- Email: [email protected]
Intrinsic connections of caudate neurons. I. Locally evoked field potentials and extracellular unitary activity
Brain Research, 53 (1973) 291-305
291
© ElsevierScientificPublishingCompany, Amsterdam- Printed in The Netherlands
INTRINSIC CONNECTIONS OF CAUDATE NEURONS. I. LOCALLY EVOKED FIELD POTENTIALS AND E X T R A C E L L U L A R UNITARY ACTIVITY
LUIS A. MARCO, PAULA COPACK, A L A N M. EDELSON AND SID G I L M A N
Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 (U.S.A.) (Accepted October 20th, 1972)
SUMMARY
The field potentials and extracellular activity within the caudate nucleus of cats were investigated by means of closely apposed fine stimulating electrodes arranged in a semicircle at 1.5 mm distant from the recording glass pipette. The studies were performed on intact animals anesthetized with Nembutal or chloralose-urethane and in unanesthetized enc6phale or cerveau isol6 preparations. All animals were paralyzed and artificially respirated. Recordings were also obtained in cats in which the caudate nucleus had been isolated from the surrounding tissues 20-90 days before recordings were taken to allow for degeneration of afferent fibers. In all types of preparations, stimulation of discrete points around the recording micropipette typically evoked field potentials of negative polarity and 3-5 msec duration at an average latency of 10 msec. Single spike unitary responses usually emerged from the falling phase or peak of the field potentials. Commonly, repetitive stimulation failed to evoke responses for 150200 msec following the first response. Spontaneous unitary activity was often arrested by single shocks, and the duration of spike suppression ranged from 150 to 200 msec. We suggest that inhibitory postsynaptic potentials are responsible for the protracted recovery cycle of the field potentials, for the long-lasting unresponsiveness of units, and for the suppression of spontaneous spikes following a stimulus. Few units were capable of responding at shorter latencies and of following high frequencies of stimulation. A few cells fired in short bursts at high frequencies in response to single shocks. We conclude that these findings reflect the synaptic properties of interconnected elements within the caudate neuropil. We suggest that the first order elements, receiving afferent impulses, may be very small and relatively inaccessible even to fine microelectrode probing, and that the present unitary recordings may reflect only the activity of larger neurons further along the chain of transmission within the caudate neuropil.
292
L.A. MARCOet al.
INTRODUCTION
The intrinsic synaptic organization of the caudate nucleus is now beginning to be unraveled due to the ultrastructural studies of Adinolfi 1-3 and particularly the series of Kemp 15-2°. These investigations have suggested that interconnections between caudate neurons must be very numerous and that consequently integration by interneurons, by far the largest population, must be considerable. Their short thin axons terminate on dendrites and somata of other caudate neurons, thus establishing short intrinsic links among themselves. On the basis of these ultrastructural and other previous studies 4,s,13, a number of suggestions have been made which can be subjected to electrophysiological testing. Albe-Fessard et al. 4 had already proposed that different neurons in intricate anatomical distribution were engaged in inhibitory interactions with interconnected neighboring elements within the caudate. The extreme thinness of intrinsic axons 1-3,15-2°,36 has prompted the suggestion that the usual long latency of responses of caudate units may be due to slow conduction through these thin intrinsic multisynaptic pathways 1,18. Bishop 8 explicitly proposed that it would be worthwhile to investigate the responses to local stimulation within the caudate in search of interference or summation of these responses. No such approach has yet been undertaken systematically, despite the large size of the caudate which makes it convenient for such a study. The head of the caudate can be exposed and identified readily by removal of the roof of the lateral ventricle. Recording microelectrodes and arrays of stimulating electrodes can then be placed within the caudate under direct visualization. Most neuronal elements in the caudate nucleus are difficult to investigate electrophysiologically due to their small size but systematic local stimulation is a powerful way of firing neurons and has in the past yielded meaningful results at the level of the cerebral cortex 7,21,22,2s,33,a4, in the cerebellum 10, and in the thalamus2a,24,3L In view of these observations, we decided to investigate the topography and properties of intranuclearly driven caudate cells. Specifically, we wished to learn what types of neuronal responses can be elicited in the caudate nucleus by stimulation through closely apposed electrodes. Are they compatible with those obtained by more conventional means of stimulation? Do cells respond with single spikes or repetitively? If repetitive responses are encountered, can they be due to the activity of cells similar to Renshaw neurons? Long latency responses in the caudate have been the rule when distant hodologically related structures were stimulated. Could these long latency responses be accounted for by extrinsic conduction or was conduction time spent mostly in intrinsic transmission? If the latter, what are the synaptic features? These questions can be answered from the characteristics of unitary responses to intranuclear stimulation and the conclusions can be reinforced by the results of parallel studies in the isolated caudate. The results of extracellular recordings will be described in this paper, and the evaluation of electron and fluorescence micrographs of isolated caudates will be given in the following paper 3~. The results of intracellular recordings will be the object of a subsequent communication (Marco et al., in preparation). The present work will show that stimulation of the caudate closely apposed to the site of recording can achieve an acceptable degree of selectiveness and provide a good basis for a first attempt at reconstructing
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
293
the intrinsic synaptic and topographical organization of caudatal elements at the electrophysiological level. Part of this work has been published in abstract form 2s. METHODS
Forty-one cats were used in this study. Most of the animals were anesthetized with 35 mg/kg of sodium pentobarbital (Nembutal) injected intraperitoneally. Five animals were anesthetized with a-chloralose plus urethane (40 and 800 mg respectively/kg) dissolved in propylene glycol and injected intraperitoneally. Seven cats were injected with a lighter dose of sodium pentobarbital or induced with a fast-acting barbiturate or ether inhalation, and all 7 were subsequently maintained as cerveau or enc6phale isol6 preparations for the recordings. Every preparation was paralyzed by means of intravenous gallamine triethiodide and artificially respired. The ear canals and infraorbital subconjunctival folds were filled with viscous xylocaine and the animals were clamped in a stereotaxic apparatus. Through a craniotomy, the dura was resected and all the brain tissue overlying the ventricular surface of one or both caudates was aspirated by suction, leaving exposed the caudate nucleus rostrally and the fimbria and hippocampus caudally. In about 50 ~ of the experiments aspiration of tissue was extended to the frontal pole. Under visual guidance stimulating and recording electrodes were placed stereotaxically in position for vertical tracking of unitary activity within the caudate. A chart of the rostrocaudal, lateral and vertical parameters used for each track was kept for every experiment. Tracking was carried out with a hydraulic microdrive capable of lowering the recording micropipette in measurable steps of 1/tm. Three stimulating electrodes were prepared from 250/~m diameter tungsten wire straightened and etched to a point with a diameter of about 1 #m by electrolysis and coated except at the tip. Under a dissecting microscope the coating was scraped off up to 0.5 mm from the tip. This procedure yielded an electrode with a DC impedance of about 50 kf2 in normal saline. These 3 wires were passed through holes drilled by the edge o f a perspex block which had been machined to a shape represented in Fig. 1A. The 3 stimulating electrodes were then cemented to the lucite block in a semicircular configuration (m, medial; c, caudal; 1, lateral stimulating electrodes). The entire assembly was clamped by means of a rigid steel strut (C) to a micromanipulator. The recording micropipette (R) was invariably positioned at the center of the semicircle formed by the stimulating electrodes before tracking was initiated so that the distance between the recording and each of the stimulating electrodes was 1.5 mm. Kemp and PowelP s have recently shown that asymmetrical intrinsic synapses extend at least 1.5 mm from the parent cell. Penetration through the depth of the caudate was carried with two independent micromanipulators, one for the stimulating 3-prong array and the other for the recording micropipette. Tracking in search of units was invariably carried out according to the following procedure: the stimulating array was lowered 0.5 mm below the point at which the tips touched the dorsal surface of the caudate and was left at that depth to ascertain that the exposed tips were buried in caudate tissue. Then tracking with the micropipette was initiated until it had been lowered 1 mm below the ependymal dorsal surface of the caudate. After this 1 mm
294
L.A. MARCOet al.
o
I 8
_
8
7
6
5
4
3
2
1
0
I
I
I
I
I
I
I
I
I
7_
4_
.
I..t
Fig. 1. A, Block of lucite with small holes drilled through its thickness close to the semicircular edge. The 3 stimulating electrodes (m, c and 1; medial, caudal and lateral) were passed through the holes and were permanently cemented in position to yield a distance of 1.5 mm from the recording glass micropipette (R) to each of the stimulating electrodes. A rigid steel strut was also cemented to the block and the stimulating assembly was clamped at the upper end of the strut (C) to an electrode holder mounted to a microdrive. B, Plotting of the first 100 unitary responses in association with field potentials at Fr +16.
penetration, the tip of the micropipette was 0.5 m m ventral to the tips of the stimulating array. Units encountered during penetration through this distance were investigated. Then the stimulating array was lowered another I mm and thus the stimulating and recording electrodes were alternately lowered at 1 m m steps until the end of the track had been reached. Glass micropipettes were pulled to yield a resistance of 5-15 Mr2 when filled with 3 M potassium chloride or 2 M potassium citrate. Every evoked response was tested with both cathodal and anodal rectangular pulses which usually ranged from 3 to 15 V, 0.1-0.3 msec duration. The indifferent electrode was placed on the temporo-occipital muscle of the contralateral side. Stimuli were delivered through a Grass stimulus isolation unit (SIU). No other measures were taken to mitigate the recording of artifacts generated by the closely apposed stimulus except in a few experiments in which the SIU was replaced by a Tektronix photon-coupled stimulus isolator without significant improvement. A switch box was interpolated between the SIU and the 3-pole stimulating array to facilitate swift checking of the effectiveness of each stimulating electrode (m, c, 1). Recorded signals were led through a negative capacitance high impedance electrometer to AC and DC coupled amplifiers in parallel and displayed on the face of an oscilloscope after appropriate amplification. U p o n completion of an experiment the tips of the stimulating array were fulgurated at stereotaxic parameters corresponding to the presumed center of the caudate, thus serving as a countercheck for the parameters recorded in the chart. The brain was then perfused with normal saline followed by 10% formalin. A few days later, a block enclosing the caudate nucleus was cut out from the rest of the brain and kept in fresh formalin for subsequent histological verification of the tracks of the stimulating electrodes.
