J. Physiol. (1975), 245, pp. 655-665 With 3 text-ftgure8 Printed in Great Britain
655
RESPONSES OF FASTIGIAL NUCLEUS NEURONES TO STIMULATION OF THE CAUDATE NUCLEUS IN THE CAT
BY M. A. GRESTY* AND D. H. PAUL From the Department of Physiology, University of Manchester, Manchester M13 9PT
(Received 12 July 1974) SUMMARY
1. Extracellular records were made of single unit activity evoked in the fastigial nucleus (FN) by electrical stimulation of the caudate nucleus (CN) in cats anaesthetized with sodium thiopentone. 2. Single shock stimulation evoked bilaterally complex responses having up to three components. These were, in temporal order, with % of units exhibiting them, (a) a short burst of evoked spikes with either a latency of < 5-5 msec and no associated field potential (6 %) or a latency of 7-5-20 msec and associated with a prominent negative-going field potential (36 %); (b) a suppression of spontaneous discharges for a period of 20-150 msec
(90 %);
(c) a resumption of spike discharges with a transient increase in frequency lasting for 25-500 msec (66 %). 3. Changes in component (c) of the response patterns of some units were noted during repetitive stimulation. The nature of the change depended on the laterality of the FN with respect to the stimulated CN. 4. Mechanisms which might account for the responses are discussed, but it is emphasized that some of the results cannot yet be satisfactorily
explained. INTRODUCTION
Johnson & Snider (1953) first reported that stimulation of the CN evoked potentials in the cerebellar cortex. This projection has been further investigated by Fox & Williams (1968), Gresty (1970) and Gresty & Paul (1970), who described short latency MFRs and long latency CFRs in Purkinje cells. In addition, Gresty (1970) reported that responses were evoked bilaterally with a much higher density of CFRs. * Present address: Division of Neurobiology, Department of Physiology and Biophysics, Iowa University, Iowa City, Iowa 52240.
M. A. GRESTY AND D. H. PAUL 656 The axons of Purkinje cells represent the output pathway of the cerebellar cortex (Cajal, 1910). Those arising in the anterior lobe vermis synapse on FN neurones (Jansen & Brodal, 1940) where they exert an inhibitory action (Ito, Yoshida & Obata, 1964; Ito, Yoshida, Obata, Kawai & Udo, 1970). The FN neurones also receive excitatory inputs via collaterals of cerebellar cortical afferent fibres. Eccles, Ito & Szentagothai (1967) considered that all cerebellar afferent systems contributed collaterals to the deep nuclei but recently Eccles, Rosen, Scheid & Taborikova (1972) have reported that fast conducting MF paths do not send collaterals to the cerebellar nuclei. To complement the investigation of the caudato-cerebellar projection, this paper presents the results of a series of experiments designed to show the existence and define the characteristics of a caudato-fastigial projection. The results confirm that such a pathway exists and show that the discharge pattern produced in the FN by CN stimulation can be complex, of long duration, and often not entirely compatible with the hypothesis that the FN output is a product solely of the balance between excitatory inputs via afferent collaterals and inhibitory inputs conveyed by Purkinje axons. Some of these results have been submitted previously to the University of Manchester in part fulfilment of the requirements for the degree of Doctor of Philosophy (Gresty, 1970). METHODS
Cats with body weights 1f5-3-5 kg were anaesthetized with i.P. injections of sodium thiopentone, 40 mg/kg. Subsequent doses of 3-7 mg/kg were administered via the cannulated cephalic vein to maintain a depth of anaesthesia which permitted sluggish pupillary and withdrawal reflexes. The trachea was cannulated and body temperature maintained at 370 C with an electric blanket thermostatically controlled by a rectal probe. Skin and musculature overlying the cranium were reflected sideways and the cerebellum exposed by craniotomy. Bleeding was controlled by diathermy, haemo. static material and bone wax. Access to the anterior portions of the cerebellum was improved by deflecting the occipital cortex upwards and forwards with cotton wool pledgets. The dura mater was removed and the exposed brain surfaces protected by warm mineral oil or 5 % Agar solution, contained in pools formed of dental acrylic. The animals' heads were fixed in a stereotaxic frame (La Precision Cinematographique) rigidly clamped to a heavy steel plate. Apparatus for stereotaxically implanting stimulating and recording electrodes was clamped to the frame and to the steel plate. Brain structures were stereotaxically specified using the atlas of Snider & Niemer (1961). The head of the CN was stimulated using bipolar parallel electrodes consisting of 0-66 mm diameter steel pins set 1 mm apart and insulated with epoxy-resin to within 0.3 mm of the tip. Stimuli were square pulses of not more than 0*5 msec duration derived from a Grass model 8 stimulator and coupled to the preparation through a stimulus isolation unit. Current flow, measured across a 100 Q series resistor was always < 0 5 mA. These upper operational limits compare favourably
CA UDATO-FASTIGIAL PROJECTION
657
