Download Intracellular study of rat substantia nigra pars reticulata neurons in

Brain Research, 437 (1987) 45-55
Elsevmr
45
BRE 13144
Intracellular study of rat substantia nigra pars reticulata neurons in
an in vitro slice preparation: electrical membrane properties and
response characteristics to subthalamic stimulation
H. Nakanish;.*, H. Kita and S.T. Kitai
Department of Anatomy and Neurobtology, The Umversity of Tennessee, Memphis, The Heahh Science Center,
Memphis, TN38163 (U.S.A.)
(Accepted 2 June 1987)
Key words" Rat substantIa mg~a neuron; Shce preparation, Intracellulat recording; Membrane property; Subthalamonlgral input
The electrical membrane properties of 5tl135tattlsa n.~,~ap~!rSreiiculata (SNR) neurons and the,r postsynapuc responses to si~.mulation of the subthalamic nucleus (STH) were studied in an m vitro slice preparation. SNR neurons were divided into two types based on
their electrical membrane properties. Type-I neurons possessed (1 } spontaneous repetmve finngs, (2) short-duration action potentials, (3) less prominent spike accommodations, and (4) a strong delayed rectification during membrane depolarization Type-II neurons had (1) no spontaneous firings, (2) Iong-du_ratiQnaction potentials, (3) a prominent spike accommodation, (4) a relatively large
post-active hyperpolarization, and (5) a less prominent delayed rectification. These membrane properties were very similar to those
observed in substantm mgra pars compacta (SNC) neurons in slice preparations. Feawures coma~on to both ,ype~ of neurons include
that (1) the input resistance was similar, (2) they showed an anomalous rectification during suoitg ~pcrp~lat~zatlous, and (3) they
were capable of generating Ca potentials Intracellular responses of both types of SNK neurons to STH shmulatIon consisted of imtial
short-duration monosynaptlc excitatory postsynaptic potentials (EPSPs) and a short-.durauon inhibitory $×~s~synapticpotential (IPSP)
followed by a long-duration depolarization The IPSP was markedly suppressed by apphcatlon of blcucuihne methlodide and the polarity was reversed by lntracellular rejection of CI-. In the preparations obtained from internal capsule-transected rats, STH-mduced
EPSPs had much longer duranons than those observed in the normal preparations, while the amphtude of IPSPs and succeeding smallamplitude long-duranon depolarizations was small. The results indicated that SNR contains two electrophysiologically different types
of neurons, and that both types of neurons receive monosynaptic EPSPs from STH and IPSPs from areas rostral to STH
INTRODUCTION
The m a j o r afferents to the substantia nigra (SN)
pars reticulata ( S N R ) originate f r o m the neostriaturn, the globus pallidus ( G P ) and the s u b t h ~ a m i c
nucleus (STH). T h e neostriatum and S T H receive
excitatory inputs f r o m the s e n s o r i m o t o r cortex 1'17'19'38. T h e r e f o r e , S N R receives cortically derived striatal input and cortically derived S T H input.
Electrophysiological studies indicated that the predominant response of S N R neurons to neostriatal
stimulation was inhibitory, while some excitatory re-
sponses were also noted 29"39. The S T H afferents to
S N R originate from neurons whose bifurcating axons
also innervate G P and the entopeduncular nucleus
(EP) 3"13'14"35. The S T H terminals form asymmetrical
synapses on the dendrites of SN neurons 2"t5. Electrophysiological study to identify the functional significance of S T H inputs to S N R is limited to one study
utilizing an extraceilular recording technique 9. T h e
study indicated that S T H stimulation evoked an excitation of nigral neurons. O n the other hand, several
reports suggested that S T H inputs to EP and G P are
inhibitory and ~,-aminobutyric acid ( G A B A ) e r -
* Present address: Department of Pharmacology. Faculty of Dentistry. Kyushu Umverslty. Fukuoka 812. Japan
Correspondence S T Kital. Department of Anatomy and Neuroblology. The Univemty of Tennessee, Memphis. The Health
Science Center, 875 Monroe Avenue. Memphis. TN 38163. U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Pubhshers B V. (Biomedical Dwision)
46
gic2s'30'31 These two opposing responses (excitatory
vs. inhibitory) of STH inputs to EP/GP and SNR are
somewhat puzzling since anatomical and electrophysiological studies clearly demonstrated that single
STH neurons project both to EP/GP and SNR with
their bifurcating axons. In order to examine the precise nature of STH inputs to SNR, we have recorded
mtracellular responses of SNR neurons to stimulation of STH m an in vitro slice preparation. We considered that thts preparation is most appropriate for
studying electrical membrane properties of SNR
neurons since one can manipulate the external chemical enwronment of the neurons studied, and also one
can make more precise measurements of membrane
phenomena with stable recordings 18. The electrical
membrane properties of the dopaminerglc cells in the
SN pars compacta (SNC) have been examined in detail usmg an m vitro preparation and mtracellular
recording techniques 16"2a. Since SNR contains both
dopaminergic and GABAergic neurons 26, our aim
was also to examine if SNR neurons can be distingmshed into two types on the basis of electrical membrane properties
Our results indicate that SNR neurons can be classified into two types based on their electrical membrane characteristics. Both types of SNR neurons
evoked monosynaptic excitatory postsynaptic potentials following stimulation of STH. The results suggest that cortically derived STH excitatory inputs and
cortically derwed neostnatal inhibitory inputs are
converging on SNR neurons.