295
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
In 7 cats the head of the caudate nucleus was isolated unilaterally from the rest of the brain except for its ventral aspect which merges with the lateral hypothalamic region and through which it receives its vascular supply from the striate arterioles. The latter originate in the base of the brain from the middle cerebral arteries 6,14. Unila teral exposure of the dorsal surface of the caudate nucleus was carried out under aseptic conditions according to the procedure outlined above for acute recordings. A sharp knife was used to cut through the tissue surrounding the caudate in all 4 planes. Isolation was completed by means of a blunt spatula which reached the ventral surface of the brain in all 4 planes. The opening was closed by anatomical layers, the animals were allowed to recover and the recording experiment, as described above, was carried out 20-90 days later. Terminal degeneration is said to be complete by the sixth day is. At the end of the experiment a wedge of caudate tissue 1 mm thick was taken in vivo at about Fr ÷ 16 from both the isolated and the contralateral control sides. The specimens were subsequently prepared for fluorescence and electron microscope evaluation 35. The essential characteristics of the responses described in this study did not differ with the type of anesthesia or preparation used. RESULTS
( A ) Field potentials Single shocks applied 1.5 mm from the recording micropipette evoked focal potentials throughout the depth of the head of the caudate nucleus. Fig. 1B shows the sites from which field potentials were recorded in combination with unitary activity at Fr + 16. Focal potentials were invariably of negative polarity and had peak latencies A
D
G
B
c ._..%-m
__F
'o Is
D
_~f
1012
C
Fig. 2. Left panel, Field potentials evoked in the dorsal portion of the head of the caudate (Fr + 15, L 5, V +6.5) by single shocks delivered through the m electrode (A-C). Stimulus strength increased from A to C which represents a maximal response. Note that the amplitude of field potentials increased and the latency correspondingly decreased with stronger stimuli. Same stimulus strengths applied to the c electrode (D-F) yielded much smaller responses and no response could be evoked by 1 stimuli (G-H), G taken at the same stimulus strength as A; H at twice that of C. Right panel, Shows that stimulation and recording in the ventral portion of the caudate nucleus (a-c) yielded responses of largest amplitude to the m stimulus (A), smaller responses to the c shock (B), and no response to the I pulse (C). Dorsally however (d) only 1 stimulation evoked responses (D). Recordings at Fr + 17, L 3.5, V +6.5 and + 1 respectively. Five sweeps superimposed in all frames. Negativity in this and all subsequent figures is signified by a downward deflection of the trace; horizontal and vertical bar calibrations represent time and amplitude in msec and mV respectively, m, c or I at the trace-break caused by the stimulus artifact indicate application of a medial, caudal or lateral stimulus respectively.
L.A. MARCO et al.
296
of 8-10 msec, amplitudes of 2.5-3 mV and durations of 3-5 msec. However, these responses could not usually be evoked by all 3 stimulating electrodes in any single placement. Responses of optimal amplitude were commonly evoked by just one stimulating electrode, the other two being either ineffective or minimally capable of eliciting responses. Fig. 2, left, is an example of focal potentials optimally evoked by the m stimulus (A-C), less effectively by the c pulse ( D - F ) and not by the I shock ( G - H ) . The orientation of the recording pipette to the stimulating electrode most effective in evoking focal potentials varied as the electrodes were lowered through the caudate. Fig. 2, right, illustrates this observation. Thus, at different levels of penetration the stimulus had to be switched from one electrode to another in order to maintain optimal or adequate focal potentials as well as a high yield of unitary evoked activity (see Section 1). Moreover, when the micropipette was left more than 2 mm dorsal to the stimulating array, or when it was lowered below the stimulating array by the same magnitude, focal potentials were lost altogether. Thus, field responses were very selectively pin-pointed at any given depth. Field potentials did not follow high frequency stimulation. Fig. 3 D - E illustrates response failure after the third and second pulses at 50 and 100/sec respectively. Occasional stimuli (0.5-1/sec) elicited responses with characteristics essentially invariant with stimulation time. Repetitive stimulation at about 20/sec completely blocked the focal potentials after the fifth or sixth pulse. Following the end of the tetanizing train there was posttetanic depression which lasted 3-5 sec, followed by gradual recovery to amplitudes somewhat above control. Fig. 3 A - C illustrates this sequence of events, i.e., the frequency-dependent attenuation and subsequent slight potentiation. A few seconds of anoxia were sufficient to suppress large field potentials (Fig. 3F-H).
(B) Unitary activity Five hundred and seven units were recorded extracellularly in this series. The
so/,
rrrrrrrr
!°I'
Fig. 3. Left, Effects of tetanization on field potentials. A, before; B, during 20/sec tetanizing train; C, responses 10 sec after the end of the tetanizing train. Center, Failure of sustained responses to 50 (D) and 100/sec (E) trains. Right, Effects of anoxia. F, before; G, after 5 sec of apnea; H, shortly after respirator was turned on again. Several sweeps in each frame.
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
297
action potentials were of 1.5-2 msec duration, 2-6 mV amplitude, positive or positivenegative in configuration. These features indicate that recordings were obtained with the pipette pore very close to the perikaryon 12. Thirty-eight per cent of the units recorded fired spontaneously. The firing rates varied widely from one neuron to another and ranged between 1 and 32/sec. Consistent differences in spontaneous activity between the various types of preparations could not be established. The mean firing rate for the entire population was l0 4- 9/sec. (1) Unitary spike responses to electrical stimulation. Twenty-five per cent of the spontaneously firing units were also driven by stimulation. Their mean latency was 11 ± 3 msec. Sixty-two per cent of all the units were silent in the absence of stimulation, but single pulses of relatively low strength (3-7 V) every 2 sec consistently evoked responses from these neurons with a mean latency of 10 4- 5. A plot of frequency distribution shows that the latencies centered around 9-10 msec. The latency histogram of Fig. 4 illustrates these findings. The percentile relative contribution to driven responses by each one of the stimulating electrodes or their combination is given in Table I. It shows that urdts responded more frequently to stimulation of 1 or all 3 electrodes independently (cml) and less often to stimulation of only 2 of the electrodes independently (cm, cl, ml). TABLE1 c
m
l
cm
cl
ml
cml
17.5
20.8
18.9
6.0
7.7
7.7
20.8
Some unitary responses were evoked only by relatively strong stimuli between 15 and 30 V, and then only 1 of the 3 electrodes could evoke the responses. Indeed, even at such high stimulus strength some units failed to respond unless a train of 2-3 pulses at 100/sec was applied. Unitary responses usually consisted of a single spike emerging and gradually growing out of the failing phase of the negative field potential in the form of initially positive notches or full spikes superimposed on the negative wave (Fig. 4C-G). Double spike responses such as those illustrated in Fig. 4A, the first spike having very short latency and the second spike the typical latency, were rarely observed. Single spike responses such as those illustrated in Fig. 4 C - G were usually observed. The stimulus location capable of evoking the largest field potential was usually the one giving the highest yield of evoked unitary activity. Unitary responses were also evoked, although less commonly, in the absence of field potentials. Nineteen per cent of the driven units were triggered by a single stimulus into a burst of 4-8 spikes at a firing rate of 115-500/sec. Shocks stronger than those required to evoke a burst response succeeded only in reducing the duration of the burst to a single spike response, but lowering the stimulus strength did not reduce the burst response to a single spike response, i.e., there was either a burst response or nothing. Fig. 5A-D shows some of these burst responses in 4 units, all from different experiments. The latencies of the first spikes in each burst ranged from 7 to 12 msec.
298
L.A. MARCO et al.
25
C 20
15
10
E I
F msec.
0
,
.
5
10
I
I
--7. 15
G
Fig. 4. Left, Frequency distribution of latency responses of the first 111 unitary recordings collected in this series. Ordinate, number of units. Abscissa, latency in msec. Inset traces represent short latency unitary responses (first spike, in A) and responses at typical latencies (B and second spike in A) elicited by stimuli delivered to the c and the m electrodes respectively. Right, Field potentials and unitary responses simultaneously evoked by m shocks as the recording micropipette was lowered about 25/~m from C to G. Note that in C only field potentials were recorded but in D the positive phase of unitary responses begins to emerge and gradually grows from E to G. Three to 5 sweeps superimposed except in G.
