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Response characteristics of subthalamic neurons to the stimulation of the sensorimotor cortex in the rat
Brain Research, 609 (1993) 185-192 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
185
BRES 18739
Response characteristics of subthalamic neurons to the stimulation of the sensorimotor cortex in the rat K. Fujimoto * and H. Kita Department of Anatomy and Neurobiology, The University of Tennessee Memphis, College of Medicine, Memphis, TN 38163 (USA) (Accepted 17 November 1992)
Key words: Sensorimotor cortex; Subthalamic nucleus; Unit and intracellular recording; Experimental brain lesion
Responses of the subthalamic nucleus (STH) neurons to the stimulation of the sensorimotor cortex (Cx) were recorded in intact rats and in those which received lesions in the pallidum, the neostriatum, the brainstem, or the corpus callosum. Most of the STH units (78%) exhibited two excitatory peaks which were interrupted by a brief period of inhibition. Some of units which were located in the peripheral part of the STH tended to lack the brief inhibitory component and exhibited a long period of excitation. These excitations were followed by a long-lasting inhibitory period. Intracellular recording indicated that these responses were EPSPs interrupted by a short IPSP and a long period of disfacilitation of Cx inputs. A quinolinic acid lesion of the neostriatum and a knife cut of the brainstem failed to alter these responses, while an ibotenic acid lesion of the globus pallidus abolished the short inhibition seen in the midst on the excitation. Stimulation of contralateral Cx also evoked excitatory responses in the STH. The responses were completely eliminated by a parasagittal knife cut of the rostral part of the corpus callosum.
INTRODUCTION 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 l o c a t e d v e n t r a l l y to t h e z o n a i n c e r t a a n d d o r s a l l y to t h e c e r e b r a l p e d u n cle. T h e i n v o l v e m e n t o f t h e S T H in m o t o r c o n t r o l is well d o c u m e n t e d 2'7'16. It has b e e n d e m o n s t r a t e d that
the
sensorimotor
c o r t e x (Cx) p r o j e c t s
to t h e
STH 1'3'4'13'15'22'24. This d i r e c t C x - S T H p a t h w a y is cons i d e r e d to play an i m p o r t a n t role in m o v e m e n t c o n t r o l 14. B a s e d o n k n o w n a n a t o m i c a l connections, t h e S T H s h o u l d also receive p o l y s y n a p t i c i n p u t s f r o m the Cx. T h e p o l y s y n a p t i c p a t h w a y s i n c l u d e t h o s e t h r o u g h t h e globus p a l l i d u s (GP), t h e d o r s a l t h a l a m u s , t h e s u b s t a n t i a nigra, a n d o t h e r b r a i n s t e m nuclei including t h e p e d u n c u l o p o n t i n e nucleus, t h e locus ceruleus, a n d t h e r a p h e n u c l e u s 3,4,6A°JT'29,3°. E l e c t r i c a l s t i m u l a t i o n o f t h e Cx evokes a shortl a t e n c y a n d relatively l o n g - d u r a t i o n excitation in t h e S T H , which was o f t e n i n t e r r u p t e d by a b r i e f inhibitory p e r i o d 5J3'24'26. A p r e v i o u s i n t r a c e l l u l a r r e c o r d i n g study i n d i c a t e d t h a t t h e excitation is d u e to t h e m o n o s y n a p tic i n p u t f r o m t h e Cx 13. H o w e v e r , t h e m e c h a n i s m un-
d e r l i n g t h e relatively l o n g - d u r a t i o n excitation a n d t h e p a t h w a y r e s p o n s i b l e for t h e b r i e f inhibition a r e unknown. W e c o n s i d e r t h e s e issues i m p o r t a n t in o r d e r to u n d e r s t a n d how t h e Cx r e g u l a t e s t h e activity o f the STH. A n e l e c t r o p h y s i o l o g i c a l study s u g g e s t e d the exist e n c e o f b i l a t e r a l m o n o s y n a p t i c C x - S T H p r o j e c t i o n s 24, while n o n e o f t h e a n a t o m i c a l studies r e p o r t e d any Cx p r o j e c t i o n to t h e c o n t r a l a t e r a l S T H 1,3.4,15,22. T h e physiological study implies that t h e Cx directly c o n t r o l s b o t h sides o f t h e STH. O u r s e c o n d objective of this study was to f u r t h e r e v a l u a t e t h e c r o s s e d C x - S T H projection. In o r d e r to study t h e s e questions, we assessed the r e s p o n s e s o f t h e S T H n e u r o n s to t h e s t i m u l a t i o n of the Cx in intact rats a n d in t h o s e which r e c e i v e d lesions in t h e GP, t h e n e o s t r i a t u m , the b r a i n s t e m , or the c o r p u s caUosum. MATERIALS AND METHODS The experiments were performed in 20 male Sprague-Dawley rats weighing 240-310 g. All rats used for the recording experiments were initially anesthetized with urethane (1.2 g/kg, i.p.) and supple-
Correspondence: H. Kita, Dept. of Anatomy and Neurobiology, The University of Tennessee Memphis, College of Medicine, 875 Monroe Ave., Memphis, TN 38163, USA. Fax: (1) (901) 528-7193. * Present address: Jichi Medical School, Department of Neurology, Mimamikawachi-machi, Tochigi-Ken 329-04, Japan.
186 mented with doses of a Ketamine (30 m g / k g , i.p.) and Xylazine (3 m g / k g ) mixture as needed. The animals were placed in a stereotaxic apparatus. Body temperature was kept at 37°C by a thermoregulated electrical heat pad. Craniotomies exposed the cortical surface and allowed access to the recording area and placement of stimulating electrodes. Bipolar stainless steel electrodes consisting of pairs of insect pins (No. 000), insulated with Epoxylite except for 0.2 mm at the tip and separated by a distance of about 0.7 ram, were used for stimulating the deep layers (layers 5 and 6) of the left Cx (3.0 mm anterior to the bregma, 2.0 mm and 2.7 mm to the midline, 1.8 m m below the cortical surface). In 2 rats, stimulating electrodes were placed on both sides of the Cx using the same coordinates (Fig. 1A is an example of the placement of a stimulating electrode). Rectangular current pulses of 0.1 ms in duration with an intensity of 50-250 > A and a frequency of 0 . 6 7 / H z were used for stimulation. All of the recordings were obtained from the left side of the brain. In order to obtain a stable recording and also to aid for searching the location of the STH, the overlying Cx tissues and the dorsal hippocampus were removed by suction to expose the dorsal surface of the thalamus prior to the recording. The cavity was filled with warm paraffin which served to reduce brain pulsations and prevent drying. The reference electrode was inserted between the bone and the cranial muscles. Unit recordings were obtained with a single glass electrode (tip diameter 0.8 # m ) filled with 2 M NaCI (10-15 M~Q). Units with initially negative peaks were isolated by a window discriminator, digitized on-line, and recorded on tape. Single
unit activity was deemed acceptable if a 2:1 signal to noise ratio or better was maintained throughout the recording. Digitized spike trains were fed to a computer (Macintosh llci) using the program Spike Train (generously provided by Dr. J.M. Tepper) to obtain the peristimulus time histogram (PSTH). The PSTH was constructed from 50 or 100 stimulation trials using 1-ms bins. The existance of excitatory or inhibitory responses was determined by a visual inspection for at least three consecutive bins with the number of spikes more than 50% more or less than prior to the stimulation. The first bin of such change was used for the estimation of onset and offset times of the responses. In order to histologically identify the recording sites, electrodes used for the last recording were kept in situ and glued on the skull with dental acrylic resin. The carefull removal of the electrodes from the skull after the aldehyde fixation of the animals left a clear impression of the electrodes in the brain. The impression was used to determine the location of the tip of the electrodes and the plane (or angle) of electrode penetration relative to brain structure. Intracellular recordings were obtained with a glass electrode filled with 2% N-(2-aminoethyl) biotinamide (Neurobiotin, Vector Labs.) dissolved in 0.05 M Tris buffer (pH 7.4) and 0.5 M potassium methylsulfate 9. The resistance of electrodes measured in Ringer's solution was 60-100 Mg2. Amplified neuronal signals were fed to an oscilloscope, an audio monitor, and a tape recorder. Intracellularly recorded neurons were injected with Neurobiotin by passing rectangular pulses of 2 nA with 250 ms duration at 2 Hz for 3-10 min.