MATERIALS AND METHODS
Male Sprague-Dawley rats (200-350 g) ~ere
used. Immediately after decapitation, the brain was
rapidly removed from the skull and trimmed tt a
block. Parasaglttal shces (350-450/~m) containing
the SN and the STH were cut usmg a Vibratome.
Slices were preincubated in oxygenated Krebs solution for about 1 h at 35 °C before recording The
recording chamber was constructed to allow Krebs
solution (35 °C) to continuously flow on the bottom
surface of the slice at a rate of 0.7-1.5 ml/mm, and
warm and moist gas mixture (95% 0 2 - 5 % CO2) to
flow over the surface of the shce 18. The Krebs solutlon was composed of (mM): NaCI 124, KCI 5 0,
KH2PO4 1 24, NaHCO 3 26, CaCI 2 2.4, MgSO4 1.3
and glucose 10. Glass pipette.~ filled with 2 M potassium methylsulfate or 1.5 M potassium chloride were
used for recording. Recording electrodes had DC resistance of 60-100 MfL Intraceilular recordings were
obtained through a high-input impedance biological
amplifier with an active bridge circuit which enabled
the measurement of membrane potentials and the injection of intracellular constant current simultaneously. The output of the amplifier was fed into an
oscilloscope. Electrical stimulation (intensity 5-30
V, duration 200/~s, 0.5 Hz) was applied through a bipolar electrode placed on the surface of the STH.
The stimulating electrode was made by twisting a
pair of 80-/~m-diameter nichrome wires, insulated except at the tip, with a tip separation of 200-400/tin.
In some animals, the internal capsule (IC) at the
level of the entopeduncular nucleus was transected
using a Halazs knife, 6-10 days prior to the recording
session, in order to eliminate afferent fibers to SNR
originating rostral to STH. In some experiments, the
Na channel blocker tetrodotoxin ( T r x , 10-s g/ml)
and a K channel blocker tetraethylammonium chloride (TEA, 2-10 raM) were added to the superfusion
medium. Cobalt chloride (2-4 raM) was used to
block Ca conductance s. Bicuculline methiodine was
dissolved in the superfusion medium with a concentration ranging from 50 to 100k~M to block GABAergic responses.
RESULTS
Intracellular recordings were obtained from 40
SNR neurons which had resting membrane potentials
of more than 50 mV and generated action potentmls
w~th an amphtude greater than 50 mV.
Membrane properttes
SNR neurons were divided into two types based on
their electrical membrane properties One group of
neurons which we named type-I (n = 26) had the following characferist~cs. They exhibited spontaneous
repetmve firings with a rate of 1-40 spikes. The duration of the action potential was less than 2 ms. Injection of a depolarizing current pulse generated repetitwe firings from these neurons. Even though the
finng frequency was highest (i.e., a maximum over
200 Hz) at the beginning of the current pulse, finngs
with a regular interval were maintained throughout
47
A
C
12om__ I
' 20
-0'4
je
-0'2/,J
/
02nA
04
e,,/e
-20
eJ e
-40
E
/
200'
H7
o
*1,'tI
B
D
20 mV
/
100"
-f
1-
/
.,(,'/
......................
o
0
0"3PA
0"6
Fig. 1. Input resistance and spike discharges of type-t neurons A membrane responses to mtraceilularly rejected hyper- and depolarrang currents of various intensities. In order to eliminate spontaneous finng, a hyperpolanzmg current of 0.06 nA was continuously injected m the neuron. B membrane responses to hyper- and depolarizing currents during appheauon of TIX (10-5 g/ml) to ehm,nate
spikes Square waves at the bottom of oscdlographic records in tlus and all subsequent figures indicate the mtensmes of rejected clepolanzmg (upward square wave) and hyperpolanzmg (downward square wave) currents Cahbrauons m A also apply to B C currentvoltage relauon for a type-I neuron recorded m the shce superfuse¢ w~th TI'X-contaming solution Note membrane recuficauon m
both hyper- and depolarizing directions D inleetion of a depolarizing current pulse m a continuously hyperpolanzed neuron produced repetmve finng followed by a small amplitude long-lasting hyperpolanzmg potentml after the termination of the current pulse
E: relattons between the discharge frequency and the intensity of current pulses obtained from the lnterspike interval of first two
spikes (1/tl) and last two spikes (l/t2) m the cell of D.