with the criteria of other authors (e.g. Fox & Williams, 1968). More intense stimuli delivered within the boundary of the caudate nucleus tended to induce movements. This was taken to indicate spread of current to the internal capsule (Laursen, 1963). The juxta fastigial (JF) region of the cerebellum was stimulated with a bipolar concentric electrode consisting of an outer steel tube (o.d. 0-457 mm) and an inner core of 0-254 mm diameter varnished tungsten wire. The wire was cemented into place with Araldite (CIBA) and the tip of the electrode ground smooth. The outer tube was insulated to the tip using a silicon based varnish. Stimulus parameters delivered through the JF electrode did not exceed 0 05 mA and 0*2 msec. The extracellular recording electrodes were 4 M-NaCl filed glass micropipettes with 1 cm of tapered shank and internal tip diameters of about 1 ,tm. The recording electrodes were coated with a fine grained ink before introduction and were controlled by means of a magnetically damped micrometer screw manipulator. Signals were coupled through a negative capacitance feed-back, unity gain amplifier (ELSAI, Electronics for Life Sciences Inc.). After further amplification (Tektronix 122, band width 0-2-10 K Hz) the signals were displayed on a CRO and recorded on film and on magnetic tape. The criteria employed to judge that the recording electrode was accurately placed in the FN were stereotaxic location, and the geometrical relationships between the nucleus and the surrounding areas of cerebellar white matter and cortex, all of which have very distinct electrophysiological characteristics. Histological investigation confirmed the validity of these criteria. At termination the entire cerebellum was removed from the skull and fixed in a solution of 10% (w/v) formalin and 1 % (w/v) calcium chloride for 2 days. It was then transferred to a solution of 30 % (w/v) sucrose and 1 % (w/v) gum acacia. The brain was freed from meninges and 50-100 ,tm sections were cut on a freezing microtome and stained with cresyl violet. It was found that the stimulating electrodes left clear marks in the brain tissue. Ink marks were clearly left by recording electrodes in about 80 % of the penetrations. RESULTS
Response patterns evoked by CN stimulation Responses were recorded from fifty-three cells in both the ipsilateral (IL) and contralateral (CL) FN relative to the ON being stimulated and were similar throughout both nuclei. This is in agreement with the observation that the CN projection to the cerebellar cortex is widespread with no evidence of zoning (Gresty & Paul, 1969; Gresty, 1970), except that CFRs in particular are about three times as dense on the CL compared to the IL side (Gresty, 1970); there was no evidence for any such disparity in the projection to the FN. The nuclear responses were usually complex, involving bursts of evoked spikes and periods of quiescence, and with considerable variation in the responses given by different units. An analysis of all responses revealed that there were three main components, some or all of which made up the individual patterns. These were, in temporal order (with % of units exhibiting each one): 27
P H Y'
245
658 M. A. GRESTY AND D. H. PAUL (a) a short burst of evoked spikes with a latency of 3-20 msec (42 %); (b) a suppression of spontaneous discharges for a period of 20-150 msec
(90 %); (c) resumption of spike discharge, often with a transient increase in frequency (compared to control levels) for several hundred msec (66 %). The initial evoked spike response. The frequency histogram for the latency of the initial spike in each burst is shown in Fig. 1 A and the mean number of spikes/burst plotted against mean latency of this first spike is shown in Fig. 1 B. Most evoked spike bursts had a latency of 7-5-10*5 msec, but 3 units had latencies between 3 and 5-5 msec (inset to Fig. 1 A). These latter units could be distinguished from all others because (a) the latency variation of the burst was always < 1 msec, compared to 1 5-3 msec for the rest; (b) they gave at least three spikes per burst, whereas the others rarely gave more than three spikes; (c) the unit wave form was characteristic of fibre spikes, being monophasic positive with a rapid rising phase; (d) they were not associated with any field potential. The other twenty-one units had diphasic wave forms probably indicating unit soma-dendritic spikes and were always seen in association with a prominent negative-going field potential (e.g. Figs. 2C, D). The suppression of spontaneous activity. This component of the response was the most frequently encountered feature, being seen in forty-eight units, although in some it did not occur after every stimulus. The suppression persisted for a minimum of 20 msec up to a maximum, in one unit, of 170 msec. The mode of the distribution was between 30 and 35 msec but 25 % of the values exceeded 50 msec. Examples of this response can be seen in Fig. 2C, D. In Fig. 2C resumption of discharge occurred between 55 and 60 msec following CN stimulation, spike suppression lasting for about 40 msec. This duration is very similar to that necessary for recovery of cortical climbing fibre responses (CFRs) following a CN stimulus (Gresty & Paul, 1969; Gresty, 1970). A suppression of spontaneous activity could be produced by a single stimulus applied to the JF region. Careful adjustment of the position of the electrode and of the stimulus current suppressed spike discharges for approximately 25 msec. Occasionally, the spontaneous discharge rate was increased above normal following the suppression. All units studied gave a similar response and the duration of the suppression showed little variation. Increases in discharge rates following spike suppression. Following the suppression of spontaneous discharge, twenty-seven units exhibited either a train of high frequency spikes if the spontaneous activity was very low (Fig. 2C) or an increase in the discharge rate which then slowly fell to control levels (Fig. 2D). Discharge rates during this phase had a mean of 114 Hz (range 53-350 Hz, mode 90 Hz); rates in excess of 150 Hz were
CA UDATO-FASTIGIAL PROJECTION
659
10 r A
6
z
5
LW5 5.0r
10 Time (msec)
15
20
8
D ._
.b CL
2-5 0
C
GD
0 I* I 0
5
10 Latency (msec)
0 I
15
20
Fig. 1. Excitatory actions on FN neurones by CN stimulation. A, latency histogram for first evoked spike; inset shows records of a short latency multiple discharge. Calibrations: horizontal, 2 msec per division; vertical, 0-5 mV/division, upwards negative. CN stimulated at arrow. B, relationship between mean number of spikes in evoked burst and latency to first spike.
