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Transcript
Bram Research. 437 ( 1987135-44
35
Elsevier
BRE 13143
Electrical membrane properties of rat subthalamic neurons
in an in vitro slice preparation
H. Nakanishi*, H. Kita and S.T. Kitai
Department of A natomy and Neurobtology. The Untverstty of Tennessee, Memphts. The Health Soence Center. Memphts. TN 38163
¢USA )
~Accepted 2 June 10871
Key words" Rat subthalamlc neuron, Slice preparation, lntracellular recordmg, Membrane propert.~
The electrical membrane properues of subthalamlc (STH) neurons and their response charactensttcs to stimulation of the mternal
capsule (IC) were studmd m an m vitro slice preparation Most STH neurons recorded exhtblted spontaneous repetmve firmg The mput reststance of STH neurons was 146 _+ 48 Mf~ and showed both an anomalous and a delayed recttfication when the membrane x~a~
hyperpolarized or depolarized by current injections In neurons with the membrane potenttal less negative than 65 mV. depolanzmg
current pulses generated repetitive finng with ~he maximum frequency of up to 500 Hz Two types of tetrodotoxm ('I'FXl-resi~tant cobait-sensitive potentials, slow depolarizing potential and slow action potential, were observed m STH neurons The slow depolanzmg
potentml had a long duration (over 500 ms in some cases) and was able to trigger repentlve tmng The slow action potenttal had a duration of about 30 ms and triggered a burst of fmng. The slow action potential was seen only whet, the neurons were hyperpolanzed to
more negative than 65 mV by a current inJection. Electrical stimulation of IC evoked monosynapttc mhibitory postsynaptm potenttals
(IPSPs) m most of the neurons examined The polarity of IPSPs was reversed in the depolarizing dlrectton by intraceUular injectton of
CI- Bath applicauon of bmueulhn¢ markedly suppressed IPSP~ and unmasked monosynaptic excitatory postsynaptlc potenttal~
{EPSPs) The EPSP was able to trigger a slow depolarization with repetitive firing or a slow action potential with burst of firing when
the neuron was hyperpolarized by a continuous current inlecuon The results demonstrated that STH neurons m an m ~ltro preparation have spontaneous discharges, high input resistance, capablhty to generate high-frequency firing, and Ca potentmls The pattern
of responses of STH neurons to synaptlc inputs ~s dependent on their membrane potentials
INTRODUCTION
a n d to the s u b s t a n t i a n i g r a (SN) m t2 15 m~.35 T h e i r
axon t e r m i n a l s f o r m a s y m m e t r i c a l s y n a p s e s m a i n l y
T h e s u b t h a l a m i c n u c l e u s ( S T H ) is a s m a l l lens-
with t h e d e n d r i t e s o f t h e t a r g e t n e u r o n s 9 tg. F u n c t i o n -
s h a p e d n u c l e u s w h i c h lies b e t w e e n t h e z o n a i n c e r t a
al significance o f S T H o u t p u t s has b e e n q u e s t i o n e d
dorsally a n d t h e c e r e b r a l p e d u n c l e v e n t r a l l y 7. M o r -
for s o m e tame, b u t o u r r e c e n t d a t a i n d i c a t e d t h a t
p h o l o g i c a l s t u d i e s using G o l g i , Nissl a n d i n t r a c e l l u l a r
electrical stimulation of STH produces monosynaptic
e x c i t a t i o n to t h e S N n e u r o n s 22 28. S T H n e u r o n s re-
labehng techniques indicated that the somatic shape
of rat S T H n e u r o n s v a r i e s f r o m f u s i f o r m t o oval o r
p o l y g o n a l , a n d 2 - 6 p r i m a r y d e n d r i t e s a r o s e f r o m the
s o m a . H o w e v e r , t h e d i s t r i b u t i o n s o f t h e s o m a size
a n d the n u m b e r o f p r i m a r y o e n d r i t e s a r e u n i m o dal I 12A6. B o t h a n a t o m t c a i a n d e l e c t r o p h y s i o l o g i c a l