the period of the current pulse (Fig. 1D). No large
changes in the amplitude nor the duration of action
potentials were observed even when relatively highfrequency (over 70 Hz) repetitive firing was generated by a strong current injection (Fig. 1D). A longlasting hyperpolarizing potential with a small amplitude (no more than 10 mV) was occasionally observed after termination of a large current pulse (Fig.
1D). SNR type neurons showed a large reduction of
the input resistance (delayed rectification) during
membrane depolarization (Fig. 1B,C).
The m e m b r a n e properties of the other group of
neurons, type-ll neurons (n = 14). ~ e r e very similar
to those observed m SNC neurons in slice preparations 16-21.
Type-ll neurons had no spontaneous firings. The
duration of action potential was usually longer than 2
ms. InJection of moderate intensity depolarizing current pulse generated low-frequency (up to 10 Hz) repetitwe firings When a strong depolarizing current
pulse was applied, the neurons showed a prominent
sptke accommodation with a decrease in the spike
amplitude and an increase in its duration, which finally led to a cessation of spike generation (Fig 2C).
The membrane response after termination of large
current pulse was a relatively large-amphtude (about
10 mV) long-lasting hyperpolanzation. The delayed
rectification which was clearly observed in type-I
neurons was less prominent in type-lI neurons Instead, type-ll neurons showed a clear skgn ot an act~ration of early K current as has been reported m SNC
neurons 16
~,8
/
B
w
0'3
-0 6 nA
./
//
./
-25
mV
./
.7
I
• -50
1
Fig 2 Input res]s ce and sp~ke discharges of a type-II neuron A membrane responses to mtracellularly rejected hyper- and depolar~.zmgcurrents of various intensities B current-voltage relation for the type-II neuron C rejection of a ,trong depolarizing current
palse produced only 4 spikes A strong spike accommodation can be seen m these responses The membrane potentml after the termination of the current pulse was a relatively large-amplitude long-lastingbyperpolanzmg potential. Calibrations in A also apply to C
tance ot type-I and type-II neurons calculated from
the slope of the current-voltage curve crossing at
zero current point was very similar; the former
Even though some membrane properties of two
types of neurons were clearly different, there were
features common to both of them. The input resis-
BII,
20_v
jj
S0mse0
_T=-~-==t. . . . . . . . . . . . . . . . . . . . . . . .
C
D
C
,,J
J
t
_/--1
Fig 3 Slow depolarizing potentials evoked m a type-I neuron A injection of a depolarizing current pulse generated repeUttve firings
and a small-amphtude membrane hyperpolanzatlon after the termmatlonofthecurrentt~td~, P, adepolartzmgcurrentpulsemjected
dunng contmuous membrane hyperpolanzatlon triggered a slow depolanzir~g p,~tential ~utla.tmg the gt~rrem oulse C and D. responses to depolarizing current pulses during apphcauon of 10 mM TEA TEA mcre,~s,.d the duration of action potentials (C) and mcreased the amphtude and the duration of slow depolarizing potential (D)
49
ranged from 5(I to 150 MQ (n = 18, e . g , Fig 1C) and
the latter ranged from 80 to 200 Mff2 (n = 10, e.g.,
Fig. 2B) The anomalous rectificatton of the cell
membrane m response to strong hyperpolartzmg current injecttons was found in both types of neurons
(Figs. 1C and 2B). Previous studies have clearly demonstrated that SNC neurons are capable of generating Ca potentials I~ 2~ We have also observed m this
study that type-I neurons are also capable of generating Ca potentials.
