Arrhythmic Firing in Dopamine Neurons of Rat Substantia Nigra Evoked by Activation of Subthalamic Neurons

Youngnam Kang1,2 and Takahiro Futami2

 1 Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan; and  2 Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee at Memphis, Memphis, Tennessee 38163


    ABSTRACT
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INTRODUCTION
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Kang, Youngnam and Takahiro Futami. Arrhythmic Firing in Dopamine Neurons of Rat Substantia Nigra Evoked by Activation of Subthalamic Neurons. J. Neurophysiol. 82: 1632-1637, 1999. Intracellular recordings were made from dopaminergic neurons of the rat substantia nigra compacta (SNc) in in vitro slice preparations to study the synaptic influence from the subthalamic nucleus (STh). After microstimulation of STh, monosynaptic excitatory postsynaptic potentials (EPSPs) were produced in dopaminergic neurons. STh-induced EPSPs were composed of 6-cyano-7-nitroquinoxalene-2,3-dione- and 2-amino-5-phosphonovaleric acid-sensitive components. Subthreshold EPSPs evoked by STh stimulation could differentially trigger pacemaker-like slow depolarization (PLSD) and low-threshold Ca2+ spike (LTS) depending on the level of baseline membrane potentials. When a subthreshold EPSP was evoked by STh stimulation during rhythmic firing, the STh-induced EPSP could shift or elevate PLSD to a more depolarized level, resulting in generation of a spike at an earlier arrhythmic timing to restart the rhythmic firing. The interspike interval after the arrhythmic spike remained almost unchanged. In contrast, when a suprathreshold EPSP for evoking spikes was produced by STh stimulation during rhythmic firing, the STh-induced spike was just interposed between two spontaneous spikes the interspike interval of which was almost the same as those seen during the preceding rhythmic firing. This ectopically induced spike did not disturb or reset rhythmic firing. It was concluded that SNc dopaminergic neurons receive monosynaptic glutamatergic inputs from STh, and subthreshold and suprathreshold EPSPs evoked by STh stimulations can induce two types of arrhythmic firing in SNc dopaminergic neurons, similar to arrhythmic occurrences of the QRS complex seen in the electrocardiogram of the atrial and ventricular arrhythmias, respectively. The former arrhythmic firing may play a crucial role in desynchronization of dopaminergic neurons.


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INTRODUCTION
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The subthalamic nucleus (STh) has been considered to be a motor nucleus that drives the final output neurons in the basal ganglia such as globus pallidum and substantia nigra pars reticulata (SNr) neurons (Deniau et al. 1978; Van der Kooy and Hattori 1980). An electrophysiological study demonstrated that STh stimulation induced glutamatergic excitatory postsynaptic potentials (EPSPs) in SNr neurons (Nakanishi et al. 1987). In an anatomic study using phaseolus vulgaris leucoagglutinin (PHA-L) immuno-electron microscopic method, synaptic terminals arising from STh were identified to form asymmetrical synapses on the dendrites within SNr (Kita and Kitai 1987). In the same anatomic study, synaptic terminals also were found to be sparsely distributed within substantia nigra pars compacta (SNc).

On the other hand, burst firing in dopamine (DA) neurons has been reported to be either suppressed by applying N-methyl-D-aspartate (NMDA)-antagonist (Chergui et al. 1991) or induced by applying NMDA (Johnson et al. 1992). Therefore it is possible to assume that rhythmic firing in SNc DA neurons can be altered to burst firing through activation of glutamate receptors. Although stimulation of the pedunculopontine tegmental nucleus (PPN) was found to produce glutamatergic as well as cholinergic EPSPs in SNc DA neurons (Futami et al. 1995), PPN stimulation never caused burst firing.

In the present study, it was examined whether or not STh stimulation induces EPSPs in SNc DA neurons. If this was the case, it also was examined whether rhythmic firing is modified or altered by STh inputs.


