-AP. C: logarithmic plots of AP delay vs. current strength in the absence (control) and presence of 4-AP (conc. in mM indicated) from the same cell as in A and B. Linear regression lines in each plot represent delay values of >30 and <10 ms, respectively. D: the magnitude of delay shift and AP half-width as function of the 4-AP concentration (averages ± SE from the same 6 cells). Open circles indicate control values. The delay shift was measured as the vertical distance between the regression lines (as in C) at the place (delay value on the bottom line) where the shift occurred in the control graph.

The possible dependency of the AP delay and threshold on the rate of depolarization was tested by varying the slope of the injected current. Under these conditions the AP delay shifted between 45 ± 1 ms and 12 ± 1 ms at an average current slope of 102 ± 15 nA/s. (range: 58-159 nA/s; holding Vm: -65 ± 2 mV; n = 7). This delay shift was inhibited or blocked by 4-AP, depending on the dose (0.05-10 mM; n = 3; Fig. 8). In control medium, the threshold of the first AP decreased gradually with increasing current slope in the range below the critical value for inducing the delay shift. Higher slopes gave less variation in threshold (Fig. 8B). In the presence of 4-AP, the threshold obtained during slow currents were markedly lowered. However, with increased rate of depolarization the threshold approximated the control values (Fig. 8B). Similar effects were seen in three cells tested with ramp depolarization. The threshold of spontaneous APs, generated at rest, was much less affected by 4-AP than the first AP during the ramp (see Fig. 8B). In neurons activated with square depolarizations, the average threshold of APs evoked at 5 ms delay in control medium (-45.7 ± 1.5 mV) was not different from those found in the presence of 5 mM 4-AP (-47.0 ± 0.7 mV, n = 6, P > 0.05). However, at 100 ms delay, the threshold was lowered by ~13 mV in the presence of 5 mM 4-AP (from -40.3 ± 1.3 to -53.5 ± 1.3 mV, P < 0.01).



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Fig. 8. Effects of current ramps on AP delay and threshold. A: action potentials generated by current ramp injections with different slope before (control) and during application of 5 mM 4-AP. Holding potential, -65 mV. B: from the same experiment the AP delay (top graph) and AP threshold (bottom graph) are plotted against the ramp slope in control medium () and in 5 mM 4-AP (open circle ). Lines in bottom graph indicate the AP threshold during spontaneous firing at rest in control recording (- - - -) and in the presence of 4-AP (···).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EPSP-induced APs

In this study, SNc neurons with membrane properties characteristic of DA cells were examined. Spontaneous APs displayed large variabilities, which decreased in the presence of CNQX. Putative non-NMDA receptor-mediated EPSPs were found in the same cells. Both observations are in line with previous demonstration of a spontaneous excitatory input in this preparation (Mereu et al. 1991). It is conceivable that the observed effects were due to activity of glutamatergic interneurons located within the confines of the slice; however, evidence for the existence of such neurons is lacking. Midbrain DA cells receive afferent glutamatergic inputs from several external sources, including the cerebral cortex (Sesack et al. 1989), the subthalamic nucleus (Hammond et al. 1978; Kita and Kitai 1987), and the pedunculopontine nucleus (Charara et al. 1996; Scarnati et al. 1986). The effects seen here could reflect some form of spontaneous release of glutamate from terminals of cut fibers from any of these structures. Assuming that a spontaneous EPSP results from transmitter release from a single terminal or terminals of a single fiber, and that the variable APs were generated directly by such EPSPs, it seems possible that activity in one afferent axon can be sufficient to activate the postsynaptic cell. In the light of the constraints on spontaneous firing exerted by intrinsic membrane conductances (Grace and Onn 1989; Nedergaard et al. 1993; Shepard and Bunney 1991), such high excitability state seems surprising. One explanation could be that these terminals were localized close to the AP initiation site at a distance from the soma. Indeed, the efficacy of an input has been shown to depend on its location relative to the initial segment of the axon, which often emerges from a dendrite (Häusser et al. 1995). Furthermore, dendritic APs are facilitated by EPSPs in this preparation (Nedergaard and Hounsgaard 1996). Whether such effects play a significant role here is unsettled. Stimulus-evoked EPSPs had similar, voltage-dependent effects on the AP shape and were found to fire the cell from a lower apparent threshold than spontaneous APs. Both effects were also seen with current pulses in the soma. This indicates that the place of origin of the depolarization is not the only critical factor. Alternatively, as discussed below, the excitability could be variable, or conditional, depending on the dynamics of certain membrane conductances.