InJection of depolarizing current pulses in type-I
neurons with a membrane potentml less negative
than 60 mV generated repetitive f~rings (Figs. 1D,
3A). When the neuron was continuously hyperpolarized more negatively than 60 mV by a current injection, the depolarizing currer ~ pulse could generate
repetitive firings followed by a long-duration slow
depolarizing potential which outlasted the duration
of the depolarizing pulse (Fig. 3B). Bath application
of TEA (10 mM) resulted in the following changes in
type-I neurons: (1) a decrease in or a termination of
the spontaneous discharge, (2) an increase in the duration of action potentials generated by depolarizing
current pulses (Fig. 3C), (3) an increase m the input
membrane resistance, and (4) a marked augmentation in the duration and amplitude of the slow depolarizing potential which tnggered a number of action
potentials (Fig. 3D). The type-I neurons were also
able to trigger slow action potentmls in response to
depolarizing current pulses when they were continuously hyperpolarized. The slow action potew~al had
a duration of 30-100 ms and was accompanied by a
burst of action potentials (Fig. 4A). A simdar burst
response was observed at the offset of a strong hyperpolarizing current pulse (Ftgs. 1A and 4B). Superfusion of the Na conductance blocker TTX (10 -5 g/ml)
abolished fast action potentials but preserved the
slow depolarizing potential and the slow action po-
O
A nil 120
J
05nA
13
B
q
I
E
]
I
Fig 4 Slow action p o t e n t i a l evoked by current pulses in a ~ype-I neuron A injection of a depolarizing current pulse in a continuously
hyperpolarized neuron reduced a slow action potentml w~th spikes B a s~mdar slow action potential was generated after the offset of a
hyperpolarizmg current pulse. C - E recordings ,during superfuslon with a medmm containing T r x (10-~ g/ml) L rejection of depolarizing current pulses generated a slow depolarizing potential (arrow) and a slow action potential D. a slow actton potenllal triggered
at the offset of a hyperpolanzing currem pulse. E: addition of Co 2÷ to the superfusing medmm abolished slow action potenttal and slow
depolarizing potential.
50
B
A8
l~v
o
m
ous depolarizing or hyperpolartz~
.
.
during injection o! co.nunu ,._a ,,,,tin short-duration EPSPs
}'
. . . . . . ded from a type [ ~euron _.,:~ STH stimmatton evott~,~ ....... I
,~'-""
. various
-two components.
"
•The arrow on the top trace points to onset of IPSPs- B:
• ca-~ mlecuv,,
stlmulat ton "induced responses
_-.nmos
with
stimulus
tnten~m,-~
mg current
appeared
to ha,Je
l'tg -~ o,,-.
. . . . . S A fast sweep recur,, ~,
slowshort.duration
sweep recordings.
These
and IPSPs were followed by a iong-durauon depolanzauon The amplitude of the intual EPSPs
and
IPSPs.
The EPSPs
tmtial EPSPs
powas decreased by depolarizing currents and increased by hyperpolanztng current mi ecti°ns' The amplitude of IPSPs was increased by
depolarmng currents and decreased by hyperpolarizing current rejections. Bottom traces in A and B represent extraee|lular f~eld
tent~als with a relatively large negative potenual
often appeared to have two components because of
an inflection on the nsmg phase (e g,, Figs 5A, 7A).
tential (Figs. 4C,D). Both of the Tl?X-reststant poThe amplitude of the initial depolarization showed
tentials were augmented by application of TEA (data
graded changes upon increase in the stimulus intensinot shown) and abolished by an addition of Co2+ (3
ty, and tt was increased during iniecti°n of hyperporaM) to the TTX-contalmng medium (Fig. 4E)
lanzing currents and decreased dunng depolarizing
currents (Fig 5A,B) STH stimulation with various
Postsyna?UCresponses evoked by STH stimulation
intensities revealed that the latency of the earliest
It was commonly observed that STH stimulation
and second components ~howed shght and graded
evoked relatively large (i.e,, up to 5 mV) negative
shortening upon increase in the stimulus intensity
field potentials with a latency of about 1 ms and a du(Fig 5A). The graded change of the latenctes was
ration of about 1.5 ms. The latency of the negative
considered to be due to an increase in the current
potential was constant in spite of changes in stimulus
spread at the stimulus site, since the change never exintensities or repeuUve stimulations of up to 500 Hz
ceeded 0 5 ms. The shortest iatency of the earliest
(data not shown). Intracellular responses of both
depolarizing potential was 1.g ms (mean; range =
types of SNR neurons to STH stimulation consisted
1.2-2.4 ms, n = 11) and that of the second compOof initial short duration (i.e. less than 10 ms) depolarnent was 4.1 ms (mean; range = 3 2-4 6 ms, n = 5).
ization and a sl-~ortduration (i.e. less than 15 ms) hyThese observations indicated that the initial depolar
perpolarizauon followed by a long duration (i.e.
20-40 ms) depolarization- The initial depolarization
51
jsmv
m
A
C
A
D
I lOmV
10 msec
A
Fig 6. Effects of blcuculhne and intracellular mlecnon of CIon the synaptlcresponses induced by STH stimulation. Recordrags m A-C are obtained from an SNR neuron. A: STH stimulation-induced EPSP~ followed 0y IPSPs. B bicuc.iline suppressed the IPSPs and increased the amphtude and the duration of the EPSPs C. extracellular field eotenuals D: the use
of KCl-fdled recording electrode rapidly reversed the polanty
of IPSPs in depolarizing direction.