Seventeen per cent o f the driven units were c a p a b l e o f following each pulse in a train o f stimuli at 30/sec; 13 ~ followed 50/sec trains; a n d only 3.7 ~ were driven at 100/sec. Fig. 5E illustrates one o f these rare units c a p a b l e o f r e s p o n d i n g to each pulse at 50/sec. The latencies o f these responses were i n v a r i a n t within each unit a n d ranged between 8 a n d 10 msec. Units which r e s p o n d e d to s t i m u l a t i o n o f m o r e t h a n one electrode site were studied with p a i r e d pulses, a p p l y i n g a c o n d i t i o n i n g pulse to one electrode a n d a testing shock to a n o t h e r electrode. F i r i n g indices (FI, n u m b e r o f r e s p o n s e s / n u m b e r o f stimuli) were o b t a i n e d for the testing electrode at various pulse intervals. R a n d o m l y , the cond i t i o n i n g pulse was t u r n e d off t h r o u g h o u t the series o f intervals to ascertain that the test spike response was still present at an F I o f 100 ~ . The results o f these experiments were as follows. (a) F a i l u r e o f response to the testing pulse ( F I ~ 0 ~o) when it was preceded by the c o n d i t i o n i n g stimulus occurred at intervals ranging from 15 to 280 msec. (b) In a n o t h e r similar set o f observations in units which were triggered by only one electrode, the test pulse was a p p l i e d t h r o u g h this electrode while the c o n d i t i o n i n g stimulus was applied to a n o t h e r electrode which did not evoke a unitary response. I n
299
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
A m
5
F
I
c
100
]
200 J
2
I
10
I
C
20
llln 11tll 111111111111 !
II~[
[ll~t
P'w [ [I~B I m
Fig. 5. A-D, High frequency barrage responses from different units evoked by single shocks. E, Pulse per pulse unitary responses to a 50/sec train delivered through the c electrode. Note doublet response to first shock. Right, Two spontaneously firing units (F and G-H) which were silenced for about 125 and 100 msec respectivelyfollowing single stimuli. Five sweeps superimposed in F. this situation, the conditioning pulse also succeeded in blocking the response to the testing pulse. Under such conditions, neuronal refractoriness, collision, occlusion, post-excitation, inhibition and after-hyperpolarization can be ruled out and the responsible process must be either active inhibition or dis facilitation. (2) Suppression of spontaneous unitary activity by electrical stimulation. An investigation of the change in behavior of spontaneously firing units following stimulation provided further support for the idea of locally generated inhibition. Thirty-three per cent of the units firing spontaneously were silenced by either single shocks or 3pulse trains at 100/see. The duration of spike suppression varied widely and ranged from 75 to 600 msec. Fig. 5 F - H shows two units firing spontaneously which were thus silenced by single shocks. After the poststimulus silent interval some units resumed firing at the prestimulus rate (F). In other units, after the silent period, firing resumed in an uneven pattern, the silent intervals between bursts often having the duration of the first period of silence induced by the shock (G), but silent intervals gradually decreased in duration in the absence of stimulation and continuous firing was eventually reestablished. When the unit was firing at a higher frequency, the interburst silent intervals were less clear-cut and of shorter duration (H).
( C) Evoked field potentials and unitary activity in chronically isolated caudates The possibility existed that the stimulus might have triggered neurons outside
300
L.A. MARCO et al.
m
I
ffl
m
C
I
5 ,
i
1
L
Fig. 6. Responses in isolated caudates. Seven units from different experiments are represented. In A-C threshold shocks evoked unitary spikes arising from the falling phase of the field potential. Unit D-F, stimuli slightly above threshold evoked similar unitary responses at 10 msec latency. Unit G, H was evoked at slightly longer latencies (12-13 msec). Unit I, response latencies varied from 10 to 13 msec. Units J, K, L exhibited response latencies of 8, 7 and 11 msec respectively. Several sweeps superimposed in frames B, E, G and I. The caudate from which unit A-C was recorded is described in the following paper 35.
the c a u d a t e which in t u r n would send their impulses to, and fire, the units r e c o r d e d in the caudate. T o test this possibility, in 7 cats the head o f the c a u d a t e was isolated f r o m the rest o f the b r a i n (see M e t h o d s ) . R e c o r d i n g s in these p r e p a r a t i o n s were m o r e difficult a n d gave a lower yield o f responses t h a n in p r e p a r a t i o n s with intact connections. Thirty-seven units were recorded in these experiments. F o r t y - t w o per cent o f them exhibited s p o n t a n e o u s activity. F i r i n g rates varied f r o m one n e u r o n to a n o t h e r and r a n g e d between 5 a n d 40/sec ( m e a n 13/sec). The rest fired only in response to local stimulation. The latencies o f field potentials a n d u n i t a r y responses were c o m p a r a b l e to those o b t a i n e d in the c o n t r o l p r e p a r a t i o n s . The usual latency was 9-10 msec (Fig. 6) b u t a few units were r e c o r d e d in which response latencies were 4 - 6 msec. The results o f electron a n d fluorescence m i c r o s c o p y o f these isolated caudates are given in the following c o m m u n i c a t i o n 35. Since some synapses exhibited all the m o r p h o l o g i c a l features o f functionally viable junctions, these can be presented as being responsible for transmission o f the e v o k e d responses e n c o u n t e r e d in our physiological experiments in the isolated caudates. N o t o p o g r a p h i c a l p a t t e r n was discernible to differentiate units which fired spont a n e o u s l y f r o m those which were silent in the absence o f stimulation, n o r was there a difference f r o m those r e s p o n d i n g in burst o r c a p a b l e of following higher frequencies
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
301
of stimulation. Maps similar to that shown in Fig. 1B were made of units recorded at more rostral and caudal planes. None of these plottings yielded a topographical characterization in terms of patterns of responses. Greater numbers of tracks were made within the mid-portion of the caudate (lateral 3-5) than on either side of this region in order to ascertain that all 4 electrodes were within the caudate for the longest tracks. DISCUSSION
Field potentials with latencies shorter than 7 msec were not observed in these experiments, indicating that the pathways involved were of slow conduction velocity. This is in keeping with the results of ultrastructural studies showing that in the caudate neuropil the mean diameter of most fibers is less than 1 /~m and that the terminal arborizations must be exceedingly fine. Moreover, axodendritic spine synapses are more numerous than axodendritic trunk synapses, and the latter in turn are much more frequently encountered than axosomatic synapses. The majority of axons in the caudate are short and slender and terminate in local arborizations close to their cell bodies, thus establishing synapses with the short, thin dendrites of other neighboring neurons. Synaptic contacts occur most frequently among the small elements of the neuropil which function as components of intrinsic chains of contact within the nucleus1-3,15-20. It follows therefore that transmission of impulses from the site of stimulation to the point of recording (1.5 mm apart) most plausibly involves a multisynaptic or at least an oligosynaptic chain of slowly conducting axodendritic junctions. Despite the typical homogeneity and absence of cell lamination in the striatum 27, formation of small clusters of neurons have been recently redescribed in the caudatO 6, 27. This feature would most certainly account for both the discreteness and the negligible stimulus-dependent growth of the focal potentials. There was a clear correlation between the characteristics of field potentials and the properties of unitary responses. Evoked unitary responses were difficult to obtain in experiments in which field potentials could not be demonstrated. The largest majority of triggered units fired during the falling phase of the field potential. Extracellular recordings were often immediately followed by penetration of the cell, leaving no doubt about the juxtasomatic position of the recording electrode. In these situations, the negative field potential consistently reversed to a positive excitatory postsynaptic potential of equal latency (Marco et al., in preparation) indicating that the field potentials reflect synaptic activation of a constellation of neurons within the receptive field of the electrode pore. The properties of the field potentials we have described are characteristic of synaptic potentials. When one stimulus site was more effective than the other two for evoking field potentials, the same site was also optimal for triggering unitary potentials. The findings summarized in Table I suggest that spread of stimulating current from one stimulus site to another was negligible. If significant spread of current had occurred one would expect, in a semicircular array, more current spread between the electrodes composing the cl and cm pairs and less spread between the electrodes of the ml pair. Accordingly, the cl and cm pairs should have recruited the largest percentages
302
L.A. MARCOet al.