ceive m a j o r a f f e r e n t s f r o m G P a n d t h e c e r e b r a l cortex 2 5 8.29.3o 30. In a d d i t i o n , S T H n e u r o n s r e c e i v e p r o j e c t i o n s from t h e p e d u n c u l o p o n t i n e t e g m e n t a l nucleus H ,,9 the d o r s a l r a p h e nucleus 27 32 a n d t h e c e n t r e
m e d i a n p a r a f a s c i c u l a r c o m p l e x 33 a4. E l e c t r o p h y s t o l -
studies i v d i c a t e d t h a t rat S T H n e u r o n s h a v e b i f u r c a t -
ogical studies d e m o n s t r a t e d that i n p u t s f r o m G P a r e
ing a x o n s w h i c h p r o j e c t to t h e g l o b u s p a l l i d u s ( G P )
mhibitory~5 31 while t h o s e f r o m the c e r e b r a l c o r t e x
* Present address Department of Pharmacology. Facultv of
KyushuUmversity, Fukuoka812. Japan
Correspondence. S T Kitai, Department of Anatomy and Neurobiology, The University of Tennessee, McmpMs. The Heahh
Science Center, 875 Monroe Avenue, Memphis. "IN 38163. U S A.
0006-8993187/$03.50 © 1987 EIsevmr Science Pubhshers B V. (Biomedical Dmslon)
36
and the pedunculopontine tegmental nucleus are exotatoryt~ _~u A review of the hterature reveals that
there is only a cursory report on the electrophysiologlcal properties of STH neurons 15. Therefore, we
have studied the electrophystologlcal characterisUcs
of STH neurons in detail using an m vitro slice preparation. The data wdl not only add to the bank of data
on the electrical membrane properties of CNS neurons but also may aid in understanding how STH neurons process their afferent inputs
MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 200-350 g
were decapitated and the brains were rapidly removed The brain was trimmed w~th a razor blade to
a block containing the STH. Parasagittal slices of 400
/~m thick were cut from the block w~th a Vibratome
and were preincubated m oxygenated Krebs solution
for about 1 h at 35 °C before recording. The recordmg chamber was constructed to allow Krebs solution
(35 °C) to continuously flow on the bottom surface of
the slice at a rate of 0 5-0.7 ml/mm and to allow a
warm and moist gas mixture (95% 0 2 - 5 % CO2) to
flow over the top surface of the shce 2~. The Krebs solution was composed of (mM): NaCI 124, KCI 5.0,
KH2PO 4 1.24, N a H C O 3 26, CaCI~ 2.4, MgSO4 1 3
and glucose 10. Glass pipettes filled with 2 M potassium methylsulfate or 1.5 M potassmm chloride with a
DC resistance between 60 and 100 Mf~ were used for
the recording. Intracellular recordings were obtained
through a high-input impedance biological amplifier
with an actwe bridge ctrcmt which enabled measurement of the membrane potential and the injection of
mtracellular constant current s~multaneously The
output of the amplifier was fed mto an osctlloscope
and a DC pen recorder. Electrical stlmulauon was
apphed through a bipolar electrode made by twisting
a pair of 80-/~m-diameter nichrome wires insulated
except at the t~ps, which were separated by 200-400
/~m The stimulating electrode was placed on the surface of the internal capsule at 0.5-1 0 mm rostral to
the STH or on the cerebral peduncle Immedmtely
ventral to the SN pars ret~culata. Stimulation parameters were 0.05-0 2 m A m Intensity and 200~s in duration delivered at 0.5/s
The drugs used were tetrodotoxln (TTX) with a
concentration of 10-5 g/mi, tetraethylammomum
chloride ( I E A ) at 10 mM, and blcuculline methiodlde at 50-100 pM. In some animals, the internal
capsule, at the level of the entopeduncular nucleus,
was transected by a Halazs knife at 6 - 1 0 days prior to
the recording. This was tc eliminate afferents to STH
ortginating from the structures rostral to STH.