,zing potentials induced by STH stimulation include
two monosynaptic excitatory postsynaptic potentials
(EPSPs) with different latencies.
The amplitude of the short-duration hyperpolarizing response was increased during intrac¢llular injection of depolarizing current and decreased during hyperpolafizing current (Fig. 5B). These data indicated
that the hyperpolarizing response is an lnh,bitory
postsynaptic potential (IPSP). Precise latencies of
IPSPs could not be determined since they were
masked by the preceding EPSPs. The latency, however, was estimated to be 2-3 ms from the records
obtained during injection of strong depolarizing current, which minimized the overlapping EPSPs (Fig.
5A, arrow on top traces). Fig. 6A shows responses of
an SNR neuron following stimulation of STH. STH
stimulation incuced EPSPs with or without spike followed by IPSPs. Application of bicuculline methiodide (100 mM) markedly suppressed STH-induced
IPSPs and at the same time increased the amplitude
and duration of EPSPs (Fig. 6B). In other experiments, the polarity of the IPSPs was rapidly reversed
in depolarizing direction when KCl-fiiled recording
electrodes were used. The duration of the depolarizing postsynaptic potentials, which include EPSPs and
the reversed IPSPs, was more than 30 ms (Fig. 6D).
As mentioned previously, STH stimulation induced a long-duration depolarizing potential which is
preceded by short-duration EPSPs and IPSPs. The
amplitude of the loo,g duration depolarization was
less than 4 mV and only slightly altered by injections
of depolarizing or hyperpolanzmg currents (Fig.
5B).
Recordings from SNR neurons in the slices obtained from IC-transected rats are shown m Fig 7
In these preparations, STH-induced EPSPs had
much longer durations than those observed in the
normal preparations, while the amplitude of IPSPs
and succeeding small-amplitude long-duration depolarizations was markedly reduced (i.e., compare
Figs. 5B and 7B). As in the normal preparation, the
latency of these EPSPs was fairly constant in spite of
changes in the stimulus intensities (Fig. 7A,B), indicaring a monosynaptic nature of the responses. The
negative field potential observed in the normal preparation was also almost totally abolished m these
preparations (compare the bottom traces in Figs. 7A
and 5A). These results would indicate that the negative field potentml recorded in SNR in the normal
preparation was caused by an activation of the descending fibers passing through or by STH.
DISCUSSION
Electrical membrane properties
It has been known that SNR contains both GABAergic and dopaminergic neurons. The GABAergic
neurons which are main representatives of SNR pro-
52
A
B
]smv
08
5mseo
-OB8
-oB
A
A
F~g 7 STH stimulation-reduced responses reeurded from Iype-I neurons m a shce preparation obtained from an IC-transected rat A:
fast-sweep recordings w~th various stimulus intensities STH st~mulatmn evoked imtml shortoduratmn FPSPs w~thtwo components. B:
slob-sweep recordings The IPSPs and the Iong-durauon depolanzatmn observed m the noLaal preparatmn were not prominent The
negative field potentml (bottom tracesj observed m the normal preparatmn was also markedly reduced (compare to Fig 5)
lect to the thalamus or the tectum and the dopaminergic neurons project to the neostriatum 26'2s'32. The
present data clearly indicate that SNR neurons could
be separated into two types based on their electrical
membrane properties. Type-I neurons were characterized by having a spontaneous &schmge, a short
duration action potential, an ability to generate sustained high-frequency discharges, and a strong
delayed rectification. They are likely to be G A B A e r glc neurons since we encountered this type of neuron more frequently in SNR, and their short duration
action potentials and ability to fire at high frequency
are characteristic properties of SNR G A B A e r g l c
neurons 7 Type-I1 neurons had no spontaneous actlvrues, a long-duration action potential, a strong spike
accommodation and a prominent long-lasting
post-active hyperpolarlzation which was reported to
be caused by a Ca-dependent K conductance and an
early K conductance 16 Type-II neurons also did not
have strong delayed rectification. Since the mere-
brane properties of type-II neurons are very similar
to the neurons recorded from SNC in the slice preparation 16'21, it m~ght be reasonable to assume that they
are dopaminergic neurons located in the SNR.