In comparison, control levels of spontaneous discharges averaged 49 Hz (range 5-200 Hz, mode 50 Hz); rates above 80 Hz were rare. The facilitated discharge rate was related directly to the control rate. In
rare.
twelve units this association was tested by calculating the product-moment correlation coefficient (r), which was 0 7, indicating a significant direct relationship at the P < 0.01 level. Duration of the facilitated discharge had a mean of 116 msec (range 27-2
660 M. A. GRESTY AND D. H. PAUL 25-500 msec; bimodal, at 60 and 125 msec). In nine units it lasted for more than 150 msec, but estimates of long duration facilitated discharges were often unreliable in spontaneously discharging units since there was no easily identified end-point (e.g. Fig. 2D). Cumulative frequencies are plotted for the times of onset and end of the facilitated discharge in twenty-four units (Fig. 2A, B). In spite of the large variation in duration of the response, times of onset and cessation showed very little overlap. 30
-
A
20 10
30
B D
20
-
0
100.
400 200 300 Time (msec)
500
+
Fig. 2. Facilitation of FN neurones by CN stimulation. A, cumulative frequency plot of times of onset of facilitation. B, cumulative frequency plot of times of cessation of facilitation. C, facilitation of unit having very low spontaneous activity. CN stimulus at arrow. Vertical calibration, 2 mV/division, negative up; horizontal calibration, 50 msec/division. D. facilitation of unit with early evoked discharge and subsequent block of activity for 40 msec. Control discharge rate was about 80 impulses/sec; maximum facilitated rate about 200/sec. Vertical calibration, 1 mV/division, negative up; horizontal calibration, 20 msec/division.
Multiple stimulation experiments Evoked spikes. The dissimilarity between the shortest latency evoked units and the rest was further emphasized in interaction experiments using a pair of stimuli. The responses with latency < 5-5 msec showed no interaction, a unit discharge being evoked by both the conditioning (CS) and testing (TS) stimuli at all intervals tested, down to 3-4 msec. In contrast, recovery of the TS response for the units with latency > 7-5 msec took up to 50 msec. This latter response resembles the 'immediate block'
661 C7A UDATO -FASTIGIAL PROJECTION described by Gresty & Paul (1970) in relation to interaction in cerebellar cortex of CN-evoked CFRs. Post-stimulus suppression. A TS applied during a period of spike suppression evoked by the CS neither prolonged it nor evoked a further suppression, but when the TS was applied after a period of spike suppression evoked by the CS had ended, then a second suppression was evoked. There was no intermediate stage between these two limiting states. Repetitive stimulation. This revealed a difference in behaviour between IL and CL-FN neurones. When CN stimuli were applied at about 7 Hz (i.e.
A4 --l l~+ l+ l I Hl 44
4_ +
1
+
4Tll1
Fig. 3. Modulation of responses of FN
neurones with repetitive CN stimulation. A, response in IL.FN. Stimulation (at arrows) at 7 Hz. Note no facilitated response after third stimulus. Early field potential unaffected. Calibrations: vertical, 2 mVlcivision; horizontal, 50 msecf division. B, response in CL-FN. Stimulation (at arrows) at 8 Hz. Note facilitated response appears only after second and subsequent stimuli. Early field potential constant throughout. Calibrations: vertical, 2 mV/division; horizontal, 50 msec/division.
662 M. A. GRESTY AND D. H. PAUL allowing time for each response to run its full course), units in IL-FN which responded with a facilitated discharge to the first stimulus ceased to do so after the third or fourth, whereas in the CL-FN, units were located which did not exhibit a facilitated discharge to the first stimulus but did so to the second or third and subsequent stimuli (Fig. 3). This result was obtained consistently in those units tested in five different preparations. DISCUSSION
Following electrical stimulation of the head of the caudate nucleus, FN cells responded in three phases. The earliest response consisted of evoked unit discharges most probably due to their excitation by collaterals of cerebellar afferent fibres. Next was a period during which unit discharge was suppressed and finally the unit discharge was sometimes facilitated for considerable periods. A similar sequence has been described by Armstrong, Cogdell & Harvey (1973) for interpositus neurones excited by peripheral afferent stimulation in chloralose-anaesthetized cats. The latter two phases were of particular significance because (a) they revealed alterations in the behaviour of the FN units for long periods after a single stimulus to the CN; (b) the late facilitation sometimes changed with successive stimuli, the effect being related to the laterality of the recording electrode relative to the stimulating electrode; (c) their origins cannot easily be explained in terms of the known mechanisms of afferent collateral excitation and Purkinje cell inhibition (see below). The experimental evidence suggests that the single unit discharges evoked in the FN by single-shock stimulation of the CN belonged to two quite distinct groups; those having a latency of < 5x5 msec and those having latencies of 7-5-20 msec. The short latency units were rarely detected and their properties indicated that they were fibre rather than somadendritic spikes. The shortness of their latency suggests activity in a MF pathway at a site either pre- or post-synaptic to the FN cells. The failure of these units to exhibit interaction in conditioning experiments is consistent with this interpretation (Gresty & Paul, 1969). The second group of units had a latency range virtually identical to that observed for CN-evoked cortical CFRs (Gresty & Paul, 1969; Gresty, 1970) and behaved in a similar fashion to CFRs in conditioning experiments (Newman & Paul, 1969; Gresty & Paul, 1970; Latham & Paul, 1971). We propose therefore that this group of units represents activation of FN cells by CF collaterals. The fact that the CF responses far exceeded the MF responses may reflect the anatomical observations of Matsushita & Ikeda (1970), that the olivo-cerebellar input to the roof nuclei appeared to be more significant, in terms of synaptic density, than the afferent collateral