Fig. 1. A: a coronal section of a rat brain shows the location of the stimulus electrodes in the sensorimotor Cx. B: a sagittal section shows an example of the quinolinic acid lesion in the n e o s t r i a t u m C: a sagittal section shows an example of the ibotenic acid lesion in the GP. D and E: two sagittal sections of the STH show neurons intracellularly recorded and subsequently labeled with N-(2-aminoethyl) biotinamide. Str, neostriatum; ic, internal capsule; ZI, zona incerta; STH, subthalamic nucleus; GP, globus pallidus; cp, cerebral peduncle.
187 In some rats, the GP or neostriatum was lesioned by injection of either ibotenic acid or quinolinic acid 10-14 days prior to recording. We found that an ibotenic acid lesion of the GP was more reliable than quinolinic acid (which is less expensive than ibotenic acid). However, both ibotenic acid and quinolinic acid lesions were reliable in the neostriatum in the anesthetic condition we used. Rats were anesthetized for the injections with a Ketamine (100 mg/kg i.p.) and Xylazine (3 mg/kg) mixture. Ibotenic acid was dissolved in 0.1 M sodium phosphate buffer (pH 7.4) in such a way that a volume of 0.4 p.1 contains 20 nM. Two injections, each of 0.2 p.1, were performed in the OP at stereotaxic coordinates 21 of -0.5 mm to bregma (AP), 2.5 mm left of the midline (L), and 6.0 mm below the surface of the Cx (DV) and -1.5 mm AP, 3.5 mm L, and 6.0 mm DV. For the neostriatal lesion, quinolinic acid 80 nM was dissolved in 0.8 ~1 of 0.1 M sodium phosphate buffer (adjusted pH 7.4 with 1 N NaOH) and injected in the left neostriatum at the coordinates of 0.5 mm AP, 2.7 mm L, and 4.5 mm DV. This dosage is less than that which was used in the previous reports 2s in order to minimize the lesion spread beyond the neostriatum. The injection of excitotoxins was accomplished by using a Hamilton syringe at a rate of 0.05 ~.l/min. Some rats were subjected to an acute (i.e., during the recording session) knife cut in the brain. In the 2 rats which received bilateral implantation of the stimulating electrodes in the Cx, the corpus callosum was cut parasagittally with a Halazs knife at the coordinates between 1.5 mm and - 3.5 mm AP, 0.5 mm right of L. In 2 other rats, knife cuts were made in the brainstem immediately caudal to the substantia nigra ( - 6.5 mm AP). At the end of the experiments, rats were deeply anesthetized and perfused intracardially with Ringer's solution followed by a fixative of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were postfixed overnight at 4 °C in the same fixative and cut into 40-/~m serial, coronal or sagittal sections on a Vibratome. Sections from brains used in the unit recording were mounted and stained with Cresyl violet to verify the placements of the stimulating and recording electrodes and also to examine the extent of the lesion made by the injection of ibotenic acid or quinolinic acid. Sections from the brains performed intracellular recordings and labelings were rinsed in 0.02 M potassium phosphate buffer saline solution (KPBS, pH 7.8), incubated for 2 h in 1.0% Avidin-Biotin Peroxide Complex (Vector Labs.) in KPBS with 0.5% of Triton X, and then rinsed with KPBS. Sections were reacted with diaminobenzidine (0.05%) and H20 2 (0.003%) for 10-30 min. The sections containing intracellularly labeled neurons were postfixed in 0.5% osmium tetroxide in 0.15 M phosphate buffer (pH 7.4) for 30 min and embedded in Epon-Araldite 812 plastic. The remaining sections were mounted and stained with Cresyl violet to verify the placement of the stimulating electrode.
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Fig. 2. A: a spike train (digitalized usmg a program Spike train) obtained from a unit in the ventral part of the zona incerta, displaying a relatively regular spontaneous firing pattern. B: a spike train obtained from a unit in the STH, showing an irregular, bursty spontaneous firing pattern. was encountered
first in t h e d o r s a l t h a l a m u s . P a s s i n g
t h r o u g h a s i l e n t z o n e o f 200 to 400 ~ m d i s t a n c e , w h i c h c o r r e s p o n d e d to t h e d o r s a l p a r t o f t h e z o n a i n c e r t a , a t h i n l a y e r ( a b o u t 100 ~ m thick) o f n e u r o n s e x h i b i t i n g a relatively regular Three
neurons
firing was encountered
in this l a y e r w e r e
(Fig. 2A).
i n t r a c e l l u l a r l y in-
jected with Neurobiotin, and the location of the neur o n s was v e r i f i e d as b e i n g w i t h i n t h e v e n t r a l p a r t o f t h e z o n a i n c e r t a i m m e d i a t e l y d o r s a l to t h e S T H . T h e n , a f t e r p a s s i n g t h r o u g h a n o t h e r e l e c t r i c a l l y silent z o n e , w h i c h w a s a b o u t 30- to 5 0 - ~ m thick, a n a r e a w i t h m a n y v i g o r o u s , i r r e g u l a r l y firing n e u r o n s
was e n c o u n t e r e d
(Fig. 2B). H i s t o l o g i c a l e x a m i n a t i o n r e v e a l e d t h a t this area corresponded
to t h e S T H . A l l t h e tracks o f t h e
final r e c o r d i n g e l e c t r o d e u s e d f o r t h e u n i t r e c o r d i n g w e r e w i t h i n t h e S T H . I n t r a c e l l u l a r r e c o r d i n g a n d stain-
RESULTS
ing w e r e a c c o m p l i s h e d in i r r e g u l a r l y f i r i n g n e u r o n s in 6 D u r i n g p e n e t r a t i o n s o f t h e r e c o r d i n g e l e c t r o d e s dir e c t e d at t h e S T H , a c h a r a c t e r i s t i c b u r s t f i r i n g o f u n i t s
rats. T h e n e u r o n s w e r e all w i t h i n t h e S T H (e.g., Fig. 1 D a n d E).
TABLE I Spontaneous firing rate and number of units which responded with single or double or excitatory peaks to the cortical stimulation of 250 iz A in intact and lesioned rats
The number in parentheses indicates the percentage of units which exhibited each response pattern. Response patterns
Intact Striatum lesioned Pailidium lesioned Brainstem cut
Spontaneous firing rates (mean 5- S.D.)
Two excitatory peaks
Single excitatory peak
No response
Total number of units
11.2 :t: 5.0 12.8 _+4.8 13.0 5- 5.7 13.8 5- 5.4
161 (78%) 45 (79%) 14 (18%) 26 (72%)
30 (14%) 8 (14%) 58 (75%) 6 (16%)
16 (8%) 4 (7%) 5 (6%) 4 (11%)
207 57 77 36
188 A total of 207 units was recorded from the STH of 10 intact rats. The spontaneous firing rate of these neurons was 11.2 _+ 5.0 (mean _+S.D.) ms. Of the 207 units, 191 units (92%) showed excitatory responses to the Cx stimulation (Table I). The threshold of these responses was usually between 50 and 80/~A. Most of the STH units (161; 78%) exhibited two excitatory peaks which were interrupted by a brief period of inhibition, as an example shows in Fig. 3A. The latency of the first excitation was 2.4 _+ 0.5 ms and that of the second excitation was 14.8 _+ 2.1 ms. Thirty units (14%) responded with a single broad excitation to the Cx stimulation. After these excitations, an inhibition of spontaneous firing followed for about 200 to 300 ms (Fig. 3B). In 3 rats, units were recorded from a large area of the STH in tracks separated by 0.1 mm, and the PSTHs of all recorded units to the Cx stimulation were obtained. The stimulation (150 p,A) was applied through a pair of electrodes placed in the sensorimotor CX. The locations of units were reconstructed and drawn on maps traced from Nissl-stained sections (Fig. 4). A large number of units in most areas of the STH re-
:~
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Caudal
Fig. 4. Schematic representation of response patterns of units to the Cx stimulation. Stimulation of 150/zA was applied through a pair of electrodes placed in the Cx. The shape of the STH was traced from Nissl-stained coronal sections and the locations of recorded units were reconstructed from the last track of a recording electrode and the measured depth. Each plane and each track were separated by 0.1 mm. Each response pattern represents a peristimulus time histogram obtained from an analysis of 50 or 100 trials. NR = no response.