The finding that CNQX alone blocked discharge related to both spontaneous and stimulus-evoked EPSPs indicates that non-NMDA receptors are involved in either type of stimulation.

Voltage-dependent contribution of ionic currents to the AP

Current-pulse-evoked attenuated APs has been noted previously (Grace 1990; Grace and Onn 1989; Nedergaard and Greenfield 1992), but not systematically characterized. The contribution of different ionic currents to the AP shape was found here to be highly dependent on the membrane potential. Near the normal threshold of -40 mV, 200 µM Cd2+ caused an accelerated repolarization and a reduced AHP, which suggests that voltage-dependent Ca2+ current underlies a depolarizing component in the late phase of the AP and mediates activation of outward currents involved in the AHP. The prolonged AP and reduced fAHP in the presence of 10 mM TEA indicates that the Ca2+-dependent K+ current Ic, and possibly a delayed rectifier (Silva et al. 1990) contribute significantly to the AP repolarization and to the fAHP.

At -40 mV threshold the response to 5 mM 4-AP involved a slowed repolarization with insignificant change in the AHP. A 4-AP-sensitive, A-type K+ current has previously been demonstrated in acutely dissociated (Silva et al. 1990) and cultured (Liu et al. 1994) DA neurons from the rat, and, recently, in slices from the mouse (Bruns et al. 1998). This current activates at potentials positive to -55 mV, and steady-state inactivation begins at -80 and is complete at -40 mV (half-maximal at -65 mV). The broadening effect found here at -40 mV threshold could therefore indicate the presence of a 4-AP-sensitive outward current, distinct from the A-type current. Depolarization-activated K+ channels are composed of alpha -subunits encoded by four gene families, Kv1-Kv4. Homomeric channels formed by Kv1.4, 3.4, 4.1, 4.2, or 4.3 subunits display rapid inactivation and are sensitive to 4-AP, properties similar to native A-type currents (Baldwin et al. 1991; Schröter et al. 1991; Serôdio et al. 1994, 1996; Stühmer et al. 1989). The Kv2.1, 2.2, 3.1, and 3.2 channels are slowly inactivating (delayed rectifier type), as are most members of the Kv1 group [Kv1.1, 1.2, and 1.5 acquire rapid inactivation when co-expressed with beta 1 subunits (Heinemann et al. 1996; Rettig et al. 1994)]. The Kv3 channels are highly sensitive to both TEA and 4-AP (Rettig et al. 1992), whereas Kv2 channels show intermediate sensitivity to TEA, and Kv2.1 is also sensitive to 4-AP (at least in the rat) (Pak et al. 1991). The involvement of Kv3 channels here is questionable, because an in situ hybridization study failed to demonstrate mRNA encoding any of the known Kv3 related proteins in rat SNc (Weiser et al. 1994). The lacking effects of 4-AP on the AHP could reflect a fast deactivation rate of the 4-AP-sensitive current. This would not compare to the relatively long deactivation time constant characteristics of Kv1 and Kv2 channels (>= 15 ms) (Grissmer et al. 1994; Martina et al. 1998). However, in the present study additional recruitment of Ca2+-activated K+ current, secondary to the AP broadening, could have obscured an effect on the AHP. Hence it is conceivable that a 4-AP-sensitive delayed rectifier could contribute significantly to the AP repolarization at -40 mV threshold. Nevertheless, this issue needs further clarification because the sustained outward current evoked from -40 mV was unaffected by 5 mM 4-AP, as reported by Silva et al. (1990). Their study was made on acutely dissociated cells from 5- to 7-day-old rats, which might deviate in channel composition from the adult guinea pig. Finally, some contribution from A-type channels at -40 mV threshold should not be discounted, because these spikes were triggered during ongoing spontaneous depolarizations from more negative potentials. The time constant of inactivation of the A-type current could be sufficiently long (between 30 and 53 ms) (Bruns et al. 1998; Silva et al. 1990) to leave an appreciable amount of current available for activation at the time of the AP.

A lack of effect of 5 mM 4-AP on the AP width has been reported in rat DA neurons (Grace 1990), which is in contrast to the present findings. However, apart from a possible species difference between rat and guinea pig, these data might be incomparable, because the APs were measured here with respect to the offset membrane potential (given the high voltage sensitivity of the AP width), shown to be lowered by 4-AP during free firing (Grace 1990; and present study).