The differences in the ionic conductance between
the two types of neurons could be summarized as follows. Type-I neurons had a strong voltage-dependent K conductance which might be involved in the
delayed rectification and short-duration action potential seen in this type of neuron. It has been recently proposed that a function of the striatonigral
pathway might be a disinhibition of tonically firing nigrothalamic neurons 5. The strong voltage-dependent
K conductance observed m type-I, presumably
GABAergic, neurons might be the basic mechanism
underlying the sustained high-frequency spike discharge, since they avoid the membrane potential to
reach the Na inactivation level even when they receive tonic excitatory inputs such as from the subthalamic nucleus 15.24. Neither Ca-dependent K conduc-
53
tance nor early K conductance were prominent in this
type of neuron. On the other hand, type-lI neurons
had a strong Ca-dependent K conductance and early
K conductance but a less prominent voltage-sensitive
K conductance. In spite of these differences between
the two types, there were some membrane characteristics common to both. They had a similar range of input resistance. The membranes of both types showed
anomalous rectification when they were hyperpolarized by current injection. Both types of neurons
were able to generate two Ca potentials (i.e., TTXresistant Ca-sensitive slow depolanzauons and slow
action potentials) (Figs. 3 and 4, also see refs. 16,21).
The Ca potentials were observed in SNR neurons
only with a membrane potential more negative than
60 mV, and both responses were markedly augmented by application of TEA to the superfusing medium. These features were very similar to those observed from the neurons of the infenor olive 22, thalamus II, spinal cord 23, SNC 16°21 and S T H 24. The observations suggest that responses of SNR neurons to
phasic synaptic inputs may differ depending on their
level of tonic inputs (i.e. the 6aembrane potential level).
Responses evoked by STH sumulation
We have demonstrated that STH stimulation
evoked a series of responses, monosynaptic EPSPs.
IPSPs, followed by a long depolanzation, in both
types of SNR neurons. The initml short-latency
(1.2-2.4 ms) EPSPs were considered to be mediated
by STH afferents since (1) STH projections to the
SNR are well demonstrated by both anatomical-" ~4
15.35 and electrophysiological studies 3 13 (2) the latency of EPSPs coincides well with the conduction
time of STH neurons to SNR t3 plus a synaptic delay.
and (3) STH-mduced EPSPs were not affected by the
chronic IC transection placed rostral to STH. A previous extracellular single-unit recording str~dy also
suggested that the subthalamonigral pathway is excitatory 9. Recently, electron m~croscop~c analysis of
STH axon terminals labeled with both mtracellular
rejection of H R P and the anterograde neurotracer
Phaseolus vulgaris leucoagglutmm revealed that they
were making asymmetric synapt~c contacts with dendntlc shafts of SN neurons-" t5
The preclse origin of the second monosynapt~c
EPSPs which had longer latencies (3.2-4.6 ms) than
the initial EPSPs could not be determined at this
time. It is most likely, however, that the EPSPs were
not induced by afferents originating rostral to STH,
since the chronic IC transection did not abolish the
response. Simdarly, relatively long-latency (2.8-4.5
ms) monosynaptlc EPSPs were evoked in STH neurons in a slice preparation after stimulation of IC, and
the EPSPs we,e not abolished by chronic IC transection at the level of the entopeduncular nucleus z4.
These observations suggest that both STH and SNR
receive common excitatory inputs originating from
midbrain nuclei (e g., from the raphe nucleus 34,
which has been shown to be excitatory to the neostnatum 27"37, and from the pedunculopontine tegmental
nucleus32.33).
STH stimulation also evoked IPSPs in SNR neurons. We consider that the IPSPs which follow monosynaptlc EPSPs must include Ci-mediated GABAergic inputs since application of bicuculline methiodide
suppressed the IPSPs, and intracellular injection of
CI- reversed their polarity in a depolarizing direction. Even though the IPSPs appeared to have a relatively short latency (2-3 ms), a precise measurement
of the latency could not be made be, ause of the overlapping EPSPs. We consider that the IPSPs were
evoked by stimulation of the striatonigral fibers
and/or pallidonigral fibers since there was a large reduction in the amplitude of IPSPs after chronic transection of the IC. Both the stnatonigral and pallidonigral projections are known to be inhibitory and
GABAerglcS.~0 13.25.29.30.36.39. In addition, an involvement of local inhibitory circuits mediated by axon
collaterals or intrinsic neurons of SNR 4"12 could also
not be dismissed in the generation of the IPSPs.
These local circuits may be responsible for small
IPSPs which persisted after chronic transection of IC.
The chronic IC transection also resulted in a large increase in the amplitude and the duration of STH-induced EPSPs. These results would indicate that the
striatonigral or palhdonigral inhibitory inputs are
very effective in suppressing the subthalamic and
other excitatory inputs to SNR neurons.