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Time (ms) Fig. 3. Peristimulus time histograms (PSTHs) of a STH unit responding to Cx stimulation of 250/~A. The responses in A and B were the same, but shown with a different time scale. Note the excitation with two peaks (A) and a long-lasting inhibition after the second excitation in B. One hundred trials were analyzed. Bin size +1 ms. Triangles mark the time of stimulation.
sponded to the stimulation. While most commonly observed responses included an initial short-latency excitation, a short period of inhibition, a second excitation, and a long-lasting inhibitory period, there was a great variability in the strength of the brief inhibitory components. Noticeably, units located in the peripheral part of the STH tend to lack the brief inhibitory component and exhibited a long period of excitation. The nature of the excitations and inhibitions was examined by intracellular recordings. The typical intracellular response of a STH neuron to Cx stimulation was a depolarizing potential with multiple humps which were often accompanied by an action potential (Fig. 5A). In 9 of 11 intracellularly recorded neurons the depolarizing potential was interrupted by a brief hyperpolarization occurring with about a 10-ms latency (e.g. Fig. 5A). The amplitude of the depolarization increased with injection of intracellular hyperpolarizing currents, and decreased by injection of depolarizing currents. The results indicate that the depolarization is composed of multiple EPSPs. The amplitude of the brief hyperpolarization was decreased and the polarity reversed by hyperpolarizing current injections, indicating that the brief hyperpolarizations were IPSPs (Fig. 5A). Injection of depolarizing currents caused a large fluctuation of the membrane potential, which appears to include large IPSPs and small spikes. The IPSP
189
A
1
1
C 10 mV 10 msec
h, Fig. 5. Intracellular responses of a subthalamic neurons to the cortical stimulation. A: an STH neuron responded to Cx stimulation with a burst of spikes. Injection of depolarizing and hyperpolarizing currents show the existence of multiple EPSP and IPSP components in the responses. B and C: intracellular responses evoked in two other neurons by the Cx stimulation at two different intensities. These neurons were continuously hyperpolarized by current injection in order to suppress spontaneous firings. The neuron shown in B exhibited a clear hyperpolarizing response of which the peak latency is slightly longer than that shown in A. The neuron shown in C had a single broad depolarization.
corresponding to the brief inhibition was observed but those corresponding to the long lasting inhibition were not (Fig. 5A). Fig. 5B shows responses to Cx stimulation with 2 different intensities recorded in a neuron which was continuously hyperpolarized by current injection to inhibit firing of spikes. The IPSPs in this neuron appear stronger and the peak latency longer than the one shown in Fig. 5A. Responses shown in Fig. 5C were obtained from another neuron at condi= tions similar to that of 5B. This neuron lacked a clear IPSP component.
Among the rats injected with ibotenic acid in the GP, 3 had neuronal cell loss and gliosis which was confined, except in the dorsal neostriatum along the needle track, in the GP (e.g. Fig. 1C). Although the lesion sites did not completely cover the entire GP, the data obtained from these 3 rats presented differences from those obtained in intact rats. A total of 77 units was recorded from the STH of these GP lesioned rats. Spontaneous firings were irregular or bursty as seen in intact rats, and the rate of the firing (13.0 + 5.7 ms) was similar to the intact rats (Table I). However, the response patterns to ipsilateral cortical stimulation were different from those of the intact rats. A much lower number of units (i.e. 14 of 77) responded with two peaks of excitation, while most.! of the remaining neurons (58 of 77) responded with a single excitation (e.g. Fig. 6C). The latency of the single broad excitation was 3.0 + 1.3 ms and it was similar to the latency of the first excitation of the intact rats (2.4 + 0.5 ms). The duration of the excitation was 20 _+ 3.8 ms and it was almost identical to the period between the beginning of the excitation and the end of the second excitation recorded in the intact rat. In 3 rats, an injection of quinolinic acid in the neostriatum resulted in a marked depletion of medium neurons in a large area of the neostriatum (e.g. Fig. 1B). A total of 57 units was recorded from the STH of these rats. Neurons in the STH fired irregularly, and the spontaneous firing rate was 12.8 + 4.8/s, which was almost the same as that of the intact rat (Table I). The pattern of response to Cx stimulation was almost the same as those of the intact rats. Of 57 units, 45 (79%) responded with two peaks of excitation and 8 (14%) responded with a single excitation to the Cx stimulation. Similarly, acute knife cuts of the brainstem failed to change the response pattern to Cx stimulation. O f 36 units recorded after the knife cut, 26 units (72%) responded with two peaks of excitation and 6 units (16%) responded to the Cx stimulation with a single excitation. The spontaneous firing rate of these units was 13.8 _+ 5 . 4 / s which was not statistically different from those intact ones. It was further observed that the long lasting inhibition, seen after the early excitations, was not affected by any of the chemical lesions or by the knife cuts performed in this study. Responses of STH neurons t o stimulation of both ipsi- and contralateral Cx to the recording site were studied in 2 rats in which stimulus electrodes were placed bilaterally at the same coordinates. The response patterns of 21 units to the ipsi- and contralateral cortical stimulation were basically the same, i.e., consisting of two excitatory peaks followed by a long period of inhibition. However, the thresholds of con-
190 10 9
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ipsilateral Cx was stimulated. After acquisition of the data, the rostral half of the corpus callosum of these two rats was cut acutely with a knife. Following the knife cut, the responses to the stimulation of the ipsilateral Cx did not change, however, the responses to the contralateral stimulation completely abolished.
7
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DISCUSSION
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Fig. 6. A and B: peristimulus time histograms of a STH unit responding to ipsi- (A) and contralateral (B) cortical stimulation with 250 p.A. Note the difference in the response amplitude and the response latency. Triangles mark the time of stimulation. B was 1.0 ms in size and 50 trials were analyzed in each histogram. C: a peristimulus time histogram of a STH unit to Cx stimulation of 250 p.A obtained from those which received an ibotenic acid lesion of the GP. B in size was I ms and 50 trials were analyzed. The response lacks the brief inhibition which were often seen in intact rat. Triangles mark the time of stimulation.