RESULTS
The results were obtained from 98 STH neurons
which had membrane potentials of more than 40 mV
and generated action potentials with an amplitude
greater than 40 mV O f the 98 neurons, 92 exhibited
spontaneous firing at a rate of 5 - 4 0 spikes/s. Although the membrane potential of neurons having
higher spontaneous activity tended to be less negative than that of neurons having lower spontaneous
activity, all the other electrical properues of these
neurons were similar.
A
14
C
f25
r v / D /
°
29rr<eC
02
---
0"2 r~A
04
j
lo5n A
"~'/
[ 50
Fig 1. Input resistance of STH neurons A membrane responses to mtraeellularly rejected hyper- and depolarizing currents of vartous mtensmes. B. membrane responses to hyperand depolarizing currents during application of TTX (10-~
g/ml) to ehmmate spikes Square waves at the bottom of oscdlographlc records m this and all subsequent figures indicate the
Intensmes of injected depolarizing (upward square wave) and
hyperpolanzmg (downward square wave) currents Calibrations in A also apply to B C current-voltage relanon for a
neuron recorded m the shce superfused with Tl~X-contalnmg
solution Note membrane recnfication in both hyper- and depolarizing directions
37
The input resistance
The input resistance of STH neurons was measured from the current-voltage relationship obtained from the membrane potential shifts to depolarizing and hyperpolanzing current pulses with a duration of 100 ms (Fig. 1A). In the neurons with spontaneous firing, the measurement of input resistance
was obtained during continuous applications of a
small hyperpolarizing current (i.e., less than 0.1 nA)
or TTX (Fig. 1B), which eliminated spontaneous
spikes. The input resistance calculated from the slope
of the current-voltage curves (Fig. IC) crossing at
zero current pulse was 146 _+ 48 MQ (mean + S.D., n
= 26). The input resistance was decreased upon apphcation of large hyperpolarizing current (an anoma-
A
o
B 530
Hzl
....
/
./7,,2"
/
/" /
./ .°
/-
c
7
0
C
0~3
0'6 nA
nA D'9
I 20 mV
n
0
Fig. 2 A- spike discharges generated by depolarizing current
pulses with different intensities. Current intensity is indicated
on the left margin of each trace In order to eliminate spontaneous firing, a hyperpolanzmg current of 0 07 nA was continuously inJected in the neuron B: relations between the discharge frequency and the intensity of current pulses obtained
from the interspike interval of first two spikes (1/tl) and last
two spikes (l/t2) in the cell of A. C: injection of depolarizing
current pulse in continuously depolarized neuron produced repetitive firing followed by a long-lastinghyperpolarizlng potenual after the termination of currenf pulses. Dotted lines in this
and all the subsequent figures indicate zero current levels D
superfusion of Ca2+-free medmm dlmimshes the long-lasting
hyperpolanzlng potential Calibration m C also applies to D
lous rectification) (Fig. 1A,B). A reduction of the input resistance was also observed during membrane
depolarization (a delayed rectification) (Fig. 1B,C)
in the slice superfused with Tl'X-contaming solution.
Direct activation by mtracellular stimulation
Injections of depolarizing current pulses to STH
neurons produced either repetitive or burst discharges. The duration of action potentials was about
1 ms. When neurons with a membrane potential of
40-65 mV were activated by the rejection of current
pulses, single or repetitive action potentials were
generated either from passive depolarization (Fig.
2A) or active slow depolarization which often outlasted the duration of the current pulses (Fig.