It has been well demonstrated that the neostriatum
and STH receive excitatory inputs from the sensorimotor cortex ~'~9"38. SNR receives inputs from both
the neostriatum and the STH. Therefore, there are
two parallel pathways between the sensorimotor cortex and SNR. The striatonigral input is known to be
54
inhibitory. The present study ind,cated that the STHntgral input ~s excitatory. From these previous and
present observations, ~t ~s conceivable that SNR neurons summate cortically derived neostrmtal inhibitory inputs and cortically derived STH excitatory inputs. The observations also ~mply that STH input to
SNR plays slgmficant roles in basal ganglia motor
control functions.
In sum, o-r present data indicate that there are
two types of SNR neurons, having different electrical
membrane characteristics. Our data also clearly indi-
cated that both of these SNR neurons receive monosynaptic excitatory inputs from STH neurons and inhibitory inputs originating from areas rostral to the
STH.
ACKNOWLEDGEMENTS
The authors express thanks to Dr. T. Kita for her
helpful comments during the experiment. This study
was supported by NIH Grants NS 20702 and NS
23886.
REFERENCES
1 Buchwald, N . A , Price, D D , Vernon, L and Hull, C D ,
Caudate intracellular response to thalamlc and cortical inputs, Exp. Neurol, 38 (1973) 311-323
2 Chang, H T , Iota, H. and Iotal, S T , The ultrastructural
morphology of the subthalamo-nlgral axon terminals intracellularly labeled with horseradish peroxidase, Brain Research, 299 (1984) 182-185.
3 Deniau, J M , Hammond, C., Chevalier, G. and Feger, J ,
Evidence for branched subthalamic nucleus projections to
substantla mgra, entopeduncular nucleus and globus pallidus, Neurosct Left, 9 (1978) 117-121
4 Dentau, J M , Kitai, S . T , Donoghue, J P and Grofova, I ,
Neuronal interactions m the substantla nlgra pars retlculata
through axon collaterals of projection neurons, Exp. Bram
Res., 47 (1982) 105-113
5 Deniau, J M and Chevalier, G , Dlslnhlbltlon as a bas,c
process in the expression of strlatal function II The stnatal-nigral influence on thalamo-cort~cal cells of the ventromedial thalamlc nucleus, Brain Research, 334 (1985)
227-233.
6 Fonnum, F., Gottesfeld, Z and Gofova, I , Distribution of
glutamate decarboxylase, choline acetyltransferase m the
basal ganglia of normal and operated rats. Evidence for
striato-palhdal, stnatoentopeduncular and striatontgral
GABAerglc fibers, Brain Research, 143 (1978) 125-138
7 Gulley, R L and Aghajanian, G K , Antldromlc Identification of dopammerg~c and other output neurons of the rat
substant,a nigra, Brain Research, 150 (1978) 69-84
8 Haglwara, S and Byerly, L , Calcmm channel, Annu Rev
Neurosct , 4 (1978) 69-125
9 Hammond, C , Denlau, J M , Rlzk, A. and Feger, J , Electrophyslologieal demonstration of an excitatory subthalamonigral pathway in the rat, Bram Research, 151 (1978)
235-244
10 Hattorl, T , McGeer, P , Fibtger, H C and McGeer, E G ,
On the source of GABA-contammg terminals m the substantla nlgra Electron m~croscoplc autoradlographic and
biochemical studies, Brmn Research. 54 (1973) 103-114
11 Jahnsen, H and Lhnas, R , Ionic basis for the electroresponslveness and oscillatory properties of guinea-pig thalarmc neurons in vitro, J Phystol (London), 349 (1984)
227-247
12 Karabelas, A and Purpura, D P , Evidence for autopsies
13
14
15
16
17
18
19
20
21
22
23
24
25
in the substantia nlgra, Brain Research, 200 (1980)
467-473
IOta, H , Chang, H.T and Iotal, S . T , Pallidal inputs to
subthalamus, intracellular analysis, Brain Research, 264
(1983) 255-265
Iota, H , Chang, H T and Kital, S T , The morphology of
intracellularly labeled rat subthalamie neurons a light microscopic analysis, J Comp. Nearol, 215 (1983) 245-257.