The inefficacy of Cd2+ and TEA, seen during hyperpolarization, indicates that Ca2+-dependent currents and TEA-sensitive delayed rectifiers play little role in the shaping of the AP and AHP evoked from potentials negative to -60 mV. The accelerated repolarization and monophasic AHP emerging from subthreshold potentials (negative to -40 mV) seems to reflect the progressive recruitment of outward current, sensitive to millimolar concentrations 4-AP and insensitive to 10 mM TEA. These characteristics correlate well to the known properties of the A-type current. It is likely that A-type channels underlie an increasing portion of the 4-AP-sensitive current at hyperpolarized potentials, and hence play a major role in the AP attenuation. Evidence from other studies indicate that Kv4 channels may contribute to this current: first, the rate of inactivation is shown to be voltage independent (Silva et al. 1990), a distinguishing characteristic of Kv4 channels (Serôdio et al. 1994), and, second, mRNA transcripts encoding Kv4.3 subunits has recently been identified in the SNc region (Serôdio and Rudy 1998). The Kv1.4 protein is abundant in the substantia nigra, but mRNA for Kv1.4 was not localized in this region and could originate from projecting neurons within the striatum (Sheng et al. 1992).

Regulation of Ca2+-dependent currents during the AP

At hyperpolarized holding potentials the broadening effect of 4-AP involved a recruitment of Ca2+ current, because a partial reversal of the effect was obtained after Cd2+ application (Fig. 5). Furthermore, the AHP evoked in the presence of 4-AP had both an early component, sensitive to Cd2+ or TEA, and a slow component sensitive to Cd2+ or apamin (Fig. 6). The latter finding corresponds to the pharmacological profile of the biphasic AHP at normal threshold (Nedergaard et al. 1993). Hence the lack of Cd2+ sensitivity of the AP and AHP could not be attributed to a direct effect of the hyperpolarization on the Ca2+ current. Instead, the most likely explanation for these findings is that the additional 4-AP-sensitive current, activated from hyperpolarized potentials, is strong enough to inhibit the Ca2+ current during the AP, and thereby prevent activation of the Ca2+-dependent K+ currents underlying the AHP. A simple shunting effect, imposed by the 4-AP-sensitive current could also mask a Ca2+-dependent component of the early AHP. However, it is difficult to see how this effect alone could account for the abolishment of the sAHP.

4-AP-sensitive delay shift

The delay shift constitutes a temporal separation between normal and attenuated APs, because the latter were confined to the initial time window (Fig. 4). The sensitivity to 4-AP of AP delays as long as 1 s indicates that a slowly inactivating current counteracts depolarization in a voltage range below firing threshold. This current is responsible for the delay shift. The higher sensitivity to 4-AP of the delay shift compared with the AP width (Fig. 7D) could indicate that these two parameters show a large difference in their dependency of the amount of 4-AP-sensitive current available. Alternatively, separate currents may coexist, with different sensitivity to 4-AP. Transient currents, distinguished from the IA by a slow inactivation and high sensitivity to 4-AP, has been shown in different neurons to mediate delayed excitation (McCormick 1991; Nisenbaum et al. 1994; Spain et al. 1991; Storm 1988).

Role of 4-AP-sensitive current in regulation of AP threshold

The threshold of the first AP was lowered by 4-AP depending on the rate of depolarization. This suggests that the effect of the 4-AP-sensitive current on the AP delay can, at least partly, be attributed to an increased threshold of APs generated at long delays. Conversely, the threshold of short-latency APs, generated within the initial time window before the delay shift, was unaffected by 4-AP. The latter observation can be explained if the activation kinetics of the 4-AP-sensitive current is slow at subthreshold potentials, and therefore has little influence on the initiation of an early AP. Considering that the early APs were precisely the ones that were maximally attenuated by the 4-AP-sensitive current, it would seem that the rate of activation increases markedly during the spike. In fact, the activation time constant of the A-current in these neurons is reported to increase from 4.5 to 0.9 ms in the voltage range between -55 and -15 mV (Bruns et al. 1998). This finding seems, at least qualitatively, to be in accordance with the above interpretation.

With 4-AP present, the AP threshold decreased with increasing delay (decreasing rate of depolarization). The reason for this was not pursued here. However, such a relationship would be expected in a cell where the site of spike initiation is at an electrotonic distance from the recording site, due to the lower degree of electrotonic filtering of slow potentials. As reported by Grace (1990), the threshold of the initial segment (IS) component of the AP, as seen from the soma, is particularly sensitive to 4-AP, possibly due to the location of a 4-AP-sensitive conductance in the segment of dendrite between the IS and the soma. The present results seem compatible with this interpretation and indicate further that activation of this conductance may obscure and oppose the frequency-dependent variation of passive filtering.