3 A - C ) . Action potentials generated from passive
depolarization had the highest frequency of firing at
the beginning of the current pulse (Fig. 2A). The relationship between the intensity of injection current
and the frequency of firing ( l - f curve) obtained from
the first interspike interval following the onset of the
current was almost linear up to 300 Hz of firing but
deviated downwards from the linearity at higher frequencies. Z~s can be seen from the graph, the STH
neuron could fire at the maximum frequency of about
500 Hz (Fig. ?B). The I - f curve obtained from the
last interspike interval was almost linear up to 200 Hz
and reached its peak at over 300 Hz. The slope of the
linear portion of the l - f curve for l/t1 (Fig. 2B) is
about 900 Hz/nA, indicating that STH neurons are
extremely sensitive to small changes in their excRatory inputs. Repetitive firings terminated by the offset
of current pulses were followed by long-lasting
(250-600 ms) hyperpo' arizing potentials with an amplitude of 5-12 mV (Fag. 2C). The long-lasting hyperpolarizing potential was not affected by the intracellular Cl- injection (not shown) but was diminished
by superfusing Ca2+-free medium (Fig. 2D).
In some STH neurons with a membrane potential
of 50-65 mV, depolarizing current pulses evoked
slow depolarizing potentials with action potentials
(Fig. 3 A - C ) . Fig. 3A, B shows responses to 3 different intensmes (Fig. 3A) and durations (Fig. 3B) of
depolarizing current pulses applied to a continuously
hyperpolarized neuron. Current pulses with stronger
intensity or longer duration evoked all-or-none slow
depolarizing potentials which clearly outlasted the
duration of the apphed current pulses. The slow de-
38
05nA
22
I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " "
I
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I
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Fig 3 Slow depolarizing potentials evoked by depolarizing current pulses in neurons continuously hyperpolanzed by current mlectlon. A and B: injection of depolarizing current pulses at 3 different mtensmes m A and 3 different durations in B C rejection of depolarizing current pulse induced slow depolarizing potentials with repetmve sp~ke firing Note that depolarizing potentmls m A - C outlast the duration of injected current pulses D recording from the same neurons as C during superfusmn with Ca2+-free medium Note
a great reductmn m the amplitude and durat'on of slow depoh, rizlng potentials after the offset of the current pulse
p o l a n z m g potential with the duration o f m o r e than
500 ms was o b s e r v e d in some neurons. A s shown m
Fig. 3C, the slow depolarization could trigger repetltwe finngs m which the frequency of firings increased
along with the d e v e l o p m e n t of the slow depolarization. The slow depolarizing potential was TTX-resis-
rant (figure not s h o w n ) but suppressed by superfusion o f Ca2÷-free m e d m m (Fig. 3D)
W h e n S T H n e u r o n s were h y p e r p o | a n z e d to a
m e m b r a n e potential m o r e negative than 65 m V by a
c o n t i n u o u s current rejection, depolarizing c u r r e n t
pulses p r o d u c e d a burst of fast actmn potentmls
which were triggered f r o m an all-or-none relatwely
large slow action potential with a duration o f a b o u t
30 ms (Figs. 4 A and B) Fig. 4 A shows responses o f a
continuously h y p e r p o l a r i z e d n e u r o n to 4 different intensities o f depolarizing current pulses with a con-
A 11 l/II 2om_
stant duration
............
_~
,......................
~
I
. . . . . . . . . . . . . . . . . .
[05nA
~
T h e lowest intensity of stimulation
I |
D
.-.I
g..__.....___
_A-
q
-..J
--4__
Fig. 4 Slow acuon potentials evoked by depolarizing current
pulses in STH neurons The neuron was continuously hyperpolanzed by current rejection m A - D A and B' rejection of depolarizing current pulses at various lntensmes m A and different duratmns m B C recordings durmg superfuslon w~th a medium containing TEA (10 mM) InJection of depolanz,ng current pulse reduced a slow action potentml w~th sp~kes Note an
mcrease m the spike duration D and E recordings during superfus~on w~th a medium containing TEA and "lq'X Injection
of depolarizing current pulses with various intensities dunng
continuous hyperpolarizatmn in D and during depolarization in
E F. addition of Co 2÷ to the superfusmg medium abohshed
~low action potentials
39
failed to evoke regenerative responses, but 3 other
sttmulations generated slow action potentials. The
rising rate of slow action potentials was increased
with the increase in stimulus intensity. Responses to
two different durations of current pulses apphed to
the same neuron are shown in F~g. 4B. The slow depolarizing potential triggered by a short-duration
pulse can be distinguished from the slow action potential triggered by a long-duration pulse (Fig. 4B).