of driven units and the ml pair the lowest. The values in Table I, however, show that all 3 groups of two electrodes have about the same percentage of driven unitary responses. On the other hand, the c, m, I and cml groups all generated about the same percentage of driven responses which is much higher than that of the combination of any two electrodes. This finding suggests, apart from the obvious random orientation of neuropil axons, that (a) appreciable spread of current from one stimulating electrode to another did not occur, and (b) direct depolarization of neuronal somata was not induced. These two conclusions would not be difficult to accept in the light of the experiments by Crain et a l ) showing that there was no non-neural spread of current in a bipolar explant from one pole to the other after midline transection even when the gap was less than l0 # m wide. In addition, the findings suggest that there are at least 4 populations of cells in terms of interconnectivity. The first is represented by those elements receiving input from more caudal regions (c-driven). The second and third receive synaptic impingement from more medial and lateral zones (m- and l-driven, respectively). The fourth population, equally large, consists of neurons having connectivity in all 3 directions (cml-driven group). The rest of the population is divided into 3 subgroups about equal in size but smaller than those described above. They are represented by those units having interconnectivity in 2 of the 3 directions investigated. The comparative macro- and microphysiological study of Albe-Fessard et al. 4,5 demonstrated that unitary latencies were much longer than the latencies of focal potentials by an average difference of about l0 msec. Such findings suggest that the first order units upon which afferents impinge are somehow not easily accessible to fine microelectrode probing, since responses from these units should have latencies only 0.5 msec longer (one synaptic delay) than the evoked field potentials contributed by the primary afferents. A conclusion similar to ours was drawn by Sedgwick and Williams 32. It seems plausible therefore that the afferents first impinge upon very small elements (7 # m diameter, Golgi type II) from which transmission is carried through a chain of short, thin axoned microneurons to larger cells from which recordings are selectively taken. Some anatomical support for our postulate was provided by Papez 27 who stated that the scarce larger cells are multipolar neurons whose dendrites synapse with the small surrounding cells. This arrangement would suggest that the larger neurons are sheltered against incoming volleys from extracaudatal structures and would insure firing of the larger cells only after the smaller surrounding elements have been triggered into activity. It is said that the smaller neurons are more difficult to record from, whether intra- or even extracellularly, since one has to bring the electrode pore much closer to discriminate their action potentials from background noise. As the probe is pushed against the cell membrane the firing capability of the neuron may be depressed or the unit may be destroyed even before penetration takes place s,31. Unitary responses in the caudate to afferent stimulation usually have longer latencies than would be anticipated by the conduction distance involved. Inordinately long latencies have also been found for cortico-caudate 29 and nigro-caudate 11,13 responses nevertheless presumed to be monosynaptic. In both situations, these latencies have been attributed to conduction along small diameter pathways. While these fibers may be of relatively
INTRINSICCONNECTIONSOF CAUDATENEURONS
303
small diameter and slow conduction velocity, the greatest reduction in diameter is known to take place at intracaudatal arborizations. The intrinsic axons of caudate neurons are also of extremely thin diameter. I f the above responses were not monosynaptic as presumed, the neuropil axons would be expected to account for a large fraction of the total conduction time. In the light of the above discussion and the present findings of relatively long latencies to local stimulation, it may be concluded that a considerable fraction of the latency of caudate unitary responses to intrinsic stimulation is due to slow conduction within the caudate. This explanation may account for the latency discrepancy between macro- and microphysiological responses 4,5. The burst responses of high frequency illustrated in Fig. 5 A - D may represent the activation of excitatory or inhibitory interneurons or cells which may have the overall role of modulating the membrane potential of other target neurons 23,24. AlbeFessard et aL ~ suggested that caudate interneurons are probably responsible for the protracted phases of post-activation hyporesponsiveness encountered in their study. McLennan and Y o r k 26 have postulated a terminal dopaminergic inhibitory interneuron lying entirely within the caudate which itself may be excited by intralaminar thalamic stimulation. More recently, K e m p and Powel120 have suggested that a large number of the neurons in the striatum, establishing symmetrical synapses with other intrinsic neurons, may function as inhibitory neurons. The fact that the latency of the first spike in our burst responses and those of others a2 was about the same as in units responding with single spikes cannot be construed as an argument against the possibility that these may represent two different populations of neurons with different physiological rolesa0, al. The number of spikes was curtailed in some high frequency burst responses when the stimulus was raised to supramaximal strength. This suggests that some other inhibitory mechanism was activated by the increase of stimulus strength, perhaps an inhibitory postsynaptic potential influencing the behavior of these interneurons. ACKNOWLEDGEMENTS We appreciated the technical help received at different stages of this work from J. M. Dessama, S. Ivany, R. Weisman, J. Hollenberg, A. Korenovsky, A. L a m m e and Mrs. T. Powell. This work was supported in part by U.S. Public Health Service Grants, NS 05184, NS 5243 and M H 10315.
REFERENCES 1 ADINOLFI,A. M., Fine Structural Organization of the Feline Caudate Nucleus and Changesfollowing Nigral Lesions, Ph.D. Thesis, Faculty of Pure Science, Columbia University, New York, 1967. 2 ADINOLrI,A.,M., Observations on the fine structure of the feline caudate nucleus, Anat. Rec., 157 (1967) 203. 3 ADINOLFI, A. M., AND PAPPAS, G. D., The fine structure of the caudate nucleus of the cat, J. comp. Neurol., 133 (1968) 167-184.
304
L.A. MARCO et al.
4 ALBE-FESSARD,D., OSWALDO-CRuz, E., ET ROCHA-MIRANDA, C. E., Activit6s 6voqu6es dans le noyau caud6 du chat en r6ponse A des types divers d'aff6rences. 1. Etude macrophysiologique, Electroenceph. clin. Neurophysiol., 12 (1960) 405-420. 5 ALBE-FESSARD, D., ROCHA-MIRANDA, C. E., ET OSWALDO-CRuz, E., Activit6s 6voqudes dans le noyau caud6 du chat en r6ponse ~ des types divers d'aff6rences. II. Etude microphysiologique, Electroenceph. clin. Neurophysiol., 12 (1960) 649-661. 6 ALEXANDER, L., The vascular supply of the strio-pallidum, Res. Publ. Ass. nerv. ment. Dis., 21 (1942) 77-132. 7 ASANUMA,H., AND ROSEN, I., Synaptic connections within the cat's motor cortex, Fed. Proc., 31 (1972) 303. 8 BISHOP, G. H., In M. D. YAHR AND D. P. PURPURA (Eds.), Neurophysiological Basis of Normal and Abnormal Motor Activities, Raven Press, New York, 1967, pp. 211-213. 9 CRAIN, S. M., PETERSON, E. R., AND BORNSTEIN, M. B., Formation of functional interneuronal connexions between explants of various mammalian central nervous tissues during development in vitro. In G. E. W. WOLSTENHOLME AND M. O'CONNOR (Eds.), Growth of the Nervous System (Ciba Symposium), Churchill, London, 1968, pp. 13-40. 10 ECCLES, J. C., LLINAS, R., AND SASAKI, K., Intracellularly recorded responses of the cerebellar Purkinje cells, Exp. Brain Res., 1 (1966) 161-183. l l FELTZ, P., AND MACKENZIE, J. S., Properties of caudate unitary responses to repetitive nigral stimulation, Brain Research, 13 (1969) 612-616. 12 FREYGANG, W. H., JR., An analysis of extracellular potentials from single neurons in the lateral geniculate nucleus of the cat, J. gen. Physiol., 41 (1958) 543-564. 13 FRIGYESI, Z. L., AND PURPURA, D. P., Electrophysiological analysis of reciprocal caudato-nigral relations, Brain Research, 6 (1967) 440-456. 14 HOLMES, R. L., NEWMAN, P. P., AND WOLSTENCROFT, J. H., The distribution of carotid and vertebral blood in the brain of the cat, J. Physiol. (Lond.), 140 (1958) 236-246. 15 KEMP, J. M., An electron microscopic study of the termination of afferent fibres in the caudate nucleus, Brain Research, 11 (1968) 464-467. 16 KEMP, J. M., AND POWELL, T. P., The structure of the caudate nucleus of the cat : light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 17 KEMP, J. M., AND POWELL, T. P., The synaptic organization of the caudate nucleus, Phil. Trans. B, 262 (1971) 403-412. 18 KEMP, J. M., AND POWELL, T. P., The site of termination of afferent fibres in the caudate nucleus, Phil. Trans. B, 262 (1971) 413-427. 19 KEMP, J. M., AND POWELL,T. P., The termination of fibres from the cerebral cortex and thalamus upon dendritic spines in the caudate nucleus: a study with the Golgi method, Phil. Trans. B, 262 (1971). 20 KEMP, J. M., AND POWELL, T. P., The connexions of the striatum and globus pallidus: synthesis and speculation, Phil. Trans. B, 262 (1971) 441-457. 21 KRNJEVI(~, K., RANDIC, M., AND STRAUGHAN, D. W., Pharmacology of cortical inhibition, J. Physiol. (Lond.), 184 0966) 78-105. 22 LI, C. L., AND CHOU, S. N., Cortical intracellular synaptic potentials and direct cortical stimulation, J. cell. comp. Physiol., 60 (1962) 1-16. 23 MARCO, L. A., BROWN, T. S., AND ROUSE, M. E., Unitary responses in ventrolateral thalamus upon intranuclear stimulation, J. Neurophysiol., 30 (1967) 482-493. 24 MARCO, L. A., BROWN, T. S., AND ROUSE, M. E., Interneuronal barrages and ripple superposed on inhibitory postsynaptic potentials, Experientia (Basel), 23 (1967) 727. 25 MARCO, L. A., COPACK, P., AND GILMAN, S., Intrinsic links of caudate neurons, Fed. Proc., 31 (1972)403. 26 MCLENNAN, H., AND YORK, O. H., The action of dopamine on neurones of the caudate nucleus, J. Physiol. (Lond.), 189 (1967) 393-402. 27 PAPEZ, J. W., A summary of fiber connections of the basal ganglia with each other and with other portions of the brain, Res. Publ. Ass. herr. ment. Dis., 21 (1942) 21-68. 28 PHILLIPS, C. G., Some properties of pyramidal neurones of the motor cortex. In G. E. W. WOESTENHOLMEAND M. O'CONNOR (Eds.), The Nature of Sleep (Ciba Symposium}, London, Churchill, 1961, pp. 4-24. 29 ROCHA-MIRANDA, C. E., Single unit analysis of cortex-caudate connections, Electroenceph. clin. Neurophysiol., 19 (1965) 237-247.
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
305
30 SCHLAG, J., In M. A. B. BRAZIER(Ed.), The Interneuron, Univ. Calif. Press, Los Angeles, 1969, pp. 519-520. 31 SCHLAG,J., AND VILLABLANCA,J., A quantitative study of temporal and spatial response patterns in a thalamic cell population electrically stimulated, Brain Research, 8 (1968) 255-270. 32 SEDGWICK, E. M., AND WILLIAMS,T. D., The response of single units in the caudate nucleus to peripheral stimulation, J. Physiol. (Lond.), 189 (1967) 281-298. 33 STOHR, e. E., GOLDRING, S., AND O'LEARY, J. L., Patterns of unit discharge associated with direct cortical response in monkey and cat, Electroenceph. clin. Neurophysiol., 15 (1963) 882-888. 34 SUGAYA, E., GOLDRING, S., AND O'LEARY, J. L., Intracellular potentials associated with direct cortical response and seizure discharge in cat, Electroenceph. clin. Neurophysiol., 17 (1964) 661669. 35 TENNYSON, V. M., AND MARCO, L. A., Intrinsic connections of caudate neurons. II. Fluorescence and electron microscopy following chronic isolation, Brain Research, 53 (1973) 307-317. 36 VERHAART,W. J. C., Fiber analysis of the basal ganglia, J. comp. Neurol., 93 (1950) 425-440.