tralateral stimulation were considerably higher, i.e., 70 and 200 ~ A , than those of the ipsilateral stimulation (i.e. 50 to 80/xA). The response latency was also much longer for stimulation of contralateral than the ipsilateral Cx (e.g. Fig. 6A and B). The latency of the first excitation was 6.6 + 0.8 ms and that of the secondary excitation was 18.6 + 1.6 ms to contralateral stimulation of 250 /zA, and 2.4 + 0.5 ms and 14.8 + 2.1 ms respectively to ipsilateral stimulation of 250 ~ A . Moreover, the latencies of the contralateral stimulation were very different depending on the intensity of the stimulation. Such p h e n o m e n o n was not observed when the
Most of the STH units exhibited irregular spontaneous firing with an average rate of about 11 per second. Spontaneous burst firing was often seen in these units. Recent reports indicate similar firing patterns and firing rates for anesthetized rat S T H units and unanesthetized monkey STH 16'26'27. RouzaireDubois et al. 23, on the other hand, described the spontaneous firing of the STH of Ketamine anesthetized rats as very regular. They used that firing pattern as one of the criteria for identification of STH units. The difference of the spontaneous firing pattern may be due to difference of recording conditions, including the anesthetics used. STH neurons in the brain slice preparation exhibit very regular spontaneous firing TM. In the monkey study and our present study, the units located in the ventral part of the zona incerta were found to exhibit regular firing. In urethane anesthetized rats and in unanesthetized monkey regular firing may not be used for identification of STH. The existence of direct C x - S T H projection is well demonstrated with anatomical techniques 1'3'4'15'22. The activation of this pathway resulted in short latency monosynaptic EPSPs with multiple spikes in STH neurons 13. In this study a large percent of the units recorded in intact rat STH showed a very strong shortlatency excitation. Stimulation applied to a single pair of electrodes in the sensorimotor Cx induced responses of units in almost the entire area of the STH. The latency of initial response was similar among the units. Since the C x - S T H projection is topographically organized 1, monosynaptic EPSPs should be induced in only a limited area of units. There are two possible disynaptic connections which might induce short latency excitatory responses. One is C x - C x connections which would produce excitations in a wide area of the Cx after an area of stimulation. The other is local axon collaterals of STH neurons 7'L2, which might also spread excitation among neurons in a wide area of the STH. Involvement of the neostriatum, the GP, and the brainstem nuclei in the wide spread response is unlikely considering the present lesion study. The origin of the short inhibition, which is identified as IPSP in this study, appears to be the globus pallidus, for the following reasons: (1) ibotenic acid lesion of the
191 GP greatly reduces the inhibition; (2) Cx stimulation induces a disynaptic excitation, mediated through the STH, in globus pallidus neurons with the latency of approximately 5 ms8; (3) stimulation of globus pallidus evokes short-latency IPSPs in STH neuronsX°; (4) the latency of the brief IPSPs matches very well to the latency of Cx-pallidal excitation plus the latency of GP-STH inhibition; and (5) a recent unit study in the rat indicated that a lesion of the GP increases the magnitude and the duration of STH excitatory responses to CX stimulation26. It remains unknown why the STH produces broad excitations after Cx stimulation. The involvement of a polysynaptic pathway such as Cx-striatal-pallidal-STH (activation of this pathway would produce disinhibition in STH neurons), and Cx-pedunculopontine-STH pathway 3'4'17'22 can be ruled out from the present lesion study. The other polysynaptic pathway is Cxcentremedian-parafascicular nuclei-STH projections 29. Our preliminary studies indicate that a quinolinic acid lesion of the dorsal thalamus preserves the long-duration excitation (Kita, unpublished data). The remaining possible causes for the long excitations are local excitatory collaterals of STH neurons ~2'25, the induction of NMDA responses which is longer duration than other glutametergic responses 19, and also in some part, the electrical membrane properties of STH neurons. STH neurons have relatively high input resistance, a short time constant, and a short refractory period 1°'18. The membrane potential of STH neurons appeared to be close to the threshold for the spike. This is based on the previous intracellular recordings in anesthetized rats and in brain slice preparations 1°'~8. These membrane characteristics are suited for producing strong excitation even when small EPSPs are evoked in the neurons. The Cx stimulation induced excitations were followed by a long-lasting inhibitory period. The existence of similar long lasting inhibition has been mentioned in a recent unit study 26. The following observations (1) that the inhibition was preserved after any of brain lesions performed in this study and (2) that intraceUular recording did not reveal long lasting hyperpolarizations, suggest disfacilitation of Cx inputs, but not IPSPs or an activation of intrinsic properties such as Ca activated K-currents, is the cause of the long lasting inhibition. In rats with a GP lesion, the spontaneous firing rate is expected to increase because the major input to the STH arises from the GP 3'4'22, and the input is GABAergic and inhibitory 1°,23,3°. However, the firing rate was only slightly increased by the lesion in the GP. Similarly, the lesion of the neostriatum failed to change
spontaneous firing of STH units in this study. These observations were different from a recent study by Ryan, et al. 26'27. They showed a lesion of the GP to increase and a lesion of the neostriatum to decrease the spontaneous firing of STH units. A possibility which accounts for the discrepancy between their resuits and ours may be that in chronically lesioned rats some mechanisms develop to compensate for the activity of STH. This possibility is mentioned because a lesion of the GP increases spontaneous activity of substantia nigra neurons only for a few days following the lesion and then return to the prelesion level 5'25. The existence of Cx projection to the contralateral STH has been indicated previously. In that study, excitation of STH induced by contralateral Cx stimulation was still present in ipsilaterally decorticated rats 24. The present study indicated that stimulation of the contralateral Cx could evoke excitatory responses in the STH. However, all the response characteristics including the threshold stimulus intensity, the latency, and the response amplitude appeared to indicate that the responses are mediated by oligosynaptic but not monosynaptic circuits. Moreover, a knife cut of the rostral part of the corpus callosum completely eliminated the STH responses to the contralateral Cx. Our results lead to several possibilities. One is Cx-STH axons emit collaterals which project to the other side of the Cx through the corpus callosum. The existence of such collaterals is not known at this point. The second possibility is that axons of some Cx neurons cross the corpus callosum to the other side and then project to the STH. This possibility is unlikely because none of the anatomical studies reported any cortical projection to the contralateral STH 1,3,4,15,22. Our anterograde neurotracing studies using highly sensitive methods (i.e. PHA-L and biotin conjugated dextran method) also failed to demonstrate the crossed Cx-STH projection (Kita unpublished observation). The third possibility which we consider most likely is the involvement of the Cx-Cx projection through the corpus callosum, which is well known to be a strong connection. Although, this possibility has been denied by previous authors, we are unable to provide another alternative. It may be possible that the responses observed by Rouzaire-Dubois et al. 24 at 15 days after ipsilateral decortication was mediated by disynaptic and bilateral, such as Cx-thalamo, projections in which the connections may strengthen after the decortication. Further studies are required to test this possibility. In sum, this study indicates that an activation of the direct Cx-STH projection produces a powerful broad excitation in the STH, in which mechanisms intrinsic to the STH appear to be responsible for the strong excita-
192 tion. Among the other afferents to the STH, the G P STH projection has a role to suppress the cortically induced excitation in the STH. Acknowledgements. This study was supported by the USPHS Grants NS-25783 and NS-26473, and the Human Frontier Science Program Grant.
REFERENCES 1 Afsharpour, S., Topographical projections of the cerebral cortex to subthalamic nucleus, J. Comp. Neurol., 236 (1985) 14-28. 2 Bergman, H., Wichmann, T. and DeLong, M.R., Reversal of experimental Parkinsonism by lesions of the subthalamic nucleus, Science, 249 (1990) 1436-1438. 3 Canteras, N.S., Shammah-Lagnado, S.J., Sliva, B.A. and Ricardo, J.A., Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat, Brain Res., 513 (1990) 43-59. 4 Carpenter, M.B., Carleton, S.C., Keller, J.T. and Conte, P., Connections of the subthalamic nucleus in the monkey, Brain Res., 224 (1981) 1-29. 5 Fujimoto, K. and Kita, H., Responses of subthalamic neurons to stimulation of the frontal cortex, Soc. Neurosci. Abstr., 17 (1991) 453. 6 Fujimoto, K. and Kita, H., Responses of rat substantia nigra pars reticulata units to cortical stimulation, Neurosci. Lett., in press. 7 Hammond, C., F~ger, J., Bioulac, B. and Souteyrand, J.P., Experimental hemiballism in the monkey produced by unilateral kainic acid lesion in corpus Luysii, Brain Res., 171 (1979) 577-580. 8 Kita, H., Responses of globus pallidus neurons to cortical stimulation; intracellular study in the rat, Brain Res., in press. 9 Kita, H. and Armstrong, W., A biotin-containing compound N(2-aminoethyl) biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin, J. Neurosci. Meth., 37 (1991) 141-150. 10 Kita, H., Chang, H.T. and Kitai, S.T., Pallidal inputs to subthalamus: intracellular analysis, Brain Res., 264 (1983) 255-265. 11 Kita, H. and Kitai, S.T., Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method, J. Comp. NeuroL, 260 (1987) 411-415. 12 Kita, H., Chang, H.T. and Kitai, S.T., The morphology of intracellularly labeled rat subthalamic neurons: a light microscopic analysis, Z Comp. Neurol., 215 (1983) 245-257. 13 Kitai, S.T. and Deniau, J.M., Cortical inputs to the subthalamus: intracellular analysis, Brain Res., 214 (1981) 411-415. 14 Kitai, S.T. and Kita H., Anatomy and physiology of the subthalamic nucleus: a driving force of the basal ganglia. In M.B. Carpenter and A. Jayaraman (Eds.), The Basal Ganglia II, Plenum, New York, 1987, pp. 357-373.
