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
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ABSTRACT |
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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.
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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 M
) 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 M
, 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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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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