291
© ElsevierScientificPublishingCompany, Amsterdam- Printed in The Netherlands
INTRINSIC CONNECTIONS OF CAUDATE NEURONS. I. LOCALLY EVOKED FIELD POTENTIALS AND E X T R A C E L L U L A R UNITARY ACTIVITY
LUIS A. MARCO, PAULA COPACK, A L A N M. EDELSON AND SID G I L M A N
Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 (U.S.A.) (Accepted October 20th, 1972)
SUMMARY
The field potentials and extracellular activity within the caudate nucleus of cats were investigated by means of closely apposed fine stimulating electrodes arranged in a semicircle at 1.5 mm distant from the recording glass pipette. The studies were performed on intact animals anesthetized with Nembutal or chloralose-urethane and in unanesthetized enc6phale or cerveau isol6 preparations. All animals were paralyzed and artificially respirated. Recordings were also obtained in cats in which the caudate nucleus had been isolated from the surrounding tissues 20-90 days before recordings were taken to allow for degeneration of afferent fibers. In all types of preparations, stimulation of discrete points around the recording micropipette typically evoked field potentials of negative polarity and 3-5 msec duration at an average latency of 10 msec. Single spike unitary responses usually emerged from the falling phase or peak of the field potentials. Commonly, repetitive stimulation failed to evoke responses for 150200 msec following the first response. Spontaneous unitary activity was often arrested by single shocks, and the duration of spike suppression ranged from 150 to 200 msec. We suggest that inhibitory postsynaptic potentials are responsible for the protracted recovery cycle of the field potentials, for the long-lasting unresponsiveness of units, and for the suppression of spontaneous spikes following a stimulus. Few units were capable of responding at shorter latencies and of following high frequencies of stimulation. A few cells fired in short bursts at high frequencies in response to single shocks. We conclude that these findings reflect the synaptic properties of interconnected elements within the caudate neuropil. We suggest that the first order elements, receiving afferent impulses, may be very small and relatively inaccessible even to fine microelectrode probing, and that the present unitary recordings may reflect only the activity of larger neurons further along the chain of transmission within the caudate neuropil.
292
L.A. MARCOet al.
INTRODUCTION
The intrinsic synaptic organization of the caudate nucleus is now beginning to be unraveled due to the ultrastructural studies of Adinolfi 1-3 and particularly the series of Kemp 15-2°. These investigations have suggested that interconnections between caudate neurons must be very numerous and that consequently integration by interneurons, by far the largest population, must be considerable. Their short thin axons terminate on dendrites and somata of other caudate neurons, thus establishing short intrinsic links among themselves. On the basis of these ultrastructural and other previous studies 4,s,13, a number of suggestions have been made which can be subjected to electrophysiological testing. Albe-Fessard et al. 4 had already proposed that different neurons in intricate anatomical distribution were engaged in inhibitory interactions with interconnected neighboring elements within the caudate. The extreme thinness of intrinsic axons 1-3,15-2°,36 has prompted the suggestion that the usual long latency of responses of caudate units may be due to slow conduction through these thin intrinsic multisynaptic pathways 1,18. Bishop 8 explicitly proposed that it would be worthwhile to investigate the responses to local stimulation within the caudate in search of interference or summation of these responses. No such approach has yet been undertaken systematically, despite the large size of the caudate which makes it convenient for such a study. The head of the caudate can be exposed and identified readily by removal of the roof of the lateral ventricle. Recording microelectrodes and arrays of stimulating electrodes can then be placed within the caudate under direct visualization. Most neuronal elements in the caudate nucleus are difficult to investigate electrophysiologically due to their small size but systematic local stimulation is a powerful way of firing neurons and has in the past yielded meaningful results at the level of the cerebral cortex 7,21,22,2s,33,a4, in the cerebellum 10, and in the thalamus2a,24,3L In view of these observations, we decided to investigate the topography and properties of intranuclearly driven caudate cells. Specifically, we wished to learn what types of neuronal responses can be elicited in the caudate nucleus by stimulation through closely apposed electrodes. Are they compatible with those obtained by more conventional means of stimulation? Do cells respond with single spikes or repetitively? If repetitive responses are encountered, can they be due to the activity of cells similar to Renshaw neurons? Long latency responses in the caudate have been the rule when distant hodologically related structures were stimulated. Could these long latency responses be accounted for by extrinsic conduction or was conduction time spent mostly in intrinsic transmission? If the latter, what are the synaptic features? These questions can be answered from the characteristics of unitary responses to intranuclear stimulation and the conclusions can be reinforced by the results of parallel studies in the isolated caudate. The results of extracellular recordings will be described in this paper, and the evaluation of electron and fluorescence micrographs of isolated caudates will be given in the following paper 3~. The results of intracellular recordings will be the object of a subsequent communication (Marco et al., in preparation). The present work will show that stimulation of the caudate closely apposed to the site of recording can achieve an acceptable degree of selectiveness and provide a good basis for a first attempt at reconstructing
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
293
the intrinsic synaptic and topographical organization of caudatal elements at the electrophysiological level. Part of this work has been published in abstract form 2s. METHODS
Forty-one cats were used in this study. Most of the animals were anesthetized with 35 mg/kg of sodium pentobarbital (Nembutal) injected intraperitoneally. Five animals were anesthetized with a-chloralose plus urethane (40 and 800 mg respectively/kg) dissolved in propylene glycol and injected intraperitoneally. Seven cats were injected with a lighter dose of sodium pentobarbital or induced with a fast-acting barbiturate or ether inhalation, and all 7 were subsequently maintained as cerveau or enc6phale isol6 preparations for the recordings. Every preparation was paralyzed by means of intravenous gallamine triethiodide and artificially respired. The ear canals and infraorbital subconjunctival folds were filled with viscous xylocaine and the animals were clamped in a stereotaxic apparatus. Through a craniotomy, the dura was resected and all the brain tissue overlying the ventricular surface of one or both caudates was aspirated by suction, leaving exposed the caudate nucleus rostrally and the fimbria and hippocampus caudally. In about 50 ~ of the experiments aspiration of tissue was extended to the frontal pole. Under visual guidance stimulating and recording electrodes were placed stereotaxically in position for vertical tracking of unitary activity within the caudate. A chart of the rostrocaudal, lateral and vertical parameters used for each track was kept for every experiment. Tracking was carried out with a hydraulic microdrive capable of lowering the recording micropipette in measurable steps of 1/tm. Three stimulating electrodes were prepared from 250/~m diameter tungsten wire straightened and etched to a point with a diameter of about 1 #m by electrolysis and coated except at the tip. Under a dissecting microscope the coating was scraped off up to 0.5 mm from the tip. This procedure yielded an electrode with a DC impedance of about 50 kf2 in normal saline. These 3 wires were passed through holes drilled by the edge o f a perspex block which had been machined to a shape represented in Fig. 1A. The 3 stimulating electrodes were then cemented to the lucite block in a semicircular configuration (m, medial; c, caudal; 1, lateral stimulating electrodes). The entire assembly was clamped by means of a rigid steel strut (C) to a micromanipulator. The recording micropipette (R) was invariably positioned at the center of the semicircle formed by the stimulating electrodes before tracking was initiated so that the distance between the recording and each of the stimulating electrodes was 1.5 mm. Kemp and PowelP s have recently shown that asymmetrical intrinsic synapses extend at least 1.5 mm from the parent cell. Penetration through the depth of the caudate was carried with two independent micromanipulators, one for the stimulating 3-prong array and the other for the recording micropipette. Tracking in search of units was invariably carried out according to the following procedure: the stimulating array was lowered 0.5 mm below the point at which the tips touched the dorsal surface of the caudate and was left at that depth to ascertain that the exposed tips were buried in caudate tissue. Then tracking with the micropipette was initiated until it had been lowered 1 mm below the ependymal dorsal surface of the caudate. After this 1 mm
294
L.A. MARCOet al.
o
I 8
_
8
7
6
5
4
3
2
1
0
I
I
I
I
I
I
I
I
I
7_
4_
.
I..t
Fig. 1. A, Block of lucite with small holes drilled through its thickness close to the semicircular edge. The 3 stimulating electrodes (m, c and 1; medial, caudal and lateral) were passed through the holes and were permanently cemented in position to yield a distance of 1.5 mm from the recording glass micropipette (R) to each of the stimulating electrodes. A rigid steel strut was also cemented to the block and the stimulating assembly was clamped at the upper end of the strut (C) to an electrode holder mounted to a microdrive. B, Plotting of the first 100 unitary responses in association with field potentials at Fr +16.
penetration, the tip of the micropipette was 0.5 m m ventral to the tips of the stimulating array. Units encountered during penetration through this distance were investigated. Then the stimulating array was lowered another I mm and thus the stimulating and recording electrodes were alternately lowered at 1 m m steps until the end of the track had been reached. Glass micropipettes were pulled to yield a resistance of 5-15 Mr2 when filled with 3 M potassium chloride or 2 M potassium citrate. Every evoked response was tested with both cathodal and anodal rectangular pulses which usually ranged from 3 to 15 V, 0.1-0.3 msec duration. The indifferent electrode was placed on the temporo-occipital muscle of the contralateral side. Stimuli were delivered through a Grass stimulus isolation unit (SIU). No other measures were taken to mitigate the recording of artifacts generated by the closely apposed stimulus except in a few experiments in which the SIU was replaced by a Tektronix photon-coupled stimulus isolator without significant improvement. A switch box was interpolated between the SIU and the 3-pole stimulating array to facilitate swift checking of the effectiveness of each stimulating electrode (m, c, 1). Recorded signals were led through a negative capacitance high impedance electrometer to AC and DC coupled amplifiers in parallel and displayed on the face of an oscilloscope after appropriate amplification. U p o n completion of an experiment the tips of the stimulating array were fulgurated at stereotaxic parameters corresponding to the presumed center of the caudate, thus serving as a countercheck for the parameters recorded in the chart. The brain was then perfused with normal saline followed by 10% formalin. A few days later, a block enclosing the caudate nucleus was cut out from the rest of the brain and kept in fresh formalin for subsequent histological verification of the tracks of the stimulating electrodes.