CA UDATO-FASTIGIAL PROJECTION 663 input from other cerebellopetal paths. Cutaneous afferent stimulation has also been observed to evoke responses in the FN more easily identified with CF inputs (Latham, Paul & Potts, 1970). However, recent experiments by Eccles et al. (1972) have shown that slowly conducting spinocerebellar paths terminating as MFs excite FN neurones so the possibility exists that slowly conducting MF paths from the CN could contribute to the excitation of FN neurones. There is no direct evidence for such a pathway although terminal degeneration of fibres from the medial segment of the globus pallidus has been described in association with neurones of the nucleus reticularis tegmenti pontis (Nauta & Mehler, 1966). Thus a small fibre, slowly conducting MF path may exist. Activity evoked by such a pathway would occur simultaneously with the massive CF input and consequently would be difficult to detect. The short latency MF responses evoked by caudate stimulation are almost certainly experimental artifacts, there being no evidence for large diameter efferents from the caudate (Gresty, 1970). Possible sources could be excitation of axon collaterals of a pathway which projects to both caudate and the FN, or stimulus spread to regions adjacent to the caudate. The latter seems less likely considering the care taken to limit the stimulus intensity. Suppression of unit discharge was the most consistent FN response evoked by stimulating the caudate nucleus. On the basis of known mechanisms, it would be predicted to result from an increased inhibitory drive from Purkinje cells or a decreased excitatory drive from afferent collaterals. Stimulation of the caudate nucleus has been shown to produce widespread activation of Purkinje cells through CF pathways, the response latencies ranging from 12 to 30 msec (Gresty, 1970). A CFR lasts for a maximum of about 15 msec and in Deiter's neurones the i.p.s.p. evoked by an impulse in a Purkinje cell axon has a duration of 5-6 msec (Ito et al. 1964). Thus a CFR mechanism might, in extreme cases, account for a maximum of about 50 msec of spike suppression; but in an extensive study of CN-evoked Purkinje cell responses (Gresty, 1970), none gave an evoked discharge long enough to account for a spike suppression in the FN exceeding 50 msec. Neither an unexpectedly long inhibitory action of Purkinje cells nor an intranuclear mechanism seems a likely explanation for these observations because JF stimulation, which might excite both Purkinje cell axons and FN neurones directly, failed to suppress spike discharge for more than 25 msec. An alternative hypothesis is that spike suppression is due in part to disfacilitation of FN cells, if the assumption is made that their main excitatory drive derives from CF collaterals. This agrees with the observation
M. A. GRESTY AND D. H. PAUL 664 that afferent collateral actions on FN neurones were principally identified with CF inputs, but implies extensive convergence of CF collaterals to maintain the spontaneous excitation of FN cells, because of the low spontaneous discharge rate of individual CFs (about 1/sec, Oscarsson, 1967; Bell & Grimm, 1969; Latham & Paul, 1971). Given such a convergence and bearing in mind that CFs themselves cannot normally be driven at rates much above 10 Hz (Eccles, Provini, Strata & Taborikov&, 1968; Gresty & Paul, 1970; Latham & Paul, 1971), the nearly simultaneous excitation of large numbers of CFs by the CN stimulus could result in disfacilitation of FN cells, thereby prolonging the suppression of spike discharge. The main objection to this hypothesis is that the 'gating' on the CF path is probably at the inferior olivary neurone level (Latham & Paul, 1971) and the 'gate' is activated by the olivary neurones discharging. Thus any FN cells which were disfacilitated should also have exhibited a preceding CF excitation (via a collateral), but this was not confirmed during the experiments. Difficulties are also encountered when trying to explain the facilitation which sometimes followed spike suppression. The obvious mechanism would be a cessation to Purkinje activity producing an enhanced (disinhibitory) discharge of the FN cell. However, in many cases the facilitation did not begin until 50-100 msec after the stimulus and sometimes persisted for more than 150 msec. The necessary reciprocal behaviour of Purkinje cells has not been observed in experiments on the caudatocerebellar path, although such a relationship between FN and anterior lobe cortex has been demonstrated following cutaneous stimulation (Latham et al. 1970), and Armstrong et al. (1973) found evidence for a similar reciprocal relationship between cerebellar Purkinje cells and interpositus neurones in chloralosed cats subjected to peripheral afferent stimulation. We have to conclude that although known cerebellar cortical and nuclear mechanisms are adequate in principle to explain the effects of CN stimulation on responses of FN cells, the patterns of activity in cortex and nucleus do not show the same apparent reciprocity when the CN is the source of afferent input as they do when peripheral afferents provide the input. Furthermore, with repetitive CN stimulation the late facilitatory effect was progressively enhanced in CL units and suppressed in IL units. Whilst this latter result might arise from some reciprocal inhibitory mechanism operating either on the projection paths or on the FN cells themselves, we know of no evidence to support such a suggestion. We are indebted for expert technical assistance to Mr P. Cheetham and Mr E. Balog. During the course of this work, M.A.G. held an M.R.C. studentship for training in research.