The slow action potential with a burst of firings was
also triggered after the offset of hyperpolarizing
pulses (Ftg. 1A).
Ionic basis of regenerattve potentials
Bath application of 10 mM TEA increased the input resistance (up to 80%) of the cell and the duration of the fast actton potenttal due to a decrease ~n
the falling rate (Fig. 4C). Application of TI'X (10 5
g/ml) to the TEA-containmg medium abolished fast
action potentials. Und"r these c,~nditions, injectton
of depolartzing curren', pulses to either continuously
hyperpolarized (Fig. aD) or depolarized (Fig. 4E)
neurons triggered Iong-duration slow action potenttals. Slow action potentials generated from the depolarized neurons were smaller in amplitude and
slower in the rising and falling rates than those from
the hyperpolarized neurons. Their duration often
outlasted the duration of the current pulse (Fig. 4E).
Both the slow depolarizing and the slow action potentials were completely abohshed by an apphcation of
Co '-+ (3 raM) to the superfusing medium (Fig. 4F) It
was also often observed that application of Co 2+ resulted in an increase of the input resistance fcompare
Fig. 4D,F) which is probably due to a blockade of the
leak Ca conductance of the neurons.
Responses to the internal capsule stimulation
StimulaUon of the mternal capsule (IC) at 0.5-1.0
mm rostral to STH evoked hyperpolarizing potentials in most of the STH neurons examined The sttmulation also evoked a negative field potential when
recordings were made from the area close to the cerebral peduncle which lies ventrolaterally to STH
(e.g., Fig. 5B). The field potential was diminished in
the slice preparation obtained from chronically ICtransected rats, indicating that the response was
caused by an activation of the descendmg fibers passing through the IC. The amplitude of the hyperpolarizing potential evoked by IC stimulation was increased by injection of depolarizing current and decreased by hyperpolarizing current. The polarity of
the hyperpolarizing potential could be reversed to
B
A
.
C
smv
o4
Fig 5 Synaptlcresponses m STH neurons followingstimulationof IC immediatelyrostral to STH. A stimulanonof IC ehctted large
IPSPs Inlecuon of depolarizingcurrent increases the amphtudeof IPSPs (top trace) and hyperpolanzmgcurrent of 0 2 nA decreases
the amphtudeof IPSPs Reversalof the IPSPswnh 0.5 nA current rejection(bottomtrace) B IPSPsevokedby variou~sumulusmtensines (top traces) Reversalof the IPSPs with CI- mlecuon(middle traces) Extracellularfield potennals (bottom tr,~ces) C stimulation of IC ehclted EPSPs with a spike potential Apphcatlonof 0 4 nA depolarizingcurrent (top traces) and hyperpolanzmgcurrent
(bottom traces).
40
A
I/Ill
!///
B
5mV_
20 msec
0 nA
.,z,..__
,-.h-_.__
Ftg. 6 Effects of blcucullme on the synaptlc responses reduced by IC sttmulatlon A. blcuculhne suppressed lC-mduced IPSPs and unmasked EPSPs Depolarizing current mjectlon produced a barrage of spikes (top trace) Hyperpolanzmg current rejection increased
EPSP amphtudes and reduced its durauons (bottom two traces). B. EPSPs evoked by vartous sumulus intensities. C. EPSPs evoked
durmg mject~on of a continuous hyperpolanzmg current trtggered slow depolarizing potenuals (top and middle traces)
the depolarizing direction by the injection of a strong
hyperpolartzmg current (Fig. 5A). The results ln&cated that hyperpolartzlng potentmls reduced by ICsttmulation were inhtbitory postsynaptic potenttals
(IPSPs). The 1PSP was constdered to be monosynaptically mduced smce the change of the latency was
very small (no more than 0.3 ms) and graded upon mcrease m the stimulus intenstty (F~g. 5B). The shortest latency observed in 19 neurons ranged between
1.2 and 2 8 ms (mean = 1.7). When KCl-filled electrodes were used, the polarity of the IPSPs reversed
itself m the depolanzmg dtrection within a few minutes after a penetration into the neuron (Ftg 5B).