295
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
In 7 cats the head of the caudate nucleus was isolated unilaterally from the rest of the brain except for its ventral aspect which merges with the lateral hypothalamic region and through which it receives its vascular supply from the striate arterioles. The latter originate in the base of the brain from the middle cerebral arteries 6,14. Unila teral exposure of the dorsal surface of the caudate nucleus was carried out under aseptic conditions according to the procedure outlined above for acute recordings. A sharp knife was used to cut through the tissue surrounding the caudate in all 4 planes. Isolation was completed by means of a blunt spatula which reached the ventral surface of the brain in all 4 planes. The opening was closed by anatomical layers, the animals were allowed to recover and the recording experiment, as described above, was carried out 20-90 days later. Terminal degeneration is said to be complete by the sixth day is. At the end of the experiment a wedge of caudate tissue 1 mm thick was taken in vivo at about Fr ÷ 16 from both the isolated and the contralateral control sides. The specimens were subsequently prepared for fluorescence and electron microscope evaluation 35. The essential characteristics of the responses described in this study did not differ with the type of anesthesia or preparation used. RESULTS
( A ) Field potentials Single shocks applied 1.5 mm from the recording micropipette evoked focal potentials throughout the depth of the head of the caudate nucleus. Fig. 1B shows the sites from which field potentials were recorded in combination with unitary activity at Fr + 16. Focal potentials were invariably of negative polarity and had peak latencies A
D
G
B
c ._..%-m
__F
'o Is
D
_~f
1012
C
Fig. 2. Left panel, Field potentials evoked in the dorsal portion of the head of the caudate (Fr + 15, L 5, V +6.5) by single shocks delivered through the m electrode (A-C). Stimulus strength increased from A to C which represents a maximal response. Note that the amplitude of field potentials increased and the latency correspondingly decreased with stronger stimuli. Same stimulus strengths applied to the c electrode (D-F) yielded much smaller responses and no response could be evoked by 1 stimuli (G-H), G taken at the same stimulus strength as A; H at twice that of C. Right panel, Shows that stimulation and recording in the ventral portion of the caudate nucleus (a-c) yielded responses of largest amplitude to the m stimulus (A), smaller responses to the c shock (B), and no response to the I pulse (C). Dorsally however (d) only 1 stimulation evoked responses (D). Recordings at Fr + 17, L 3.5, V +6.5 and + 1 respectively. Five sweeps superimposed in all frames. Negativity in this and all subsequent figures is signified by a downward deflection of the trace; horizontal and vertical bar calibrations represent time and amplitude in msec and mV respectively, m, c or I at the trace-break caused by the stimulus artifact indicate application of a medial, caudal or lateral stimulus respectively.
L.A. MARCO et al.
296
of 8-10 msec, amplitudes of 2.5-3 mV and durations of 3-5 msec. However, these responses could not usually be evoked by all 3 stimulating electrodes in any single placement. Responses of optimal amplitude were commonly evoked by just one stimulating electrode, the other two being either ineffective or minimally capable of eliciting responses. Fig. 2, left, is an example of focal potentials optimally evoked by the m stimulus (A-C), less effectively by the c pulse ( D - F ) and not by the I shock ( G - H ) . The orientation of the recording pipette to the stimulating electrode most effective in evoking focal potentials varied as the electrodes were lowered through the caudate. Fig. 2, right, illustrates this observation. Thus, at different levels of penetration the stimulus had to be switched from one electrode to another in order to maintain optimal or adequate focal potentials as well as a high yield of unitary evoked activity (see Section 1). Moreover, when the micropipette was left more than 2 mm dorsal to the stimulating array, or when it was lowered below the stimulating array by the same magnitude, focal potentials were lost altogether. Thus, field responses were very selectively pin-pointed at any given depth. Field potentials did not follow high frequency stimulation. Fig. 3 D - E illustrates response failure after the third and second pulses at 50 and 100/sec respectively. Occasional stimuli (0.5-1/sec) elicited responses with characteristics essentially invariant with stimulation time. Repetitive stimulation at about 20/sec completely blocked the focal potentials after the fifth or sixth pulse. Following the end of the tetanizing train there was posttetanic depression which lasted 3-5 sec, followed by gradual recovery to amplitudes somewhat above control. Fig. 3 A - C illustrates this sequence of events, i.e., the frequency-dependent attenuation and subsequent slight potentiation. A few seconds of anoxia were sufficient to suppress large field potentials (Fig. 3F-H).
(B) Unitary activity Five hundred and seven units were recorded extracellularly in this series. The
so/,
rrrrrrrr
!°I'
Fig. 3. Left, Effects of tetanization on field potentials. A, before; B, during 20/sec tetanizing train; C, responses 10 sec after the end of the tetanizing train. Center, Failure of sustained responses to 50 (D) and 100/sec (E) trains. Right, Effects of anoxia. F, before; G, after 5 sec of apnea; H, shortly after respirator was turned on again. Several sweeps in each frame.
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
297
action potentials were of 1.5-2 msec duration, 2-6 mV amplitude, positive or positivenegative in configuration. These features indicate that recordings were obtained with the pipette pore very close to the perikaryon 12. Thirty-eight per cent of the units recorded fired spontaneously. The firing rates varied widely from one neuron to another and ranged between 1 and 32/sec. Consistent differences in spontaneous activity between the various types of preparations could not be established. The mean firing rate for the entire population was l0 4- 9/sec. (1) Unitary spike responses to electrical stimulation. Twenty-five per cent of the spontaneously firing units were also driven by stimulation. Their mean latency was 11 ± 3 msec. Sixty-two per cent of all the units were silent in the absence of stimulation, but single pulses of relatively low strength (3-7 V) every 2 sec consistently evoked responses from these neurons with a mean latency of 10 4- 5. A plot of frequency distribution shows that the latencies centered around 9-10 msec. The latency histogram of Fig. 4 illustrates these findings. The percentile relative contribution to driven responses by each one of the stimulating electrodes or their combination is given in Table I. It shows that urdts responded more frequently to stimulation of 1 or all 3 electrodes independently (cml) and less often to stimulation of only 2 of the electrodes independently (cm, cl, ml). TABLE1 c
m
l
cm
cl
ml
cml
17.5
20.8
18.9
6.0
7.7
7.7
20.8
Some unitary responses were evoked only by relatively strong stimuli between 15 and 30 V, and then only 1 of the 3 electrodes could evoke the responses. Indeed, even at such high stimulus strength some units failed to respond unless a train of 2-3 pulses at 100/sec was applied. Unitary responses usually consisted of a single spike emerging and gradually growing out of the failing phase of the negative field potential in the form of initially positive notches or full spikes superimposed on the negative wave (Fig. 4C-G). Double spike responses such as those illustrated in Fig. 4A, the first spike having very short latency and the second spike the typical latency, were rarely observed. Single spike responses such as those illustrated in Fig. 4 C - G were usually observed. The stimulus location capable of evoking the largest field potential was usually the one giving the highest yield of evoked unitary activity. Unitary responses were also evoked, although less commonly, in the absence of field potentials. Nineteen per cent of the driven units were triggered by a single stimulus into a burst of 4-8 spikes at a firing rate of 115-500/sec. Shocks stronger than those required to evoke a burst response succeeded only in reducing the duration of the burst to a single spike response, but lowering the stimulus strength did not reduce the burst response to a single spike response, i.e., there was either a burst response or nothing. Fig. 5A-D shows some of these burst responses in 4 units, all from different experiments. The latencies of the first spikes in each burst ranged from 7 to 12 msec.
298
L.A. MARCO et al.
25
C 20
15
10
E I
F msec.
0
,
.
5
10
I
I
--7. 15
G
Fig. 4. Left, Frequency distribution of latency responses of the first 111 unitary recordings collected in this series. Ordinate, number of units. Abscissa, latency in msec. Inset traces represent short latency unitary responses (first spike, in A) and responses at typical latencies (B and second spike in A) elicited by stimuli delivered to the c and the m electrodes respectively. Right, Field potentials and unitary responses simultaneously evoked by m shocks as the recording micropipette was lowered about 25/~m from C to G. Note that in C only field potentials were recorded but in D the positive phase of unitary responses begins to emerge and gradually grows from E to G. Three to 5 sweeps superimposed except in G.
Seventeen per cent o f the driven units were c a p a b l e o f following each pulse in a train o f stimuli at 30/sec; 13 ~ followed 50/sec trains; a n d only 3.7 ~ were driven at 100/sec. Fig. 5E illustrates one o f these rare units c a p a b l e o f r e s p o n d i n g to each pulse at 50/sec. The latencies o f these responses were i n v a r i a n t within each unit a n d ranged between 8 a n d 10 msec. Units which r e s p o n d e d to s t i m u l a t i o n o f m o r e t h a n one electrode site were studied with p a i r e d pulses, a p p l y i n g a c o n d i t i o n i n g pulse to one electrode a n d a testing shock to a n o t h e r electrode. F i r i n g indices (FI, n u m b e r o f r e s p o n s e s / n u m b e r o f stimuli) were o b t a i n e d for the testing electrode at various pulse intervals. R a n d o m l y , the cond i t i o n i n g pulse was t u r n e d off t h r o u g h o u t the series o f intervals to ascertain that the test spike response was still present at an F I o f 100 ~ . The results o f these experiments were as follows. (a) F a i l u r e o f response to the testing pulse ( F I ~ 0 ~o) when it was preceded by the c o n d i t i o n i n g stimulus occurred at intervals ranging from 15 to 280 msec. (b) In a n o t h e r similar set o f observations in units which were triggered by only one electrode, the test pulse was a p p l i e d t h r o u g h this electrode while the c o n d i t i o n i n g stimulus was applied to a n o t h e r electrode which did not evoke a unitary response. I n
299
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
A m
5
F
I
c
100
]
200 J
2
I
10
I
C
20
llln 11tll 111111111111 !