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REFERENCES
ARMSTRONG, D. M., COGDELL, B. & HARVEY, R. J. (1973). Responses of interpositus neurons to nerve stimulation in chloralose anaesthetized cats. Brain Res. 54, 461-466. BELL, C. C. & GRIMM, R. J. (1969). Discharge properties of Purkinje cells recorded on single and double microelectrodes. J. Neurophysiol. 32, 1044-1055. CAJAL, R. Y S. (1910). Histologie du systerne nerveux de l'homme et des animaux, vol. II. Paris: A. Maloine. ECCLES, J. C., ITO, M. & SZENTAGOTHAI, J. (1967). The Cerebellum as a Neuronal Machine. New York, London, Heidelberg: Springer. ECCLES, J. C., PROVINI, L., STRATA, P. & TkBOMKOVX, H. (1968). Analysis of electrical potentials evoked in the cerebellar anterior lobe by stimulation of hindlimb and forelimb nerves. Expl Brain Res. 6, 171-194. ECCLES, J. C., ROSEN, I., SCHEID, P. & TABOAIfKOvk, H. (1972). Cutaneous afferent responses in interpositus neurons of the cat. Brain Res. 42, 207-211. Fox, M. & WILLIAMS, T. D. (1968). Responses evoked in the cerebellar cortex by stimulation of the caudate nucleus. J. Physiol. 198, 435-449. GRESTY, M. A. (1970). The electrophysiological projection of the caudate nucleus on to the anterior lobe and fastigial nucleus of the cat. Doctoral Thesis, University of Manchester. GRESTY, M. A. & PAUL, D. H. (1969). Projection of the caudate nucleus on the anterior lobe of the cerebellum of the cat. J. Physiol. 204, 81-82P. GRESTY, M. A. & PAUL, D. H. (1970). Input-output relations of the inferior olive. J. Physiol. 208, 92-93 P. ITO, M., YOSHIDA, M. & OBATA, K. (1964). Monosynaptic inhibition of the intracerebellar nuclei induced from the cerebellar cortex. Experientia 20, 575-576. ITO, M., YOSHIDA, M., OBATA, K., KAWAI, N. & UDO, M. (1970). Inhibitory control of intracerebellar nuclei by the Purkinje cell axons. Expl Brain Res. 10, 64-80. JANSEN, J. & BRODAL, A. (1940). Experimental studies on the intrinsic fibres of the cerebellum. II. The cortico-nuclear projection. J. comp. Neurol. 73, 267-321. JOHNSON, B. & SNIDER, R. S. (1953). Projections of the basal ganglia to cerebrum and cerebellum. Anat. Rec. 115, 327. LATHAM, ANN & PAUL, D. H. (1971). Spontaneous activity of cerebellar Purkinje cells and their responses to impulses in climbing fibres. J. Physiol. 213, 135-156. LATHAM, ANN, PAUL, D. H. & POTTS, A. J. (1970). Responses of fastigial nucleus neurones to stimulation of a peripheral nerve. J. Physiol. 206, 15-17P. LAURSEN, A. M. (1963). Corpus striatum. Acta physiol. scand. 59, suppl. 211. MATSUSHITA, M. & IKEDA, M. (1970). Olivary projections to the cerebellar nuclei in the cat. Expl Brain Res. 10, 488-500. NAUTA, W. J. H. & MEHLER, W. R. (1966). Projections of the lentiform nucleus in the monkey. Brain Res. 1, 3-42. NEWMAN, P. P. & PAUL, D. H. (1969). The projection of splanchnic afferents on the cerebellum of the cat. J. Physiol. 202, 223-237. OSCARSSON, 0. (1967). Termination and functional organization of a dorsal spinoolivocerebellar path. Brain Res. 5, 531-534. SNIDER, R. S. & NIEMER, W. T. (1961). A Stereotaxic Atlas of the Cat Brain. Chicago: University of Chicago Press.
655
RESPONSES OF FASTIGIAL NUCLEUS NEURONES TO STIMULATION OF THE CAUDATE NUCLEUS IN THE CAT
BY M. A. GRESTY* AND D. H. PAUL From the Department of Physiology, University of Manchester, Manchester M13 9PT
(Received 12 July 1974) SUMMARY
1. Extracellular records were made of single unit activity evoked in the fastigial nucleus (FN) by electrical stimulation of the caudate nucleus (CN) in cats anaesthetized with sodium thiopentone. 2. Single shock stimulation evoked bilaterally complex responses having up to three components. These were, in temporal order, with % of units exhibiting them, (a) a short burst of evoked spikes with either a latency of < 5-5 msec and no associated field potential (6 %) or a latency of 7-5-20 msec and associated with a prominent negative-going field potential (36 %); (b) a suppression of spontaneous discharges for a period of 20-150 msec
(90 %);
(c) a resumption of spike discharges with a transient increase in frequency lasting for 25-500 msec (66 %). 3. Changes in component (c) of the response patterns of some units were noted during repetitive stimulation. The nature of the change depended on the laterality of the FN with respect to the stimulated CN. 4. Mechanisms which might account for the responses are discussed, but it is emphasized that some of the results cannot yet be satisfactorily
explained. INTRODUCTION
Johnson & Snider (1953) first reported that stimulation of the CN evoked potentials in the cerebellar cortex. This projection has been further investigated by Fox & Williams (1968), Gresty (1970) and Gresty & Paul (1970), who described short latency MFRs and long latency CFRs in Purkinje cells. In addition, Gresty (1970) reported that responses were evoked bilaterally with a much higher density of CFRs. * Present address: Division of Neurobiology, Department of Physiology and Biophysics, Iowa University, Iowa City, Iowa 52240.