Bath apphcauon of blcuculhne methlodide (50-100
uM) markedly or even completely suppressed the
IPSPs (Ftg 6A). These results indicate that the IPSPs
were GABAerglc and Ci-mediated responses It was
observed during bicuculline apphcatlon that IC sumulation evoked depolarizing potentials in STH neurons The latency of depolartzmg potentials (mean =
3 5 ms, n = 6) did not change more than 0.3 ms upon
mcrease in stimulus intensities (Fig. 6B). The amplitude of the depolartzmg responses was decreased by
continuous mlection of depolarizing current and increased by hyperpolarizing current (Fig. 6A) These
results would indtcate that the depolarizing re-
sponses include monosynaptic excitatory postsynaptic potentials (EPSPs). The duration of the depolarizmg response could be largely altered with changes in
the stimulus mtensity (i.e., less than 20 ms to over
400 ms). Fig. 6C shows responses of a continuously
hvperpolartzing neuron to IC stimulations with 3 &fferent intensities. The lowest-intensity sttmulation
evoked depolarizing potential with a relatwely small
amplitude and a short duration. A slight increase m
stimulus mtensity, on the other hand, led to depolarizations with much larger amphtudes and longer durations The duration of depolarizing responses was
also found to be related to the membrane potential of
the neuron recorded. As can be seen in Fig. 6A,
membrane hyperpolartzattons by constant current mjecttons reduce the duration of depolarizing responses.
In most of the neurons the EPSPs were masked by
preceding larger IPSPs. In 5 neurons, however, IC
stimulation evoked EPSPs followed by IPSPs (Fig.
5C). During mjection of continuous hyperpolanzmg
current, a burst of firing was triggered from the
EPSPs (Fig. 5C). In the slice preparauon obtained
from animals which recewed chromc kmfe cuts at the
level of EP, stimulatton of IC immediately rostral to
STH evoked monosynapttc EPSPs overlapping wtth
41
small IPSPs m all the neurons tested (n = 8L These
responses were very similar to those shown in Fig
5C. In some experiments the stimulation electrode
was placed on the cerebral peduncle ventral to the
SN m the slice obtained from normal ammals Stimulation of the cerebral peduncle in this preparation
also evoked EPSPs similar to those shown m Fig. 5C,
but the latency (1.0-2.5 ms; n = 4) of EPSPs was
considerably shorter than that evoked by IC stimulation.
DISCUSSION
Shce preparations
We employed an in vitro slice preparation since
STH is small and located deep in the brain making it
difficult to reach the nucleus and to obtain a stable intracellular recording in in vivo preparations The m
vitro slice preparations, on the other hand, allow
easy placement of the recording and stimulating electrodes under visual guidance and stable intracellular
recordings without pulsations. Slice preparations
also allow an investigator to manipulate the chemical
environment of the recording neurons by changmg
the chemical composition of superfusing medm 2~.
Passivemembrane properties
The mean input resistance (146 MQ) of rat STH
neurons obtamed from the present in vitro slice preparatlon was about 8 time~ h~gher than the value obtained from rat STH neurons in an in vivo preparation 15 Factors that could be responsible for the differences in the values obtained between in vitro and
in vivo preparations include the following. The compositron of the extracellular fired is different in the in
vitro preparation from that in the in vivo preparation. The nucleus is isolated from the extrinsic circuitries which results m a lack of tonic synaptic inputs in
in wtro preparation. Another difference is that the
neurona,~ processes (e.g., dendrites and axons) may
be severed in the slice preparation, reducing the area
of the surface membrane which would influence the
input resistance The resting membrane potentials of
the neurons analyzed in the in vivo preparatmn
vaned from 15 to 40 mV. while tho-e of the neurons
analyzed in thts study were more than 40 mV. The
difference in the membrane potential also would influence the input resistance since the present in vitro
study revealed that the membrane of STH neurons
has strong delayed rectification.