    METHODS
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The experimental procedures for preparation of solutions and recording was essentially the same as those described in the previous study (Kang and Kitai 1993a). Male Sprague-Dawley rats weighing 50-250 g were decapitated and the brains were removed. The brain first was blocked with a razor blade to contain SNc and STh and then sectioned parasagittally with a Vibratome (Oxford) for 400- to 500-µm-thick slices. Microstimulation (<5 µA) was applied to various sites within STh through a single tungsten microelectrode (DC resistance, 1 MOmega ) by changing its position within STh to search responses as long as intracellular recordings in SNc DA neurons were maintained. To exclude the possibility of stimulating passing fibers, microstimulation also was applied to various sites in STh and its surrounding area. To more directly reveal monosynaptic connection from STh to SNc, simultaneous intracellular and extracellular recordings were obtained from SNc and STh, respectively, and a spike triggered averaging method was performed. Glutamate-induced and/or spontaneous extracellular spikes of STh neurons were used to trigger a computer, which averaged intracellular potentials of simultaneously recorded SNc neurons. Extracellular spikes were regarded as antidromic if they could follow high-frequency repetitive stimulation (>= 125 Hz). Two types of glass microelectrodes filled with 0.5 M KCl and 2 M NaCl (DC resistances, 60-120 and 5-10 MOmega , respectively) were used for intracellular and extracellular recording, respectively. An intracellular double labeling technique with immunocytochemistry was used to identify the transmitter phenotype (dopamine) of the recorded neurons as described previously (Kang and Kitai 1993a).


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Nature of synaptic inputs from STh to SNc DA neurons

After microstimulation applied at sites within STh, EPSPs were produced in 68 SNc DA neurons. As shown in Fig. 1A, during injection of depolarizing and hyperpolarizing current pulses in a DA neuron, microstimulation was applied at a site of STh. STh-induced EPSPs increased in amplitude and decayed more slowly with membrane hyperpolarization [compare ** (top trace) * (bottom trace)]. Monosynaptic nature of STh-induced EPSPs was examined by double shock stimulation. Figure 1Ba shows control responses to single shock stimulations. As seen in Fig. 1Bb, in response to double-shock stimulations with an interstimulus interval of 2 ms, two EPSPs were successively induced. The second EPSPs were almost identical with the first EPSPs in the rise time and amplitude, although the latency was slightly longer in the second EPSPs (1.7 ms) than in the first EPSPs (1.4 ms; Fig. 1Bc). Therefore EPSPs were likely to be produced monosynaptically. No apparent EPSPs were induced in DA neurons by stimulation (<5 µA) of the rostral or surrounding parts of STh. The latency of monosynaptic EPSPs produced in SNc DA neurons by STh stimulation was 2.1 ± 0.7 (mean ± SD) ms (n = 38).



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Fig. 1. Monosynaptic nature of subthalamic nucleus (STh)-induced excitatory postsynaptic potentials (EPSPs). A: EPSPs induced in a DA neuron by STh stimulation applied during injection of depolarizing and hyperpolarizing current pulses at -60 mV (horizontal arrow). EPSPs decayed more rapidly when evoked at more depolarized membrane potentials. Compare the top trace (**) with the bottom trace (*). B, a and b: EPSPs (top) and extracellular field potentials (gray traces) evoked in response to single-and double-shock stimulations of STh at a membrane potential of -60 mV. Four traces of EPSPs were superimposed. Interstimulus interval was 2 ms. Bc: superimposed traces of B, a and b, showing similarities in amplitude, rise-time and latency between the 1st and 2nd EPSPs. Ca: extracellular spikes of an STh neuron with an antidromic latency of 3.4 ms in response to a paired stimulation separated by 8 ms applied at a site in substantia nigra compacta (SNc). Cb: an averaged trace of glutamate-induced or spontaneous extracellular spike of the same STh neuron. Cc: an averaged individual EPSP (500 responses) with a latency of 3.8 ms obtained in a SNc DA neuron at a membrane potential of -60 mV by the activity of the simultaneously recorded STh neuron (Cb).