M. A. GRESTY AND D. H. PAUL 656 The axons of Purkinje cells represent the output pathway of the cerebellar cortex (Cajal, 1910). Those arising in the anterior lobe vermis synapse on FN neurones (Jansen & Brodal, 1940) where they exert an inhibitory action (Ito, Yoshida & Obata, 1964; Ito, Yoshida, Obata, Kawai & Udo, 1970). The FN neurones also receive excitatory inputs via collaterals of cerebellar cortical afferent fibres. Eccles, Ito & Szentagothai (1967) considered that all cerebellar afferent systems contributed collaterals to the deep nuclei but recently Eccles, Rosen, Scheid & Taborikova (1972) have reported that fast conducting MF paths do not send collaterals to the cerebellar nuclei. To complement the investigation of the caudato-cerebellar projection, this paper presents the results of a series of experiments designed to show the existence and define the characteristics of a caudato-fastigial projection. The results confirm that such a pathway exists and show that the discharge pattern produced in the FN by CN stimulation can be complex, of long duration, and often not entirely compatible with the hypothesis that the FN output is a product solely of the balance between excitatory inputs via afferent collaterals and inhibitory inputs conveyed by Purkinje axons. Some of these results have been submitted previously to the University of Manchester in part fulfilment of the requirements for the degree of Doctor of Philosophy (Gresty, 1970). METHODS
Cats with body weights 1f5-3-5 kg were anaesthetized with i.P. injections of sodium thiopentone, 40 mg/kg. Subsequent doses of 3-7 mg/kg were administered via the cannulated cephalic vein to maintain a depth of anaesthesia which permitted sluggish pupillary and withdrawal reflexes. The trachea was cannulated and body temperature maintained at 370 C with an electric blanket thermostatically controlled by a rectal probe. Skin and musculature overlying the cranium were reflected sideways and the cerebellum exposed by craniotomy. Bleeding was controlled by diathermy, haemo. static material and bone wax. Access to the anterior portions of the cerebellum was improved by deflecting the occipital cortex upwards and forwards with cotton wool pledgets. The dura mater was removed and the exposed brain surfaces protected by warm mineral oil or 5 % Agar solution, contained in pools formed of dental acrylic. The animals' heads were fixed in a stereotaxic frame (La Precision Cinematographique) rigidly clamped to a heavy steel plate. Apparatus for stereotaxically implanting stimulating and recording electrodes was clamped to the frame and to the steel plate. Brain structures were stereotaxically specified using the atlas of Snider & Niemer (1961). The head of the CN was stimulated using bipolar parallel electrodes consisting of 0-66 mm diameter steel pins set 1 mm apart and insulated with epoxy-resin to within 0.3 mm of the tip. Stimuli were square pulses of not more than 0*5 msec duration derived from a Grass model 8 stimulator and coupled to the preparation through a stimulus isolation unit. Current flow, measured across a 100 Q series resistor was always < 0 5 mA. These upper operational limits compare favourably
CA UDATO-FASTIGIAL PROJECTION
657
with the criteria of other authors (e.g. Fox & Williams, 1968). More intense stimuli delivered within the boundary of the caudate nucleus tended to induce movements. This was taken to indicate spread of current to the internal capsule (Laursen, 1963). The juxta fastigial (JF) region of the cerebellum was stimulated with a bipolar concentric electrode consisting of an outer steel tube (o.d. 0-457 mm) and an inner core of 0-254 mm diameter varnished tungsten wire. The wire was cemented into place with Araldite (CIBA) and the tip of the electrode ground smooth. The outer tube was insulated to the tip using a silicon based varnish. Stimulus parameters delivered through the JF electrode did not exceed 0 05 mA and 0*2 msec. The extracellular recording electrodes were 4 M-NaCl filed glass micropipettes with 1 cm of tapered shank and internal tip diameters of about 1 ,tm. The recording electrodes were coated with a fine grained ink before introduction and were controlled by means of a magnetically damped micrometer screw manipulator. Signals were coupled through a negative capacitance feed-back, unity gain amplifier (ELSAI, Electronics for Life Sciences Inc.). After further amplification (Tektronix 122, band width 0-2-10 K Hz) the signals were displayed on a CRO and recorded on film and on magnetic tape. The criteria employed to judge that the recording electrode was accurately placed in the FN were stereotaxic location, and the geometrical relationships between the nucleus and the surrounding areas of cerebellar white matter and cortex, all of which have very distinct electrophysiological characteristics. Histological investigation confirmed the validity of these criteria. At termination the entire cerebellum was removed from the skull and fixed in a solution of 10% (w/v) formalin and 1 % (w/v) calcium chloride for 2 days. It was then transferred to a solution of 30 % (w/v) sucrose and 1 % (w/v) gum acacia. The brain was freed from meninges and 50-100 ,tm sections were cut on a freezing microtome and stained with cresyl violet. It was found that the stimulating electrodes left clear marks in the brain tissue. Ink marks were clearly left by recording electrodes in about 80 % of the penetrations. RESULTS