Functional implications

The delay shift is likely to be exploited under physiological conditions, because the EPSP rise time (2.8 ms) was well below the lower limit of the shift (7-12 ms). The high efficacy of EPSPs at hyperpolarized potentials could be due to their ability to depolarize the membrane rapidly and thereby "escape" the delay shift. Judged from the effect of ramp injections, a somewhat slower depolarization would fail to cause direct excitation, unless sustained for several tens of milliseconds. It seems that this unlinear response property would function as a filter discriminating between fast and slow events. An obvious consequence of such a mechanism is that fast EPSPs are allowed to mediate precisely timed outputs (in millisecond scale) with respect to presynaptic discharge. Because direct AP generation could occur over the range of membrane potentials (-40 to -65 mV) normally experienced by the cell during spontaneous firing, such activation might be more or less independent on the background activity. The pacemaker potential is rising slowly compared with an EPSP, which may explain why APs in the presence of CNQX are unlikely to be fired from hyperpolarized thresholds (Fig. 2). Therefore a discriminatory function based on the slope could be an effective way of keeping a high sensitivity to fast afferent signals, while at the same time maintaining a low intrinsic firing rate. The influence of afferent connections on the discharge may involve both a direct spike activation by fast EPSPs and an indirect modification of the ongoing activity by slow inputs. The shifts in firing patterns of nigral neurons, seen to be implicated in changes in motor behavior (Romo and Schultz 1990), could be a reflection of transitions between slow and fast afferent drive.

In SNc cells the AHP influences the firing properties, in particular the sAHP component, which has a dampening and regularizing effect on the discharge (Shepard and Bunney 1991). Inhibition of the sAHP seems, from this study, to be a general consequence of activating these cells from low membrane potentials. The exact role of this inhibition for the firing properties under various conditions was not examined. It is possible that a shortened AHP could enhance the excitability during repetitive activation. This would further dissociate the properties of EPSP-driven discharge from the intrinsic pacemaker mechanism.

Calcium influx during the AP may have several functions. Because the Ca2+ component of the AP causes prolongation of the depolarizing phase, it may help backpropagation of unattenuated APs into the dendrites as suggested by Häusser et al. (1995). Consequently, inhibition of the Ca2+ component could reduce the dendritic AP and perhaps shorten the distance over which it propagates. Attenuated APs, driven by EPSPs, may therefore be less effective in invading the dendrites than intrinsic APs. Dendritic voltage-dependent Ca2+ conductances (Hounsgaard et al. 1992), co-activated during the AP, may influence intracellular processes, including those involved in dendritic release of dopamine (Geffen et al. 1976; Rice et al. 1994, 1997). Several studies have shown a lack of correlation between the discharge rate and dopamine release in substantia nigra (Cheramy et al. 1981; Nieoullon et al. 1977; Nissbrandt et al. 1985), which questions the role of APs in the release process. However, a discrepancy between the discharge rate and the amount of released dopamine might be expected, if dendritic Ca2+ influx depends on the AP size. It is possible that dendritic dopamine release, for this reason, is facilitated or inhibited, depending on the mode of spike activation, rather than on the overall discharge rate.

In conclusion, this study suggests that the excitability and action potential size are regulated during excitatory synaptic transmission, depending on activation of 4-AP-sensitive current in the postsynaptic cell. This current may be composed of both delayed rectifier-type and A-type current, the latter of which becomes dominant at subthreshold membrane potentials. A regulatory role of IA on the membrane response properties has previously been shown in cerebellar Purkinje cells and hippocampal pyramidal neurons (Andreasen and Lambert 1995; Hoffman et al. 1997; Midtgaard 1994), where its dampening effect on dendritic excitability appears to be a means by which synaptic integration can be regulated locally. The present study adds to these findings and suggests that another important function of such current is to provide spontaneously active neurons with the ability to discriminate between different afferent inputs and their own intrinsic activity.


    ACKNOWLEDGMENTS

I thank Drs. M. Andreasen and J. Hounsgaard for helpful comments on the manuscript.

This work was supported by a grant from the Aarhus University Research Foundation.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 December 1998; accepted in final form 23 July 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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