To more directly reveal the monosynaptic connection from STh to SNc DA neurons, a spike-triggered averaging was performed. Twenty-one pairs of simultaneous intracellular and extracellular recordings were made from SNc DA and STh neurons, respectively. Among them, only three pairs of positive connections could be detected. By applying a paired microstimulation separated by 8 ms at a site of SNc, an extracellular spike with an antidromic latency of 3.4 ms was recorded from an STh neuron (Fig. 1Ca). Glutamate-induced and/or spontaneous extracellular spikes of the STh neuron were used to trigger the computer which averaged the extracellular spike of the STh neuron (Fig. 1Cb) and the membrane potential of the intracellularly recorded DA neuron (Fig. 1Cc). An individual EPSP with a latency of 3.8 ms, which was preceded by a capacitative coupling potential reflecting the extracellular spike potential of the STh neuron, was obtained at -60 mV. The latency of the individual EPSP was longer by 0.4 ms than the antidromic latency of the STh neuron (3.4 ms; axonal conduction time), presumably due to the synaptic delay. Thus monosynaptic individual EPSPs were obtained in three pairs of STh and SNc DA neurons. The latency, rise time, and amplitude for the three individual EPSPs were 3.6 ± 0.3 ms, 0.7 ± 0.2 ms, and 45 ± 15 µV, respectively.

Fast and slow components of STh-induced EPSPs

As seen in Fig. 2A, kynurenic acid (1 mM) reversibly abolished STh-induced EPSPs in DA neurons (n = 3). A bath application of 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX; 30 µM) also reversibly abolished fast components of STh-induced EPSPs (Fig. 2B) in seven DA neurons examined. It is also seen in Fig. 2B that STh-induced EPSPs were followed further by slow depolarizations similar to a pacemaker-like slow depolarization (PLSD). Although this slow depolarization also became smaller after application of CNQX, this may be because CNQX-sensitive fast component was reduced to such a degree that voltage-dependent activation of PLSD did not occur. Thus PLSD may follow STh-induced EPSPs when evoked at membrane potentials more positive than -60 mV (Kang and Kitai 1993a,b). Therefore at hyperpolarized membrane potentials around -70 mV in a low-Mg2+ (0.3 mM) Ringer solution, we have examined effects of 2-amino-5-phosphonovaleric acid (APV) on STh-induced EPSPs that were not contaminated with PLSDs. As shown in Fig. 2C, the decay of EPSPs evoked by stimulation of STh at a membrane potential of -71 mV was accelerated by applying APV (50 µM). APV significantly shortened the half-width of STh-induced EPSPs from 24.5 ± 5.3 to 15.7 ± 3.9 ms (P < 0.05; n = 6). This indicates that APV-sensitive components were involved in the falling phase of STh-induced EPSPs. Thus STh-induced EPSPs involved both non-NMDA and NMDA components. As seen in Fig. 2B, STh-induced EPSPs appeared to trigger PLSDs. Therefore the relationship between STh-induced EPSPs and PLSDs was analyzed further.



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Fig. 2. Effects of glutamatergic antagonists on STh-induced EPSPs and differential activation of pacemaker-like slow depolarization (PLSD) and low-threshold spike (LTS) by STh-induced EPSPs. A: kynurenic acid (1 mM) reversibly abolished EPSPs evoked at -64 mV. B: 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX; 30 µM) reversibly depressed fast EPSPs evoked at -55 mV. C: the decay of EPSPs evoked at -71 mV was reversibly accelerated by APV (50 µM). This experiment was performed in a low-Mg2+ (0.3 mM) and high Ca2+ (3.2 mM) Ringer solution. Da: at -65 mV, an EPSP induced by STh stimulation did not decay but elevated PLSD (down-arrow ), from which 2 spikes eventually were triggered. Db: at -70 mV, an STh-induced EPSP triggered PLSD and a spike at a latency longer than that at -65 mV. Rebound activation of LTS (black-down-triangle ) was caused by an afterhyperpolarization (AHP). Dc: at -73 mV, an STh-induced EPSP only triggered PLSD (down-arrow ). Dd: at -87 mV, an STh-induced EPSP followed by an LTS-like response (black-down-triangle ). E: PLSD (down-arrow ) and LTS (black-down-triangle ) were differentially triggered at the offset of hyperpolarizing current pulses applied at -73 and -85 mV, respectively, in the same DA neuron as in D. Calibrations in Da also apply in D, b-d and E, a-c.