Kita, H and Iotal, S T . Subthalamic afferents to the globus palhdus and the substantia nlgra In the rat light and
electron microscopic studies using PHA-L methods, J
Comp NeuroL, 260 (1987) 435-452
Iota, T., Ktta, H. and Iotal, S T., Electrical membrane
properties of rat substantla nigra compacta neurones in an
In vitro preparation, Brain Research, 372 (1986) 21-30
Kltal, S T and Denlau, J M., Cortical inputs to the subtha!amus" intracellular analysis, Brain Research, 214 (1981)
411-415
Kital, S.T. and Klta. H., Electrophyslologlcal stt,dy of the
neostrlatum in brain slice preparation In R Dmgledlne,
( E d ) , Brain Shce, Plenum, New York, 1984, pp 285-296
Kocsls, J . D , Sugimori, M and Kttai, S T , Convergence of
excitatory synapUc inputs to caudate spiny neurons, Brain
Research, 124 (1976) 403-413
Larsen, K D and Sutm, J , Output orgamzatlon of the feline entopeduncular and subthalamic nuclei, Brain Research, 157 (1978) 21-31
LIlnas, R., Greenfield, S.A. and Jahnsen, H , Electrophysiology of pars compacta cells in the in vitro substantla mgra'
a possible mechanism for dendritic release, Brain Research,
294 (1984) 127-132.
Llinas, R and Yarom, Y , Properties and distribution of
Ionic conductances generating electroresponslveness of
mammahan inferior ollvary neurons in vitro, J. Physiol.
(London), 315 (1981) 569-584
Murase, K and Randlc, M , Electrophyslological propertles of rat ,.,plnal dorsal horn neurones m vitro, calciumdependent action potentials, J Phystol (London), 334
(1983) 141-153.
Nakanlshl, H . Kita, H and Kltal. S T , Electrical membrane properties of rat subthalam~c neurons in an in vitro
shce preparation, Brain Research, 437 (1987) 35-44
Oertel, W H and Mugnaml, E., Immunocytochemlcal
studies of GABAerglc neurons in rat basal ganglia and their
relations to other neuronal systems, Neurosct Lett, 47
55
(1984) 233-238
26 PalkovRs, M and Jacobowltz, D M , Topographic atlas of
catechalamme and achetylcholinesterase-contaimng neurons m the rat brain II. hmdbrain (mesencephalon, rhombeneephalon), J Comp. Neurol, 157 (1974) 29-42
27 Park, M . R , Gonzales-Vegas, J.A and Kital, S.T., Serotonerglc exotation from dorsal raphe ~timulation recorded
mtracellularly from rat caudate-putan'ten, Brain Research,
243 (1982) 49-58
28 Perkins, M ~ and Stone, T W , Iontophoretlc studies on
palhdal h~arons and the projection from the subthalamlc
nucleus, Q J. Exp Phystol , 66 (1981) 225-236.
29 Preston, R.J., McCrea, R.A., Chang, H T. and Kitm, S.T.,
Anatomy and physiology of substantla nigra and retrorubral neurons studied by extra- and intracellular recording
and by horseradish peroxidase labehng, NeuroscIence, 6
(1981) 331-344.
30 Riback. C.E . Vaughm, J.E and Roberts. E., GABAergic
nerve terminals decrease m the substantla mgra following
hemltransectlons of the stnatonlgral and pallidonigral pathways, Brain Research, 192 (1980) 413-420
31 Rouzalre-Dubois, B , Scarnati, E., Hammond, C , Crossman, A.R and Shlbazaki, T , Microiontophoretlc studies
on the nature of neurotransmitter in the subthalamo-entopeduncular pathway of the rat, Brain Research, 271 (1983)
11-20.
32 Saper, C.B. and Loewy, A . B , Projections of the peduncu-
lopontine tegmental nucleus m the rat: evidence for additional extrapyranudal circuitry, Brain Research, 252 (1982)
367-372.
33 Scarnatl, E., Campana, E. and Pacmi, C , Pedunculopontree-evoked excitation of substantla nigra neurons m the
rat, Bran Research, 304 (1984) 351-361
34 Stembusch, H . W . M , D~stnbution of serotomn-immunoreactwity in the central nervous system of the rat-cell bodies
and terminals lVeurosclence, 6 (1981) 557-618.
35 Van der Kooy, D. and Hattori, T., Smgle subthalamlc nucleus neurons project to both globus palhdus and substantla
mgra in the rat, J Comp Neurol, 192 (1980) 751-768.
36 Van der Kooy, D., Hatton, T., Shannak, K and Hornyklew~cz, O., The palhdosubthalam~c projection m rat: anatomical and biochemical studies, Brain Research, 204 (1981)
253-268
37 Van der Maelen, C.P., Bonduki, A.C. and Kitai, S . T , Excitation of caudate-putamen neurons following stimulatmon
of the dorsal raphe nucleus m the rat, Brain Research, 175
(1979) 356-361.
38 Wilson, C J., Postsynaptic potentials evoked m spiny neostnatal projecuon neurons by stimulation of ipsflaterai and
contralateral neocortex, Bran Research, 367 (1986)
201-213.
39 Yoshida, M and Precht, W Monosynaptic mlubltaon of
neurons of the substantia nigra by caudato-mgral fibers,
Brain Research, 32 (1971) 225-228.