Response patterns evoked by CN stimulation Responses were recorded from fifty-three cells in both the ipsilateral (IL) and contralateral (CL) FN relative to the ON being stimulated and were similar throughout both nuclei. This is in agreement with the observation that the CN projection to the cerebellar cortex is widespread with no evidence of zoning (Gresty & Paul, 1969; Gresty, 1970), except that CFRs in particular are about three times as dense on the CL compared to the IL side (Gresty, 1970); there was no evidence for any such disparity in the projection to the FN. The nuclear responses were usually complex, involving bursts of evoked spikes and periods of quiescence, and with considerable variation in the responses given by different units. An analysis of all responses revealed that there were three main components, some or all of which made up the individual patterns. These were, in temporal order (with % of units exhibiting each one): 27
P H Y'
245
658 M. A. GRESTY AND D. H. PAUL (a) a short burst of evoked spikes with a latency of 3-20 msec (42 %); (b) a suppression of spontaneous discharges for a period of 20-150 msec
(90 %); (c) resumption of spike discharge, often with a transient increase in frequency (compared to control levels) for several hundred msec (66 %). The initial evoked spike response. The frequency histogram for the latency of the initial spike in each burst is shown in Fig. 1 A and the mean number of spikes/burst plotted against mean latency of this first spike is shown in Fig. 1 B. Most evoked spike bursts had a latency of 7-5-10*5 msec, but 3 units had latencies between 3 and 5-5 msec (inset to Fig. 1 A). These latter units could be distinguished from all others because (a) the latency variation of the burst was always < 1 msec, compared to 1 5-3 msec for the rest; (b) they gave at least three spikes per burst, whereas the others rarely gave more than three spikes; (c) the unit wave form was characteristic of fibre spikes, being monophasic positive with a rapid rising phase; (d) they were not associated with any field potential. The other twenty-one units had diphasic wave forms probably indicating unit soma-dendritic spikes and were always seen in association with a prominent negative-going field potential (e.g. Figs. 2C, D). The suppression of spontaneous activity. This component of the response was the most frequently encountered feature, being seen in forty-eight units, although in some it did not occur after every stimulus. The suppression persisted for a minimum of 20 msec up to a maximum, in one unit, of 170 msec. The mode of the distribution was between 30 and 35 msec but 25 % of the values exceeded 50 msec. Examples of this response can be seen in Fig. 2C, D. In Fig. 2C resumption of discharge occurred between 55 and 60 msec following CN stimulation, spike suppression lasting for about 40 msec. This duration is very similar to that necessary for recovery of cortical climbing fibre responses (CFRs) following a CN stimulus (Gresty & Paul, 1969; Gresty, 1970). A suppression of spontaneous activity could be produced by a single stimulus applied to the JF region. Careful adjustment of the position of the electrode and of the stimulus current suppressed spike discharges for approximately 25 msec. Occasionally, the spontaneous discharge rate was increased above normal following the suppression. All units studied gave a similar response and the duration of the suppression showed little variation. Increases in discharge rates following spike suppression. Following the suppression of spontaneous discharge, twenty-seven units exhibited either a train of high frequency spikes if the spontaneous activity was very low (Fig. 2C) or an increase in the discharge rate which then slowly fell to control levels (Fig. 2D). Discharge rates during this phase had a mean of 114 Hz (range 53-350 Hz, mode 90 Hz); rates in excess of 150 Hz were
CA UDATO-FASTIGIAL PROJECTION
659
10 r A
6
z
5
LW5 5.0r
10 Time (msec)
15
20
8
D ._
.b CL
2-5 0
C
GD
0 I* I 0
5
10 Latency (msec)
0 I
15
20
Fig. 1. Excitatory actions on FN neurones by CN stimulation. A, latency histogram for first evoked spike; inset shows records of a short latency multiple discharge. Calibrations: horizontal, 2 msec per division; vertical, 0-5 mV/division, upwards negative. CN stimulated at arrow. B, relationship between mean number of spikes in evoked burst and latency to first spike.
In comparison, control levels of spontaneous discharges averaged 49 Hz (range 5-200 Hz, mode 50 Hz); rates above 80 Hz were rare. The facilitated discharge rate was related directly to the control rate. In
rare.
twelve units this association was tested by calculating the product-moment correlation coefficient (r), which was 0 7, indicating a significant direct relationship at the P < 0.01 level. Duration of the facilitated discharge had a mean of 116 msec (range 27-2
660 M. A. GRESTY AND D. H. PAUL 25-500 msec; bimodal, at 60 and 125 msec). In nine units it lasted for more than 150 msec, but estimates of long duration facilitated discharges were often unreliable in spontaneously discharging units since there was no easily identified end-point (e.g. Fig. 2D). Cumulative frequencies are plotted for the times of onset and end of the facilitated discharge in twenty-four units (Fig. 2A, B). In spite of the large variation in duration of the response, times of onset and cessation showed very little overlap. 30
-
A
20 10
30
B D
20
-
0
100.
400 200 300 Time (msec)
500
+
Fig. 2. Facilitation of FN neurones by CN stimulation. A, cumulative frequency plot of times of onset of facilitation. B, cumulative frequency plot of times of cessation of facilitation. C, facilitation of unit having very low spontaneous activity. CN stimulus at arrow. Vertical calibration, 2 mV/division, negative up; horizontal calibration, 50 msec/division. D. facilitation of unit with early evoked discharge and subsequent block of activity for 40 msec. Control discharge rate was about 80 impulses/sec; maximum facilitated rate about 200/sec. Vertical calibration, 1 mV/division, negative up; horizontal calibration, 20 msec/division.