Spontaneousfirings
Most of the STH neurons studied had spontaneous
firings of 5-40 Hz. Spontaneous extraceilular umt
discharges (23.3 _+ 9.8 Hz; mean _+ S.D.; n = 26)
were also frequently encountered in STH of slice
preparations (Nakamshi et al., unpubhshed observation). We considered that the spontaneous firing was
caused by a relatively low resting membrane potential of STH neurons, low threshold for Na-spike and a
strong delayed rectification, probably due to voltagedependent K-conductance, which may prevent spike
accommodation. The membrane potential of STH
neurons reported in this study falls between 40 and 65
mV. These relatively low resting membrane potentials of STH neurons, however, are not likely to be a
result of damage to the neuron by electrode penetrauon since (1) the recording was stable and could be
maintained for at least 1 h, (2) the input resistance
was relatively high (70-250 M~), and (3) no large
changes in the rate of spontaneous discharge were
noted after penetratmn of the neuron. Llimis and Yarom have described a sequence of events that causes
the slow rhythmic discharge (4-10 Hz) of inferior olivary neurons 26. The events include Na spike, highthreshold Ca spike, Ca-dependent K conductance
and low-threshold Ca-spike. However, spontaneous
firings of STH neurons may not be due to Ca-dependent potentials since (1) the finng frequency is relatively high and has a wide range (5-40 Hz), (2) substitution of Mg2÷ in the Krebs solution for Ca -`÷ does
not cause a big change in the frequency nor in the pattern of spontaneous unit firing (Nakamshl et al., unpublished observation), and (3) no spike after depolarization which might correspond to a high-threshold Ca spike can be detected (i e., the duration of the
action potential was about 1 ms).
Ca-potennals
Recent electrophyslological studies revealed that
the neurons in many areas of the central nervous system are able to generate two or 3 different types of
Ca_potentials~-6 13 14.171823-26.39 The Ca potentmls
are thought to subserve a variety of functions in neuronal actwiv.es such as an intrinsic oscillatory mechanism, a generation of burst firings, an integration ,f
42
synapuc Inputs and a release of neuroactive substances. In this study we have revealed that STH neurons possess two distinct Tl'X-resistant potentials,
namely the slow depolarizing potential and the slow
action potential. These potentials were considered to
represent activation of inward Ca currents since their
generation was blocked by superfusion of Ca2+-free
medium or application of Co -'+ which is known to
block Ca conductance 13 The slow depolarizing potential, with the characteristics of slow rising, long
duration and relatively small amplitude, had similar
characteristics to the chick sensory neurons 5 6 and the
Ca-dependent plateau potential recorded in the dendrltes of cerebellar Purkinje cells~-5. The slow depolarizing potentials are generated by depolarizing current pulses In many STH neurons having a membrane
potential of more than 50 mV The present study suggested that EPSPs could trigger the slow depolarizing
potential, since the duration of depolarizing responses induced by IC stimulation was greatly altered by the changes in the stimulus intensities or
membrane potential levels. Then, the slow depolarizing potential may play an essential role in the response to excitatory inputs since it could strongly enhance the duration and the amphtude of postsynapUc
excitation which leads to triggering repetitive spikes.
It has been reported that stimulation of the cerebral
cortex evoked a large, long-lasting depolarizing potential accompanied by a multiple firing of spikes in
STH neurons 2° These response patterns are clearly
in contrast to those of excitatory responses recorded
in neostnatal neurons w'as The response of the neostrlatal neurons after stimulation of the cerebral cortex is a short-duration depolarizing potential with
one or two spikes. It may be that the difference in the
response pattern in the two nuclei is a result of the
generation of slow depolarizing potentials in STH
neurons.