PLSD triggered by STh-induced EPSPs

When membrane potentials in DA neurons were changed to various levels by injection of DC, STh-induced EPSPs were followed by various responses. As seen in Fig. 2D, when the membrane potential in a DA neuron was held at -65 mV where no rhythmic firing was seen, STh stimulation induced a subthreshold EPSP that decayed only slightly and was followed by a voltage-dependent PLSD, leading to a generation of a spike that also was followed by another spike (Fig. 2Da). Thus STh stimulation transiently induced a rhythmic firing at around -65 mV. When membrane potential was hyperpolarized to -70 mV, the same STh stimulation triggered only one spike through a sequential response of an STh-induced EPSP and the following PLSD (Fig. 2Db). At this membrane potential (-70 mV), the spike afterhyperpolarization (AHP) led to a rebound generation of low-threshold Ca2+ spike (LTS), which did not trigger any further responses unlike the case shown in Fig. 2Da. At a further hyperpolarized membrane potential (-73 mV), an STh-induced EPSP only triggered a PLSD which was followed by a prominent AHP (Fig. 2Dc). This AHP may be mediated by a Ca2+-dependent and apamin-sensitive K+ conductance (Shepard and Bunney 1991). At -87 mV, an STh-induced EPSP triggered a response similar to LTS, whereas PLSD was no longer triggered (Fig. 2Dd). Thus various responses were triggered from STh-induced EPSPs depending on the membrane potential. Similarly, PLSD and LTS were also differentially triggered at the offset of hyperpolarizing current pulses (Fig. 2E), without STh stimulation. The threshold for activation of LTS was invariably lower than that of PLSD, in agreement with the previous observation on the difference in the threshold between transient and persistent low-voltage-activated (LVA) Ca2+ currents (Kang and Kitai 1993b).

Arrhythmic firing in DA neurons caused by STh inputs

When STh was stimulated during rhythmic firing, STh-induced EPSPs were able to alter rhythmic firing. As seen in Fig. 3A, an STh-induced EPSP did not decay but elevated PLSD to a more depolarized level (compare with the gray trace in Fig. 3A), reaching the spike threshold at a timing earlier than that expected from the interspike interval (ISI) during the preceding rhythmic firing. However, the ISI after the spike triggered indirectly by subthreshold STh stimulation was almost the same as that during the preceding rhythmic firing. Thus STh-induced EPSPs evoked by subthreshold STh stimulation were able to restart or reset the rhythmic firing. Even when STh stimulation was applied immediately after an action potential, rhythmic firing was reset (figure not shown). Similar observations were made in 12 DA neurons.



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Fig. 3. Effects of STh stimulation on rhythmic firing. A: a subthreshold STh-induced EPSP (upward arrow) elevated PLSD, resulted in a spike generation at a timing earlier than that expected from the interspike interval (ISI) seen during the preceding rhythmic firing. Note that ISI after the subthreshold STh stimulation remained almost unchanged. Gray trace shows the assumed rhythmic firing that would have continued to occur unless STh had been stimulated. *, PLSD, toward which EPSPs would have decayed if voltage-dependent activation of PLSD had not been caused by EPSPs. Ba: suprathreshold STh stimulation directly evoked a spike without waiting for PLSD. This spike was just interposed between the 2 spontaneous spikes the ISI of which remained unchanged from those during rhythmic firing. Bb: superimposed traces of spikes evoked by suprathreshold STh stimulations applied at 2 different timings during rhythmic firing in the same DA neuron as in Ba. Note that rhythmic firing was not reset by spike AHP although the membrane potential was hyperpolarized to the same level due to the spike-AHP. Calibrations in Bb also apply in Ba. C, a and b: 2 different types of arrhythmic firing generated in the same DA neurons by suprathreshold and subthreshold STh stimulations. Calibrations in Cb also apply in Ca.