II~[
[ll~t
P'w [ [I~B I m
Fig. 5. A-D, High frequency barrage responses from different units evoked by single shocks. E, Pulse per pulse unitary responses to a 50/sec train delivered through the c electrode. Note doublet response to first shock. Right, Two spontaneously firing units (F and G-H) which were silenced for about 125 and 100 msec respectivelyfollowing single stimuli. Five sweeps superimposed in F. this situation, the conditioning pulse also succeeded in blocking the response to the testing pulse. Under such conditions, neuronal refractoriness, collision, occlusion, post-excitation, inhibition and after-hyperpolarization can be ruled out and the responsible process must be either active inhibition or dis facilitation. (2) Suppression of spontaneous unitary activity by electrical stimulation. An investigation of the change in behavior of spontaneously firing units following stimulation provided further support for the idea of locally generated inhibition. Thirty-three per cent of the units firing spontaneously were silenced by either single shocks or 3pulse trains at 100/see. The duration of spike suppression varied widely and ranged from 75 to 600 msec. Fig. 5 F - H shows two units firing spontaneously which were thus silenced by single shocks. After the poststimulus silent interval some units resumed firing at the prestimulus rate (F). In other units, after the silent period, firing resumed in an uneven pattern, the silent intervals between bursts often having the duration of the first period of silence induced by the shock (G), but silent intervals gradually decreased in duration in the absence of stimulation and continuous firing was eventually reestablished. When the unit was firing at a higher frequency, the interburst silent intervals were less clear-cut and of shorter duration (H).
( C) Evoked field potentials and unitary activity in chronically isolated caudates The possibility existed that the stimulus might have triggered neurons outside
300
L.A. MARCO et al.
m
I
ffl
m
C
I
5 ,
i
1
L
Fig. 6. Responses in isolated caudates. Seven units from different experiments are represented. In A-C threshold shocks evoked unitary spikes arising from the falling phase of the field potential. Unit D-F, stimuli slightly above threshold evoked similar unitary responses at 10 msec latency. Unit G, H was evoked at slightly longer latencies (12-13 msec). Unit I, response latencies varied from 10 to 13 msec. Units J, K, L exhibited response latencies of 8, 7 and 11 msec respectively. Several sweeps superimposed in frames B, E, G and I. The caudate from which unit A-C was recorded is described in the following paper 35.
the c a u d a t e which in t u r n would send their impulses to, and fire, the units r e c o r d e d in the caudate. T o test this possibility, in 7 cats the head o f the c a u d a t e was isolated f r o m the rest o f the b r a i n (see M e t h o d s ) . R e c o r d i n g s in these p r e p a r a t i o n s were m o r e difficult a n d gave a lower yield o f responses t h a n in p r e p a r a t i o n s with intact connections. Thirty-seven units were recorded in these experiments. F o r t y - t w o per cent o f them exhibited s p o n t a n e o u s activity. F i r i n g rates varied f r o m one n e u r o n to a n o t h e r and r a n g e d between 5 a n d 40/sec ( m e a n 13/sec). The rest fired only in response to local stimulation. The latencies o f field potentials a n d u n i t a r y responses were c o m p a r a b l e to those o b t a i n e d in the c o n t r o l p r e p a r a t i o n s . The usual latency was 9-10 msec (Fig. 6) b u t a few units were r e c o r d e d in which response latencies were 4 - 6 msec. The results o f electron a n d fluorescence m i c r o s c o p y o f these isolated caudates are given in the following c o m m u n i c a t i o n 35. Since some synapses exhibited all the m o r p h o l o g i c a l features o f functionally viable junctions, these can be presented as being responsible for transmission o f the e v o k e d responses e n c o u n t e r e d in our physiological experiments in the isolated caudates. N o t o p o g r a p h i c a l p a t t e r n was discernible to differentiate units which fired spont a n e o u s l y f r o m those which were silent in the absence o f stimulation, n o r was there a difference f r o m those r e s p o n d i n g in burst o r c a p a b l e of following higher frequencies
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
301
of stimulation. Maps similar to that shown in Fig. 1B were made of units recorded at more rostral and caudal planes. None of these plottings yielded a topographical characterization in terms of patterns of responses. Greater numbers of tracks were made within the mid-portion of the caudate (lateral 3-5) than on either side of this region in order to ascertain that all 4 electrodes were within the caudate for the longest tracks. DISCUSSION
Field potentials with latencies shorter than 7 msec were not observed in these experiments, indicating that the pathways involved were of slow conduction velocity. This is in keeping with the results of ultrastructural studies showing that in the caudate neuropil the mean diameter of most fibers is less than 1 /~m and that the terminal arborizations must be exceedingly fine. Moreover, axodendritic spine synapses are more numerous than axodendritic trunk synapses, and the latter in turn are much more frequently encountered than axosomatic synapses. The majority of axons in the caudate are short and slender and terminate in local arborizations close to their cell bodies, thus establishing synapses with the short, thin dendrites of other neighboring neurons. Synaptic contacts occur most frequently among the small elements of the neuropil which function as components of intrinsic chains of contact within the nucleus1-3,15-20. It follows therefore that transmission of impulses from the site of stimulation to the point of recording (1.5 mm apart) most plausibly involves a multisynaptic or at least an oligosynaptic chain of slowly conducting axodendritic junctions. Despite the typical homogeneity and absence of cell lamination in the striatum 27, formation of small clusters of neurons have been recently redescribed in the caudatO 6, 27. This feature would most certainly account for both the discreteness and the negligible stimulus-dependent growth of the focal potentials. There was a clear correlation between the characteristics of field potentials and the properties of unitary responses. Evoked unitary responses were difficult to obtain in experiments in which field potentials could not be demonstrated. The largest majority of triggered units fired during the falling phase of the field potential. Extracellular recordings were often immediately followed by penetration of the cell, leaving no doubt about the juxtasomatic position of the recording electrode. In these situations, the negative field potential consistently reversed to a positive excitatory postsynaptic potential of equal latency (Marco et al., in preparation) indicating that the field potentials reflect synaptic activation of a constellation of neurons within the receptive field of the electrode pore. The properties of the field potentials we have described are characteristic of synaptic potentials. When one stimulus site was more effective than the other two for evoking field potentials, the same site was also optimal for triggering unitary potentials. The findings summarized in Table I suggest that spread of stimulating current from one stimulus site to another was negligible. If significant spread of current had occurred one would expect, in a semicircular array, more current spread between the electrodes composing the cl and cm pairs and less spread between the electrodes of the ml pair. Accordingly, the cl and cm pairs should have recruited the largest percentages
302
L.A. MARCOet al.