In this study, we have ob ,erred another Ca-dependent potential different from the slow depolarizing
potential. We call this potential 'slow action potential', distinguished from the slow depolarizing potential by its fast rate of rise, a short duration and a large
peak amplitude. In the preparation superfused with
Krebs solution, generation of this po,entla, gas seen
only in the neurons with a memb~ ane potential more
negative than 65 mV. These phenomena indicate
that the Ca conductance responsible for the slow ac-
tlon potential as inactivated m the depolarized membrane, as has been previously observed in other central nervous system neurons 5 6 ~3.17.,3-26. In preparations superfused with TEA-containing Ringer, however, the slow action potential was generated from
neurons having membrane potentials less negative
than 65 mV. The experiment with TEA revealed that
the termination of the slow action potential involves
activation of K conductances (i.e., probably both
voltage- and Ca-dependent K conductances) since
T E A effectively prolonged the duration of the potenual. Th~s result was consistent with the reports tor
other CNS neurons 3"4"17"25"26 As in the case of slow
depolarizing potential, the slow spike potential could
also enhance excitatory synaptlc inputs (Fig. 6B).
The enhancement of excitation by the slow spike potential differs from that by the slow depolarizing potential, since the slow sp~ke potential occurs only in
the neuron which has a resting membrane potential
of more than 65 mV and can trigger bursts of spikes.
Origins of IC sttmulatton-induced IPSPs and EPSPs
Stimulation of IC evoked short-latency monosynaptlc IPSPs with overlapping EPSPs in STH neurons.
The IPSPs were considered to be GABAerglc and
Cl-mediated since they were blocked by application
of bicuculline and their polarity was reversed by intracellular injections of CI-. These IPSPs were consldered to be induced by stimulation of the axons
originating from the GP and/or the entopeduncular
nucleus (EP) since a chromc transection of the IC at
the level of the EP resulted in a drastic reduction m
the IPSPs reduced by IC sttmulation. A support for
this interpretation also comes from a recent in vwo
electrophysloioglcal study which showed that stimulation of GP produced monosynaptic large-amphtude
and short-duration IPSPs in STH neurons 15. These
IPSPs are similar to those obtained m this study The
origin of EPSPs evoked by IC stimulation, however.
remains unclear. Kltai and Deniau 2° reported that
stlmulauon of the cerebral cortex evokes monosynaptlc EPSPs in STH neurons. However, we consider
that the EPSPs observed in the present study are not
cortical in origin for the following reasons" (1) the
EPSPs evoked by cortical sumulatlon had much
shorter latency (mean 2.5 ms) than the latency (mean
3.5 ms) of the EPSPs observed after IC stimulation,
and (2) the EPSPs to IC stimulation were not
43
a b o l i s h e d by c h r o m c IC t r a n s e c t i o n . Possible sources
self-regulate a n d m t e n s f f y their excitatory i n p u t s
for the E P S P s are the acttvation of a x o n collaterals o f
(e.g., cortical origin) t h r o u g h C a - d e p e n d e n t p o t e n tials.
S T H n e u r o n s p r o j e c t m g to G P I" 16 the p e d u n c u l o p o n t l n e t e g m e n t a l n u c l e u s ~-~9 a n d the r a p h e n u cleus27 32 M o r e studies are n e e d e d to clarify the origin of these E P S P s .
ACKNOWLEDGEMENTS
In s u m m a r y , the p r e s e n t study d e m o n s t r a t e d that
S T H n e u r o n s are u n d e r an m f l u e n c e o f G A B A e r g i c
T h e a u t h o r s express t h a n k s to Dr. T. K~ta for her
and C I - m e d i a i e d i n h i b i t o r y i n p u t s o r i g i n a t i n g from
helpful c o m m e n t s d u r i n g the e x p e r i m e n t . This study
the globus ,~alildus. T h e s e n e u r o n s , h o w e v e r , are ca-
was s u p p o r t e d by N I H G r a n t s NS 20702 a n d NS
23886.
pable of h i g h - f r e q u e n c y firing a n d possess features to
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