By contrast, rhythmic firing was not reset by suprathreshold STh stimulation that produced large EPSPs to directly trigger a spike. As seen in Fig. 3Ba, a suprathreshold EPSP evoked by STh stimulation directly triggered a spike without waiting for PLSD. The ISI between the STh-induced action potential and the succeeding one was shorter than the original ISI during the preceding rhythmic firing. The STh-evoked spike was just interposed between the two spontaneous spikes the ISI of which was almost the same as that between the two spikes preceding the interposed one. This is probably because the LVA Ca2+ current underlying PLSD might have been deactivated due to an AHP after the ectopic spike to almost the same level as that just before the STh-induced spike. However, it is not clear whether PLSDs were deactivated to the same constant level even when STh-induced spike AHPs were evoked at different timings. If this was the case, the ISI between the STh-evoked spike and the following one would have been constant, thereby resetting the rhythmic firing. As shown in the superimposed records in Fig. 3Bb, when STh-evoked spikes were evoked at different timings during rhythmic firing, the spontaneous spike after the STh-induced one occurred at an earlier timing as the potential level prior to the STh stimulation was more positive. Thus even though the PLSD might have been deactivated to almost the same level by STh-induced spike-AHP, STh-evoked spike did not reset rhythmic firing. Similar observations were made in nine DA neurons. Figure 3C, a and b, shows the two different types of arrhythmic firing generated in the same DA neuron by suprathreshold and subthreshold STh stimulations, respectively.


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It was demonstrated in the present study that when STh was stimulated during rhythmic firing, subthreshold and suprathreshold EPSPs could induce two types of arrhythmic firing in SNc DA neurons, similar to arrhythmic occurrences of QRS complexes seen in the electrocardiogram of the atrial and ventricular arrhythmias, respectively (Ganong 1980). If the subthreshold STh-induced EPSPs was simply summated with PLSD, EPSPs would have decayed in 50-100 ms (see Fig. 2, A and C) toward the trajectory of spontaneous PLSD of the assumed rhythmic firing (indicated with * under the gray trace in Fig. 3A), which would have continued to occur unless STh had been stimulated. In our previous study, it has been demonstrated that the Ca2+ current underlying PLSD is activated and deactivated depending on the membrane potential (Kang and Kitai 1993a,b). Voltage-dependent activation of PLSD might have been caused by subthreshold EPSPs, resulting in an elevation of PLSD to a more depolarized level. On the other hand, voltage-dependent deactivation of PLSD might have been caused by an AHP after the spike evoked by suprathreshold EPSPs. This is consistent with the previous observation that PLSD was invariably and promptly deactivated at the offset of depolarizing current pulses (see Fig. 8D in Kang and Kitai 1993a). However, the slow decay of STh-induced EPSPs might hamper the deactivation of PLSD that was activated by STh-induced EPSPs. The rate of the regenerative activation of PLSD could have been faster than the rate of the deactivation of PLSD during the decay time course of STh-induced EPSPs.

Suprathreshold STh stimulation neither reset nor markedly affected rhythmic firing, in spite of voltage-dependent deactivation of PLSD by the spike AHP. This is probably because the pacemaker activity is dependent not only on the membrane potential but also on the [Ca2+]i level (Kang and Kitai 1993b). In fact, Ca2+-dependent activation of SK channels and Ca2+-dependent inactivation of PLSD play crucial roles on rhythmic firing (Kang and Kitai 1993b; Shepard and Bunney 1991). Then to reset rhythmic firing, not only the membrane potential but also the [Ca2+]i level must be reset. However, the spike AHP can reset only the membrane potential but cannot reset or remove Ca2+-dependent activation or inactivation. This may be the reason why suprathreshold STh stimulation did not reset rhythmic firing. Possible Ca2+ influx after an STh-evoked ectopic spike may not have markedly affected the Ca2+ concentration at the pacemaker region. Then there may be a compartmentalization of [Ca2+]i between the pacemaker region and the spike generation site.

At present, physiological significances of these arrhythmic firings are not clear. DA neurons are known to display synchronized rhythmic firing (Grace and Bunney 1983). STh inputs may be able to break synchronized firing. Desynchronization can be caused by arrhythmic firings evoked at different timings in different DA neurons depending on the amplitude and latency of subthreshold STh-induced EPSPs.


    ACKNOWLEDGMENTS

The authors thank Dr. S. T. Kitai for much valuable discussion and criticism.

This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-20702 and NS-26473 to S. T. Kitai and Grant-in-Aid for General Scientific Research (C) 10680765 to Y. Kang.

Present address of T. Futami: Dept. of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.


    FOOTNOTES

Address for reprint requests: Y. Kang, Dept. of Physiology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan.

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 16 February 1999; accepted in final form 10 May 1999.


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