15 Kunzel, H. and Akert, A., Efferent connections of cortical area S (frontal eye field) in Macaca fascicularis: a reinvestigation using the autoradiographic technique, J. Comp. NeuroL, 173 (1977) 147-164. 16 Matsumura, M., Kojima, J. Gardiner, T.W. and Hikosaka, O., Visual and oculomotor functions of monkey subthalamic nucleus, J. NeurophysioL, 67 (1992) 1615-1632. 17 Moon-Edley, S. and Graybiel, A.M., The afferent and efferent connections of the feline nucleus tegmenti pedunculopontinus pars compacta, Z Comp. NeuroL, 217 (1983) 187-215. 18 Nakanishi, H., Kita, H. and Kitai, S.T., Electrical membrane properties of rat subthalamic neurons in an in vitro slice preparation, Brain Res., 437 (1987) 35-44. 19 Nakanishi, H., Kita, H. and Kitai, S.T., An N-methyl-o-aspartate receptor mediated excitatory postsynaptic potential evoked in subthalamic neurons in an in t,itro slice preparation of the rat. Neurosci. Lett., 95 (1988) 130-136. 20 Nauta, W.J.H. and Cole, M., Efferent projections of the subthalamic nucleus: an autoradiographic study in monkey and cat, Z Comp. Neurol., 180 (1978) 1-16. 21 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press Australia, Sydney, 1986. 22 Rinvik, E., Grofova, I., Hammond, C., F~ger, J., and Deniau, J.M., A study of the afferent connections to the subthalamic nucleus in the monkey and the cat using the horseradish peroxidase technique, AdL,. NeuroL, 24 (1979) 53-69. 23 Rouzaire-Dubois, B., Hammond, C., Hamn, B. and F~ger, J., Pharmacological blockade of the globus pallidus-induced inhibitory response of subthalamic cells in the rat, Brain Res., 200 (1980) 321-329. 24 Rouzaire-Dubois, B. and Scarnati, E., Bilateral corticosubthalamic nucleus projections; an electrophysiological study in rats with chronic cerebral lesions, Neuroscience, 15 (1985) 69-79. 25 Robledo, P. and F6ger, J., Excitatory influence of rat subthalamic nucleus to substantia pars reticulata and the pallidal complex: electrophysiological data, Brain Res., 518 (1990) 47-54. 26 Ryan, L.J. and Clark, K.B., Alteration of neuronal responses in the subthalamic nucleus following globus pallidus and neostriatal lesion in rats, Brain Res. Bull., 29 (1992) 319-327. 27 Ryan, L.J., Sanders, D.J. and Clark, K.B., Auto- and cross-correlation analysis of subthalamic nucleus neuronal activity in neostriatal- and globus pallidal-lesioned rats, Brain Res., 583 (1992) 253-261. 28 Schwarcz, R. and Kohler, C., Differential vulnerability of central neurons of the rat to quinolinic acid, Neurosci. Lett., 38 (1983) 85 -90. 29 Sugimoto, T., Hattori, T., Mizuno, N., Itoh, R. and Sato, M., Direct projections from the centre median-parafascicular complex to subthalamic nucleus in the cat and rat, Z Comp. Neurol, 214 (1983) 209-216. 30 van der Kooy, D., Hattori, T., Shannak, K. and Hornykewicz, O., The pallido-subthalamic projection in rat; anatomical and biochemical studies, Brain Res., 204 (1981) 253-268.
185
BRES 18739
Response characteristics of subthalamic neurons to the stimulation of the sensorimotor cortex in the rat K. Fujimoto * and H. Kita Department of Anatomy and Neurobiology, The University of Tennessee Memphis, College of Medicine, Memphis, TN 38163 (USA) (Accepted 17 November 1992)
Key words: Sensorimotor cortex; Subthalamic nucleus; Unit and intracellular recording; Experimental brain lesion
Responses of the subthalamic nucleus (STH) neurons to the stimulation of the sensorimotor cortex (Cx) were recorded in intact rats and in those which received lesions in the pallidum, the neostriatum, the brainstem, or the corpus callosum. Most of the STH units (78%) exhibited two excitatory peaks which were interrupted by a brief period of inhibition. Some of units which were located in the peripheral part of the STH tended to lack the brief inhibitory component and exhibited a long period of excitation. These excitations were followed by a long-lasting inhibitory period. Intracellular recording indicated that these responses were EPSPs interrupted by a short IPSP and a long period of disfacilitation of Cx inputs. A quinolinic acid lesion of the neostriatum and a knife cut of the brainstem failed to alter these responses, while an ibotenic acid lesion of the globus pallidus abolished the short inhibition seen in the midst on the excitation. Stimulation of contralateral Cx also evoked excitatory responses in the STH. The responses were completely eliminated by a parasagittal knife cut of the rostral part of the corpus callosum.
INTRODUCTION 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 l o c a t e d v e n t r a l l y to t h e z o n a i n c e r t a a n d d o r s a l l y to t h e c e r e b r a l p e d u n cle. T h e i n v o l v e m e n t o f t h e S T H in m o t o r c o n t r o l is well d o c u m e n t e d 2'7'16. It has b e e n d e m o n s t r a t e d that
the
sensorimotor
c o r t e x (Cx) p r o j e c t s
to t h e
STH 1'3'4'13'15'22'24. This d i r e c t C x - S T H p a t h w a y is cons i d e r e d to play an i m p o r t a n t role in m o v e m e n t c o n t r o l 14. B a s e d o n k n o w n a n a t o m i c a l connections, t h e S T H s h o u l d also receive p o l y s y n a p t i c i n p u t s f r o m the Cx. T h e p o l y s y n a p t i c p a t h w a y s i n c l u d e t h o s e t h r o u g h t h e globus p a l l i d u s (GP), t h e d o r s a l t h a l a m u s , t h e s u b s t a n t i a nigra, a n d o t h e r b r a i n s t e m nuclei including t h e p e d u n c u l o p o n t i n e nucleus, t h e locus ceruleus, a n d t h e r a p h e n u c l e u s 3,4,6A°JT'29,3°. E l e c t r i c a l s t i m u l a t i o n o f t h e Cx evokes a shortl a t e n c y a n d relatively l o n g - d u r a t i o n excitation in t h e S T H , which was o f t e n i n t e r r u p t e d by a b r i e f inhibitory p e r i o d 5J3'24'26. A p r e v i o u s i n t r a c e l l u l a r r e c o r d i n g study i n d i c a t e d t h a t t h e excitation is d u e to t h e m o n o s y n a p tic i n p u t f r o m t h e Cx 13. H o w e v e r , t h e m e c h a n i s m un-
d e r l i n g t h e relatively l o n g - d u r a t i o n excitation a n d t h e p a t h w a y r e s p o n s i b l e for t h e b r i e f inhibition a r e unknown. W e c o n s i d e r t h e s e issues i m p o r t a n t in o r d e r to u n d e r s t a n d how t h e Cx r e g u l a t e s t h e activity o f the STH. A n e l e c t r o p h y s i o l o g i c a l study s u g g e s t e d the exist e n c e o f b i l a t e r a l m o n o s y n a p t i c C x - S T H p r o j e c t i o n s 24, while n o n e o f t h e a n a t o m i c a l studies r e p o r t e d any Cx p r o j e c t i o n to t h e c o n t r a l a t e r a l S T H 1,3.4,15,22. T h e physiological study implies that t h e Cx directly c o n t r o l s b o t h sides o f t h e STH. O u r s e c o n d objective of this study was to f u r t h e r e v a l u a t e t h e c r o s s e d C x - S T H projection. In o r d e r to study t h e s e questions, we assessed the r e s p o n s e s o f t h e S T H n e u r o n s to t h e s t i m u l a t i o n of the Cx in intact rats a n d in t h o s e which r e c e i v e d lesions in t h e GP, t h e n e o s t r i a t u m , the b r a i n s t e m , or the c o r p u s caUosum. MATERIALS AND METHODS The experiments were performed in 20 male Sprague-Dawley rats weighing 240-310 g. All rats used for the recording experiments were initially anesthetized with urethane (1.2 g/kg, i.p.) and supple-
Correspondence: H. Kita, Dept. of Anatomy and Neurobiology, The University of Tennessee Memphis, College of Medicine, 875 Monroe Ave., Memphis, TN 38163, USA. Fax: (1) (901) 528-7193. * Present address: Jichi Medical School, Department of Neurology, Mimamikawachi-machi, Tochigi-Ken 329-04, Japan.