of driven units and the ml pair the lowest. The values in Table I, however, show that all 3 groups of two electrodes have about the same percentage of driven unitary responses. On the other hand, the c, m, I and cml groups all generated about the same percentage of driven responses which is much higher than that of the combination of any two electrodes. This finding suggests, apart from the obvious random orientation of neuropil axons, that (a) appreciable spread of current from one stimulating electrode to another did not occur, and (b) direct depolarization of neuronal somata was not induced. These two conclusions would not be difficult to accept in the light of the experiments by Crain et a l ) showing that there was no non-neural spread of current in a bipolar explant from one pole to the other after midline transection even when the gap was less than l0 # m wide. In addition, the findings suggest that there are at least 4 populations of cells in terms of interconnectivity. The first is represented by those elements receiving input from more caudal regions (c-driven). The second and third receive synaptic impingement from more medial and lateral zones (m- and l-driven, respectively). The fourth population, equally large, consists of neurons having connectivity in all 3 directions (cml-driven group). The rest of the population is divided into 3 subgroups about equal in size but smaller than those described above. They are represented by those units having interconnectivity in 2 of the 3 directions investigated. The comparative macro- and microphysiological study of Albe-Fessard et al. 4,5 demonstrated that unitary latencies were much longer than the latencies of focal potentials by an average difference of about l0 msec. Such findings suggest that the first order units upon which afferents impinge are somehow not easily accessible to fine microelectrode probing, since responses from these units should have latencies only 0.5 msec longer (one synaptic delay) than the evoked field potentials contributed by the primary afferents. A conclusion similar to ours was drawn by Sedgwick and Williams 32. It seems plausible therefore that the afferents first impinge upon very small elements (7 # m diameter, Golgi type II) from which transmission is carried through a chain of short, thin axoned microneurons to larger cells from which recordings are selectively taken. Some anatomical support for our postulate was provided by Papez 27 who stated that the scarce larger cells are multipolar neurons whose dendrites synapse with the small surrounding cells. This arrangement would suggest that the larger neurons are sheltered against incoming volleys from extracaudatal structures and would insure firing of the larger cells only after the smaller surrounding elements have been triggered into activity. It is said that the smaller neurons are more difficult to record from, whether intra- or even extracellularly, since one has to bring the electrode pore much closer to discriminate their action potentials from background noise. As the probe is pushed against the cell membrane the firing capability of the neuron may be depressed or the unit may be destroyed even before penetration takes place s,31. Unitary responses in the caudate to afferent stimulation usually have longer latencies than would be anticipated by the conduction distance involved. Inordinately long latencies have also been found for cortico-caudate 29 and nigro-caudate 11,13 responses nevertheless presumed to be monosynaptic. In both situations, these latencies have been attributed to conduction along small diameter pathways. While these fibers may be of relatively
INTRINSICCONNECTIONSOF CAUDATENEURONS
303
small diameter and slow conduction velocity, the greatest reduction in diameter is known to take place at intracaudatal arborizations. The intrinsic axons of caudate neurons are also of extremely thin diameter. I f the above responses were not monosynaptic as presumed, the neuropil axons would be expected to account for a large fraction of the total conduction time. In the light of the above discussion and the present findings of relatively long latencies to local stimulation, it may be concluded that a considerable fraction of the latency of caudate unitary responses to intrinsic stimulation is due to slow conduction within the caudate. This explanation may account for the latency discrepancy between macro- and microphysiological responses 4,5. The burst responses of high frequency illustrated in Fig. 5 A - D may represent the activation of excitatory or inhibitory interneurons or cells which may have the overall role of modulating the membrane potential of other target neurons 23,24. AlbeFessard et aL ~ suggested that caudate interneurons are probably responsible for the protracted phases of post-activation hyporesponsiveness encountered in their study. McLennan and Y o r k 26 have postulated a terminal dopaminergic inhibitory interneuron lying entirely within the caudate which itself may be excited by intralaminar thalamic stimulation. More recently, K e m p and Powel120 have suggested that a large number of the neurons in the striatum, establishing symmetrical synapses with other intrinsic neurons, may function as inhibitory neurons. The fact that the latency of the first spike in our burst responses and those of others a2 was about the same as in units responding with single spikes cannot be construed as an argument against the possibility that these may represent two different populations of neurons with different physiological rolesa0, al. The number of spikes was curtailed in some high frequency burst responses when the stimulus was raised to supramaximal strength. This suggests that some other inhibitory mechanism was activated by the increase of stimulus strength, perhaps an inhibitory postsynaptic potential influencing the behavior of these interneurons. ACKNOWLEDGEMENTS We appreciated the technical help received at different stages of this work from J. M. Dessama, S. Ivany, R. Weisman, J. Hollenberg, A. Korenovsky, A. L a m m e and Mrs. T. Powell. This work was supported in part by U.S. Public Health Service Grants, NS 05184, NS 5243 and M H 10315.
REFERENCES 1 ADINOLFI,A. M., Fine Structural Organization of the Feline Caudate Nucleus and Changesfollowing Nigral Lesions, Ph.D. Thesis, Faculty of Pure Science, Columbia University, New York, 1967. 2 ADINOLrI,A.,M., Observations on the fine structure of the feline caudate nucleus, Anat. Rec., 157 (1967) 203. 3 ADINOLFI, A. M., AND PAPPAS, G. D., The fine structure of the caudate nucleus of the cat, J. comp. Neurol., 133 (1968) 167-184.
304
L.A. MARCO et al.
4 ALBE-FESSARD,D., OSWALDO-CRuz, E., ET ROCHA-MIRANDA, C. E., Activit6s 6voqu6es dans le noyau caud6 du chat en r6ponse A des types divers d'aff6rences. 1. Etude macrophysiologique, Electroenceph. clin. Neurophysiol., 12 (1960) 405-420. 5 ALBE-FESSARD, D., ROCHA-MIRANDA, C. E., ET OSWALDO-CRuz, E., Activit6s 6voqudes dans le noyau caud6 du chat en r6ponse ~ des types divers d'aff6rences. II. Etude microphysiologique, Electroenceph. clin. Neurophysiol., 12 (1960) 649-661. 6 ALEXANDER, L., The vascular supply of the strio-pallidum, Res. Publ. Ass. nerv. ment. Dis., 21 (1942) 77-132. 7 ASANUMA,H., AND ROSEN, I., Synaptic connections within the cat's motor cortex, Fed. Proc., 31 (1972) 303. 8 BISHOP, G. H., In M. D. YAHR AND D. P. PURPURA (Eds.), Neurophysiological Basis of Normal and Abnormal Motor Activities, Raven Press, New York, 1967, pp. 211-213. 9 CRAIN, S. M., PETERSON, E. R., AND BORNSTEIN, M. B., Formation of functional interneuronal connexions between explants of various mammalian central nervous tissues during development in vitro. In G. E. W. WOLSTENHOLME AND M. O'CONNOR (Eds.), Growth of the Nervous System (Ciba Symposium), Churchill, London, 1968, pp. 13-40. 10 ECCLES, J. C., LLINAS, R., AND SASAKI, K., Intracellularly recorded responses of the cerebellar Purkinje cells, Exp. Brain Res., 1 (1966) 161-183. l l FELTZ, P., AND MACKENZIE, J. S., Properties of caudate unitary responses to repetitive nigral stimulation, Brain Research, 13 (1969) 612-616. 12 FREYGANG, W. H., JR., An analysis of extracellular potentials from single neurons in the lateral geniculate nucleus of the cat, J. gen. Physiol., 41 (1958) 543-564. 13 FRIGYESI, Z. L., AND PURPURA, D. P., Electrophysiological analysis of reciprocal caudato-nigral relations, Brain Research, 6 (1967) 440-456. 14 HOLMES, R. L., NEWMAN, P. P., AND WOLSTENCROFT, J. H., The distribution of carotid and vertebral blood in the brain of the cat, J. Physiol. (Lond.), 140 (1958) 236-246. 15 KEMP, J. M., An electron microscopic study of the termination of afferent fibres in the caudate nucleus, Brain Research, 11 (1968) 464-467. 16 KEMP, J. M., AND POWELL, T. P., The structure of the caudate nucleus of the cat : light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 17 KEMP, J. M., AND POWELL, T. P., The synaptic organization of the caudate nucleus, Phil. Trans. B, 262 (1971) 403-412. 18 KEMP, J. M., AND POWELL, T. P., The site of termination of afferent fibres in the caudate nucleus, Phil. Trans. B, 262 (1971) 413-427. 19 KEMP, J. M., AND POWELL,T. P., The termination of fibres from the cerebral cortex and thalamus upon dendritic spines in the caudate nucleus: a study with the Golgi method, Phil. Trans. B, 262 (1971). 20 KEMP, J. M., AND POWELL, T. P., The connexions of the striatum and globus pallidus: synthesis and speculation, Phil. Trans. B, 262 (1971) 441-457. 21 KRNJEVI(~, K., RANDIC, M., AND STRAUGHAN, D. W., Pharmacology of cortical inhibition, J. Physiol. (Lond.), 184 0966) 78-105. 22 LI, C. L., AND CHOU, S. N., Cortical intracellular synaptic potentials and direct cortical stimulation, J. cell. comp. Physiol., 60 (1962) 1-16. 23 MARCO, L. A., BROWN, T. S., AND ROUSE, M. E., Unitary responses in ventrolateral thalamus upon intranuclear stimulation, J. Neurophysiol., 30 (1967) 482-493. 24 MARCO, L. A., BROWN, T. S., AND ROUSE, M. E., Interneuronal barrages and ripple superposed on inhibitory postsynaptic potentials, Experientia (Basel), 23 (1967) 727. 25 MARCO, L. A., COPACK, P., AND GILMAN, S., Intrinsic links of caudate neurons, Fed. Proc., 31 (1972)403. 26 MCLENNAN, H., AND YORK, O. H., The action of dopamine on neurones of the caudate nucleus, J. Physiol. (Lond.), 189 (1967) 393-402. 27 PAPEZ, J. W., A summary of fiber connections of the basal ganglia with each other and with other portions of the brain, Res. Publ. Ass. herr. ment. Dis., 21 (1942) 21-68. 28 PHILLIPS, C. G., Some properties of pyramidal neurones of the motor cortex. In G. E. W. WOESTENHOLMEAND M. O'CONNOR (Eds.), The Nature of Sleep (Ciba Symposium}, London, Churchill, 1961, pp. 4-24. 29 ROCHA-MIRANDA, C. E., Single unit analysis of cortex-caudate connections, Electroenceph. clin. Neurophysiol., 19 (1965) 237-247.
INTRINSIC CONNECTIONS OF CAUDATE NEURONS
305
30 SCHLAG, J., In M. A. B. BRAZIER(Ed.), The Interneuron, Univ. Calif. Press, Los Angeles, 1969, pp. 519-520. 31 SCHLAG,J., AND VILLABLANCA,J., A quantitative study of temporal and spatial response patterns in a thalamic cell population electrically stimulated, Brain Research, 8 (1968) 255-270. 32 SEDGWICK, E. M., AND WILLIAMS,T. D., The response of single units in the caudate nucleus to peripheral stimulation, J. Physiol. (Lond.), 189 (1967) 281-298. 33 STOHR, e. E., GOLDRING, S., AND O'LEARY, J. L., Patterns of unit discharge associated with direct cortical response in monkey and cat, Electroenceph. clin. Neurophysiol., 15 (1963) 882-888. 34 SUGAYA, E., GOLDRING, S., AND O'LEARY, J. L., Intracellular potentials associated with direct cortical response and seizure discharge in cat, Electroenceph. clin. Neurophysiol., 17 (1964) 661669. 35 TENNYSON, V. M., AND MARCO, L. A., Intrinsic connections of caudate neurons. II. Fluorescence and electron microscopy following chronic isolation, Brain Research, 53 (1973) 307-317. 36 VERHAART,W. J. C., Fiber analysis of the basal ganglia, J. comp. Neurol., 93 (1950) 425-440.