186 mented with doses of a Ketamine (30 m g / k g , i.p.) and Xylazine (3 m g / k g ) mixture as needed. The animals were placed in a stereotaxic apparatus. Body temperature was kept at 37°C by a thermoregulated electrical heat pad. Craniotomies exposed the cortical surface and allowed access to the recording area and placement of stimulating electrodes. Bipolar stainless steel electrodes consisting of pairs of insect pins (No. 000), insulated with Epoxylite except for 0.2 mm at the tip and separated by a distance of about 0.7 ram, were used for stimulating the deep layers (layers 5 and 6) of the left Cx (3.0 mm anterior to the bregma, 2.0 mm and 2.7 mm to the midline, 1.8 m m below the cortical surface). In 2 rats, stimulating electrodes were placed on both sides of the Cx using the same coordinates (Fig. 1A is an example of the placement of a stimulating electrode). Rectangular current pulses of 0.1 ms in duration with an intensity of 50-250 > A and a frequency of 0 . 6 7 / H z were used for stimulation. All of the recordings were obtained from the left side of the brain. In order to obtain a stable recording and also to aid for searching the location of the STH, the overlying Cx tissues and the dorsal hippocampus were removed by suction to expose the dorsal surface of the thalamus prior to the recording. The cavity was filled with warm paraffin which served to reduce brain pulsations and prevent drying. The reference electrode was inserted between the bone and the cranial muscles. Unit recordings were obtained with a single glass electrode (tip diameter 0.8 # m ) filled with 2 M NaCI (10-15 M~Q). Units with initially negative peaks were isolated by a window discriminator, digitized on-line, and recorded on tape. Single
unit activity was deemed acceptable if a 2:1 signal to noise ratio or better was maintained throughout the recording. Digitized spike trains were fed to a computer (Macintosh llci) using the program Spike Train (generously provided by Dr. J.M. Tepper) to obtain the peristimulus time histogram (PSTH). The PSTH was constructed from 50 or 100 stimulation trials using 1-ms bins. The existance of excitatory or inhibitory responses was determined by a visual inspection for at least three consecutive bins with the number of spikes more than 50% more or less than prior to the stimulation. The first bin of such change was used for the estimation of onset and offset times of the responses. In order to histologically identify the recording sites, electrodes used for the last recording were kept in situ and glued on the skull with dental acrylic resin. The carefull removal of the electrodes from the skull after the aldehyde fixation of the animals left a clear impression of the electrodes in the brain. The impression was used to determine the location of the tip of the electrodes and the plane (or angle) of electrode penetration relative to brain structure. Intracellular recordings were obtained with a glass electrode filled with 2% N-(2-aminoethyl) biotinamide (Neurobiotin, Vector Labs.) dissolved in 0.05 M Tris buffer (pH 7.4) and 0.5 M potassium methylsulfate 9. The resistance of electrodes measured in Ringer's solution was 60-100 Mg2. Amplified neuronal signals were fed to an oscilloscope, an audio monitor, and a tape recorder. Intracellularly recorded neurons were injected with Neurobiotin by passing rectangular pulses of 2 nA with 250 ms duration at 2 Hz for 3-10 min.
Fig. 1. A: a coronal section of a rat brain shows the location of the stimulus electrodes in the sensorimotor Cx. B: a sagittal section shows an example of the quinolinic acid lesion in the n e o s t r i a t u m C: a sagittal section shows an example of the ibotenic acid lesion in the GP. D and E: two sagittal sections of the STH show neurons intracellularly recorded and subsequently labeled with N-(2-aminoethyl) biotinamide. Str, neostriatum; ic, internal capsule; ZI, zona incerta; STH, subthalamic nucleus; GP, globus pallidus; cp, cerebral peduncle.
187 In some rats, the GP or neostriatum was lesioned by injection of either ibotenic acid or quinolinic acid 10-14 days prior to recording. We found that an ibotenic acid lesion of the GP was more reliable than quinolinic acid (which is less expensive than ibotenic acid). However, both ibotenic acid and quinolinic acid lesions were reliable in the neostriatum in the anesthetic condition we used. Rats were anesthetized for the injections with a Ketamine (100 mg/kg i.p.) and Xylazine (3 mg/kg) mixture. Ibotenic acid was dissolved in 0.1 M sodium phosphate buffer (pH 7.4) in such a way that a volume of 0.4 p.1 contains 20 nM. Two injections, each of 0.2 p.1, were performed in the OP at stereotaxic coordinates 21 of -0.5 mm to bregma (AP), 2.5 mm left of the midline (L), and 6.0 mm below the surface of the Cx (DV) and -1.5 mm AP, 3.5 mm L, and 6.0 mm DV. For the neostriatal lesion, quinolinic acid 80 nM was dissolved in 0.8 ~1 of 0.1 M sodium phosphate buffer (adjusted pH 7.4 with 1 N NaOH) and injected in the left neostriatum at the coordinates of 0.5 mm AP, 2.7 mm L, and 4.5 mm DV. This dosage is less than that which was used in the previous reports 2s in order to minimize the lesion spread beyond the neostriatum. The injection of excitotoxins was accomplished by using a Hamilton syringe at a rate of 0.05 ~.l/min. Some rats were subjected to an acute (i.e., during the recording session) knife cut in the brain. In the 2 rats which received bilateral implantation of the stimulating electrodes in the Cx, the corpus callosum was cut parasagittally with a Halazs knife at the coordinates between 1.5 mm and - 3.5 mm AP, 0.5 mm right of L. In 2 other rats, knife cuts were made in the brainstem immediately caudal to the substantia nigra ( - 6.5 mm AP). At the end of the experiments, rats were deeply anesthetized and perfused intracardially with Ringer's solution followed by a fixative of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4). The brains were postfixed overnight at 4 °C in the same fixative and cut into 40-/~m serial, coronal or sagittal sections on a Vibratome. Sections from brains used in the unit recording were mounted and stained with Cresyl violet to verify the placements of the stimulating and recording electrodes and also to examine the extent of the lesion made by the injection of ibotenic acid or quinolinic acid. Sections from the brains performed intracellular recordings and labelings were rinsed in 0.02 M potassium phosphate buffer saline solution (KPBS, pH 7.8), incubated for 2 h in 1.0% Avidin-Biotin Peroxide Complex (Vector Labs.) in KPBS with 0.5% of Triton X, and then rinsed with KPBS. Sections were reacted with diaminobenzidine (0.05%) and H20 2 (0.003%) for 10-30 min. The sections containing intracellularly labeled neurons were postfixed in 0.5% osmium tetroxide in 0.15 M phosphate buffer (pH 7.4) for 30 min and embedded in Epon-Araldite 812 plastic. The remaining sections were mounted and stained with Cresyl violet to verify the placement of the stimulating electrode.
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Fig. 2. A: a spike train (digitalized usmg a program Spike train) obtained from a unit in the ventral part of the zona incerta, displaying a relatively regular spontaneous firing pattern. B: a spike train obtained from a unit in the STH, showing an irregular, bursty spontaneous firing pattern. was encountered
first in t h e d o r s a l t h a l a m u s . P a s s i n g
t h r o u g h a s i l e n t z o n e o f 200 to 400 ~ m d i s t a n c e , w h i c h c o r r e s p o n d e d to t h e d o r s a l p a r t o f t h e z o n a i n c e r t a , a t h i n l a y e r ( a b o u t 100 ~ m thick) o f n e u r o n s e x h i b i t i n g a relatively regular Three
neurons
firing was encountered
in this l a y e r w e r e
(Fig. 2A).
i n t r a c e l l u l a r l y in-
jected with Neurobiotin, and the location of the neur o n s was v e r i f i e d as b e i n g w i t h i n t h e v e n t r a l p a r t o f t h e z o n a i n c e r t a i m m e d i a t e l y d o r s a l to t h e S T H . T h e n , a f t e r p a s s i n g t h r o u g h a n o t h e r e l e c t r i c a l l y silent z o n e , w h i c h w a s a b o u t 30- to 5 0 - ~ m thick, a n a r e a w i t h m a n y v i g o r o u s , i r r e g u l a r l y firing n e u r o n s
was e n c o u n t e r e d
(Fig. 2B). H i s t o l o g i c a l e x a m i n a t i o n r e v e a l e d t h a t this area corresponded
to t h e S T H . A l l t h e tracks o f t h e
final r e c o r d i n g e l e c t r o d e u s e d f o r t h e u n i t r e c o r d i n g w e r e w i t h i n t h e S T H . I n t r a c e l l u l a r r e c o r d i n g a n d stain-
RESULTS
ing w e r e a c c o m p l i s h e d in i r r e g u l a r l y f i r i n g n e u r o n s in 6 D u r i n g p e n e t r a t i o n s o f t h e r e c o r d i n g e l e c t r o d e s dir e c t e d at t h e S T H , a c h a r a c t e r i s t i c b u r s t f i r i n g o f u n i t s
rats. T h e n e u r o n s w e r e all w i t h i n t h e S T H (e.g., Fig. 1 D a n d E).
TABLE I Spontaneous firing rate and number of units which responded with single or double or excitatory peaks to the cortical stimulation of 250 iz A in intact and lesioned rats
The number in parentheses indicates the percentage of units which exhibited each response pattern. Response patterns
Intact Striatum lesioned Pailidium lesioned Brainstem cut
Spontaneous firing rates (mean 5- S.D.)
Two excitatory peaks
Single excitatory peak
No response
Total number of units
11.2 :t: 5.0 12.8 _+4.8 13.0 5- 5.7 13.8 5- 5.4
161 (78%) 45 (79%) 14 (18%) 26 (72%)
30 (14%) 8 (14%) 58 (75%) 6 (16%)
16 (8%) 4 (7%) 5 (6%) 4 (11%)
207 57 77 36
188 A total of 207 units was recorded from the STH of 10 intact rats. The spontaneous firing rate of these neurons was 11.2 _+ 5.0 (mean _+S.D.) ms. Of the 207 units, 191 units (92%) showed excitatory responses to the Cx stimulation (Table I). The threshold of these responses was usually between 50 and 80/~A. Most of the STH units (161; 78%) exhibited two excitatory peaks which were interrupted by a brief period of inhibition, as an example shows in Fig. 3A. The latency of the first excitation was 2.4 _+ 0.5 ms and that of the second excitation was 14.8 _+ 2.1 ms. Thirty units (14%) responded with a single broad excitation to the Cx stimulation. After these excitations, an inhibition of spontaneous firing followed for about 200 to 300 ms (Fig. 3B). In 3 rats, units were recorded from a large area of the STH in tracks separated by 0.1 mm, and the PSTHs of all recorded units to the Cx stimulation were obtained. The stimulation (150 p,A) was applied through a pair of electrodes placed in the sensorimotor CX. The locations of units were reconstructed and drawn on maps traced from Nissl-stained sections (Fig. 4). A large number of units in most areas of the STH re-
:~
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Fig. 4. Schematic representation of response patterns of units to the Cx stimulation. Stimulation of 150/zA was applied through a pair of electrodes placed in the Cx. The shape of the STH was traced from Nissl-stained coronal sections and the locations of recorded units were reconstructed from the last track of a recording electrode and the measured depth. Each plane and each track were separated by 0.1 mm. Each response pattern represents a peristimulus time histogram obtained from an analysis of 50 or 100 trials. NR = no response.
45
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Time (ms) Fig. 3. Peristimulus time histograms (PSTHs) of a STH unit responding to Cx stimulation of 250/~A. The responses in A and B were the same, but shown with a different time scale. Note the excitation with two peaks (A) and a long-lasting inhibition after the second excitation in B. One hundred trials were analyzed. Bin size +1 ms. Triangles mark the time of stimulation.
sponded to the stimulation. While most commonly observed responses included an initial short-latency excitation, a short period of inhibition, a second excitation, and a long-lasting inhibitory period, there was a great variability in the strength of the brief inhibitory components. Noticeably, units located in the peripheral part of the STH tend to lack the brief inhibitory component and exhibited a long period of excitation. The nature of the excitations and inhibitions was examined by intracellular recordings. The typical intracellular response of a STH neuron to Cx stimulation was a depolarizing potential with multiple humps which were often accompanied by an action potential (Fig. 5A). In 9 of 11 intracellularly recorded neurons the depolarizing potential was interrupted by a brief hyperpolarization occurring with about a 10-ms latency (e.g. Fig. 5A). The amplitude of the depolarization increased with injection of intracellular hyperpolarizing currents, and decreased by injection of depolarizing currents. The results indicate that the depolarization is composed of multiple EPSPs. The amplitude of the brief hyperpolarization was decreased and the polarity reversed by hyperpolarizing current injections, indicating that the brief hyperpolarizations were IPSPs (Fig. 5A). Injection of depolarizing currents caused a large fluctuation of the membrane potential, which appears to include large IPSPs and small spikes. The IPSP
189
A
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h, Fig. 5. Intracellular responses of a subthalamic neurons to the cortical stimulation. A: an STH neuron responded to Cx stimulation with a burst of spikes. Injection of depolarizing and hyperpolarizing currents show the existence of multiple EPSP and IPSP components in the responses. B and C: intracellular responses evoked in two other neurons by the Cx stimulation at two different intensities. These neurons were continuously hyperpolarized by current injection in order to suppress spontaneous firings. The neuron shown in B exhibited a clear hyperpolarizing response of which the peak latency is slightly longer than that shown in A. The neuron shown in C had a single broad depolarization.
corresponding to the brief inhibition was observed but those corresponding to the long lasting inhibition were not (Fig. 5A). Fig. 5B shows responses to Cx stimulation with 2 different intensities recorded in a neuron which was continuously hyperpolarized by current injection to inhibit firing of spikes. The IPSPs in this neuron appear stronger and the peak latency longer than the one shown in Fig. 5A. Responses shown in Fig. 5C were obtained from another neuron at condi= tions similar to that of 5B. This neuron lacked a clear IPSP component.
Among the rats injected with ibotenic acid in the GP, 3 had neuronal cell loss and gliosis which was confined, except in the dorsal neostriatum along the needle track, in the GP (e.g. Fig. 1C). Although the lesion sites did not completely cover the entire GP, the data obtained from these 3 rats presented differences from those obtained in intact rats. A total of 77 units was recorded from the STH of these GP lesioned rats. Spontaneous firings were irregular or bursty as seen in intact rats, and the rate of the firing (13.0 + 5.7 ms) was similar to the intact rats (Table I). However, the response patterns to ipsilateral cortical stimulation were different from those of the intact rats. A much lower number of units (i.e. 14 of 77) responded with two peaks of excitation, while most.! of the remaining neurons (58 of 77) responded with a single excitation (e.g. Fig. 6C). The latency of the single broad excitation was 3.0 + 1.3 ms and it was similar to the latency of the first excitation of the intact rats (2.4 + 0.5 ms). The duration of the excitation was 20 _+ 3.8 ms and it was almost identical to the period between the beginning of the excitation and the end of the second excitation recorded in the intact rat. In 3 rats, an injection of quinolinic acid in the neostriatum resulted in a marked depletion of medium neurons in a large area of the neostriatum (e.g. Fig. 1B). A total of 57 units was recorded from the STH of these rats. Neurons in the STH fired irregularly, and the spontaneous firing rate was 12.8 + 4.8/s, which was almost the same as that of the intact rat (Table I). The pattern of response to Cx stimulation was almost the same as those of the intact rats. Of 57 units, 45 (79%) responded with two peaks of excitation and 8 (14%) responded with a single excitation to the Cx stimulation. Similarly, acute knife cuts of the brainstem failed to change the response pattern to Cx stimulation. O f 36 units recorded after the knife cut, 26 units (72%) responded with two peaks of excitation and 6 units (16%) responded to the Cx stimulation with a single excitation. The spontaneous firing rate of these units was 13.8 _+ 5 . 4 / s which was not statistically different from those intact ones. It was further observed that the long lasting inhibition, seen after the early excitations, was not affected by any of the chemical lesions or by the knife cuts performed in this study. Responses of STH neurons t o stimulation of both ipsi- and contralateral Cx to the recording site were studied in 2 rats in which stimulus electrodes were placed bilaterally at the same coordinates. The response patterns of 21 units to the ipsi- and contralateral cortical stimulation were basically the same, i.e., consisting of two excitatory peaks followed by a long period of inhibition. However, the thresholds of con-
190 10 9
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ipsilateral Cx was stimulated. After acquisition of the data, the rostral half of the corpus callosum of these two rats was cut acutely with a knife. Following the knife cut, the responses to the stimulation of the ipsilateral Cx did not change, however, the responses to the contralateral stimulation completely abolished.
7
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Fig. 6. A and B: peristimulus time histograms of a STH unit responding to ipsi- (A) and contralateral (B) cortical stimulation with 250 p.A. Note the difference in the response amplitude and the response latency. Triangles mark the time of stimulation. B was 1.0 ms in size and 50 trials were analyzed in each histogram. C: a peristimulus time histogram of a STH unit to Cx stimulation of 250 p.A obtained from those which received an ibotenic acid lesion of the GP. B in size was I ms and 50 trials were analyzed. The response lacks the brief inhibition which were often seen in intact rat. Triangles mark the time of stimulation.
tralateral stimulation were considerably higher, i.e., 70 and 200 ~ A , than those of the ipsilateral stimulation (i.e. 50 to 80/xA). The response latency was also much longer for stimulation of contralateral than the ipsilateral Cx (e.g. Fig. 6A and B). The latency of the first excitation was 6.6 + 0.8 ms and that of the secondary excitation was 18.6 + 1.6 ms to contralateral stimulation of 250 /zA, and 2.4 + 0.5 ms and 14.8 + 2.1 ms respectively to ipsilateral stimulation of 250 ~ A . Moreover, the latencies of the contralateral stimulation were very different depending on the intensity of the stimulation. Such p h e n o m e n o n was not observed when the
Most of the STH units exhibited irregular spontaneous firing with an average rate of about 11 per second. Spontaneous burst firing was often seen in these units. Recent reports indicate similar firing patterns and firing rates for anesthetized rat S T H units and unanesthetized monkey STH 16'26'27. RouzaireDubois et al. 23, on the other hand, described the spontaneous firing of the STH of Ketamine anesthetized rats as very regular. They used that firing pattern as one of the criteria for identification of STH units. The difference of the spontaneous firing pattern may be due to difference of recording conditions, including the anesthetics used. STH neurons in the brain slice preparation exhibit very regular spontaneous firing TM. In the monkey study and our present study, the units located in the ventral part of the zona incerta were found to exhibit regular firing. In urethane anesthetized rats and in unanesthetized monkey regular firing may not be used for identification of STH. The existence of direct C x - S T H projection is well demonstrated with anatomical techniques 1'3'4'15'22. The activation of this pathway resulted in short latency monosynaptic EPSPs with multiple spikes in STH neurons 13. In this study a large percent of the units recorded in intact rat STH showed a very strong shortlatency excitation. Stimulation applied to a single pair of electrodes in the sensorimotor Cx induced responses of units in almost the entire area of the STH. The latency of initial response was similar among the units. Since the C x - S T H projection is topographically organized 1, monosynaptic EPSPs should be induced in only a limited area of units. There are two possible disynaptic connections which might induce short latency excitatory responses. One is C x - C x connections which would produce excitations in a wide area of the Cx after an area of stimulation. The other is local axon collaterals of STH neurons 7'L2, which might also spread excitation among neurons in a wide area of the STH. Involvement of the neostriatum, the GP, and the brainstem nuclei in the wide spread response is unlikely considering the present lesion study. The origin of the short inhibition, which is identified as IPSP in this study, appears to be the globus pallidus, for the following reasons: (1) ibotenic acid lesion of the
191 GP greatly reduces the inhibition; (2) Cx stimulation induces a disynaptic excitation, mediated through the STH, in globus pallidus neurons with the latency of approximately 5 ms8; (3) stimulation of globus pallidus evokes short-latency IPSPs in STH neuronsX°; (4) the latency of the brief IPSPs matches very well to the latency of Cx-pallidal excitation plus the latency of GP-STH inhibition; and (5) a recent unit study in the rat indicated that a lesion of the GP increases the magnitude and the duration of STH excitatory responses to CX stimulation26. It remains unknown why the STH produces broad excitations after Cx stimulation. The involvement of a polysynaptic pathway such as Cx-striatal-pallidal-STH (activation of this pathway would produce disinhibition in STH neurons), and Cx-pedunculopontine-STH pathway 3'4'17'22 can be ruled out from the present lesion study. The other polysynaptic pathway is Cxcentremedian-parafascicular nuclei-STH projections 29. Our preliminary studies indicate that a quinolinic acid lesion of the dorsal thalamus preserves the long-duration excitation (Kita, unpublished data). The remaining possible causes for the long excitations are local excitatory collaterals of STH neurons ~2'25, the induction of NMDA responses which is longer duration than other glutametergic responses 19, and also in some part, the electrical membrane properties of STH neurons. STH neurons have relatively high input resistance, a short time constant, and a short refractory period 1°'18. The membrane potential of STH neurons appeared to be close to the threshold for the spike. This is based on the previous intracellular recordings in anesthetized rats and in brain slice preparations 1°'~8. These membrane characteristics are suited for producing strong excitation even when small EPSPs are evoked in the neurons. The Cx stimulation induced excitations were followed by a long-lasting inhibitory period. The existence of similar long lasting inhibition has been mentioned in a recent unit study 26. The following observations (1) that the inhibition was preserved after any of brain lesions performed in this study and (2) that intraceUular recording did not reveal long lasting hyperpolarizations, suggest disfacilitation of Cx inputs, but not IPSPs or an activation of intrinsic properties such as Ca activated K-currents, is the cause of the long lasting inhibition. In rats with a GP lesion, the spontaneous firing rate is expected to increase because the major input to the STH arises from the GP 3'4'22, and the input is GABAergic and inhibitory 1°,23,3°. However, the firing rate was only slightly increased by the lesion in the GP. Similarly, the lesion of the neostriatum failed to change
spontaneous firing of STH units in this study. These observations were different from a recent study by Ryan, et al. 26'27. They showed a lesion of the GP to increase and a lesion of the neostriatum to decrease the spontaneous firing of STH units. A possibility which accounts for the discrepancy between their resuits and ours may be that in chronically lesioned rats some mechanisms develop to compensate for the activity of STH. This possibility is mentioned because a lesion of the GP increases spontaneous activity of substantia nigra neurons only for a few days following the lesion and then return to the prelesion level 5'25. The existence of Cx projection to the contralateral STH has been indicated previously. In that study, excitation of STH induced by contralateral Cx stimulation was still present in ipsilaterally decorticated rats 24. The present study indicated that stimulation of the contralateral Cx could evoke excitatory responses in the STH. However, all the response characteristics including the threshold stimulus intensity, the latency, and the response amplitude appeared to indicate that the responses are mediated by oligosynaptic but not monosynaptic circuits. Moreover, a knife cut of the rostral part of the corpus callosum completely eliminated the STH responses to the contralateral Cx. Our results lead to several possibilities. One is Cx-STH axons emit collaterals which project to the other side of the Cx through the corpus callosum. The existence of such collaterals is not known at this point. The second possibility is that axons of some Cx neurons cross the corpus callosum to the other side and then project to the STH. This possibility is unlikely because none of the anatomical studies reported any cortical projection to the contralateral STH 1,3,4,15,22. Our anterograde neurotracing studies using highly sensitive methods (i.e. PHA-L and biotin conjugated dextran method) also failed to demonstrate the crossed Cx-STH projection (Kita unpublished observation). The third possibility which we consider most likely is the involvement of the Cx-Cx projection through the corpus callosum, which is well known to be a strong connection. Although, this possibility has been denied by previous authors, we are unable to provide another alternative. It may be possible that the responses observed by Rouzaire-Dubois et al. 24 at 15 days after ipsilateral decortication was mediated by disynaptic and bilateral, such as Cx-thalamo, projections in which the connections may strengthen after the decortication. Further studies are required to test this possibility. In sum, this study indicates that an activation of the direct Cx-STH projection produces a powerful broad excitation in the STH, in which mechanisms intrinsic to the STH appear to be responsible for the strong excita-
192 tion. Among the other afferents to the STH, the G P STH projection has a role to suppress the cortically induced excitation in the STH. Acknowledgements. This study was supported by the USPHS Grants NS-25783 and NS-26473, and the Human Frontier Science Program Grant.
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