Multiple stimulation experiments Evoked spikes. The dissimilarity between the shortest latency evoked units and the rest was further emphasized in interaction experiments using a pair of stimuli. The responses with latency < 5-5 msec showed no interaction, a unit discharge being evoked by both the conditioning (CS) and testing (TS) stimuli at all intervals tested, down to 3-4 msec. In contrast, recovery of the TS response for the units with latency > 7-5 msec took up to 50 msec. This latter response resembles the 'immediate block'
661 C7A UDATO -FASTIGIAL PROJECTION described by Gresty & Paul (1970) in relation to interaction in cerebellar cortex of CN-evoked CFRs. Post-stimulus suppression. A TS applied during a period of spike suppression evoked by the CS neither prolonged it nor evoked a further suppression, but when the TS was applied after a period of spike suppression evoked by the CS had ended, then a second suppression was evoked. There was no intermediate stage between these two limiting states. Repetitive stimulation. This revealed a difference in behaviour between IL and CL-FN neurones. When CN stimuli were applied at about 7 Hz (i.e.
A4 --l l~+ l+ l I Hl 44
4_ +
1
+
4Tll1
Fig. 3. Modulation of responses of FN
neurones with repetitive CN stimulation. A, response in IL.FN. Stimulation (at arrows) at 7 Hz. Note no facilitated response after third stimulus. Early field potential unaffected. Calibrations: vertical, 2 mVlcivision; horizontal, 50 msecf division. B, response in CL-FN. Stimulation (at arrows) at 8 Hz. Note facilitated response appears only after second and subsequent stimuli. Early field potential constant throughout. Calibrations: vertical, 2 mV/division; horizontal, 50 msec/division.
662 M. A. GRESTY AND D. H. PAUL allowing time for each response to run its full course), units in IL-FN which responded with a facilitated discharge to the first stimulus ceased to do so after the third or fourth, whereas in the CL-FN, units were located which did not exhibit a facilitated discharge to the first stimulus but did so to the second or third and subsequent stimuli (Fig. 3). This result was obtained consistently in those units tested in five different preparations. DISCUSSION
Following electrical stimulation of the head of the caudate nucleus, FN cells responded in three phases. The earliest response consisted of evoked unit discharges most probably due to their excitation by collaterals of cerebellar afferent fibres. Next was a period during which unit discharge was suppressed and finally the unit discharge was sometimes facilitated for considerable periods. A similar sequence has been described by Armstrong, Cogdell & Harvey (1973) for interpositus neurones excited by peripheral afferent stimulation in chloralose-anaesthetized cats. The latter two phases were of particular significance because (a) they revealed alterations in the behaviour of the FN units for long periods after a single stimulus to the CN; (b) the late facilitation sometimes changed with successive stimuli, the effect being related to the laterality of the recording electrode relative to the stimulating electrode; (c) their origins cannot easily be explained in terms of the known mechanisms of afferent collateral excitation and Purkinje cell inhibition (see below). The experimental evidence suggests that the single unit discharges evoked in the FN by single-shock stimulation of the CN belonged to two quite distinct groups; those having a latency of < 5x5 msec and those having latencies of 7-5-20 msec. The short latency units were rarely detected and their properties indicated that they were fibre rather than somadendritic spikes. The shortness of their latency suggests activity in a MF pathway at a site either pre- or post-synaptic to the FN cells. The failure of these units to exhibit interaction in conditioning experiments is consistent with this interpretation (Gresty & Paul, 1969). The second group of units had a latency range virtually identical to that observed for CN-evoked cortical CFRs (Gresty & Paul, 1969; Gresty, 1970) and behaved in a similar fashion to CFRs in conditioning experiments (Newman & Paul, 1969; Gresty & Paul, 1970; Latham & Paul, 1971). We propose therefore that this group of units represents activation of FN cells by CF collaterals. The fact that the CF responses far exceeded the MF responses may reflect the anatomical observations of Matsushita & Ikeda (1970), that the olivo-cerebellar input to the roof nuclei appeared to be more significant, in terms of synaptic density, than the afferent collateral
CA UDATO-FASTIGIAL PROJECTION 663 input from other cerebellopetal paths. Cutaneous afferent stimulation has also been observed to evoke responses in the FN more easily identified with CF inputs (Latham, Paul & Potts, 1970). However, recent experiments by Eccles et al. (1972) have shown that slowly conducting spinocerebellar paths terminating as MFs excite FN neurones so the possibility exists that slowly conducting MF paths from the CN could contribute to the excitation of FN neurones. There is no direct evidence for such a pathway although terminal degeneration of fibres from the medial segment of the globus pallidus has been described in association with neurones of the nucleus reticularis tegmenti pontis (Nauta & Mehler, 1966). Thus a small fibre, slowly conducting MF path may exist. Activity evoked by such a pathway would occur simultaneously with the massive CF input and consequently would be difficult to detect. The short latency MF responses evoked by caudate stimulation are almost certainly experimental artifacts, there being no evidence for large diameter efferents from the caudate (Gresty, 1970). Possible sources could be excitation of axon collaterals of a pathway which projects to both caudate and the FN, or stimulus spread to regions adjacent to the caudate. The latter seems less likely considering the care taken to limit the stimulus intensity. Suppression of unit discharge was the most consistent FN response evoked by stimulating the caudate nucleus. On the basis of known mechanisms, it would be predicted to result from an increased inhibitory drive from Purkinje cells or a decreased excitatory drive from afferent collaterals. Stimulation of the caudate nucleus has been shown to produce widespread activation of Purkinje cells through CF pathways, the response latencies ranging from 12 to 30 msec (Gresty, 1970). A CFR lasts for a maximum of about 15 msec and in Deiter's neurones the i.p.s.p. evoked by an impulse in a Purkinje cell axon has a duration of 5-6 msec (Ito et al. 1964). Thus a CFR mechanism might, in extreme cases, account for a maximum of about 50 msec of spike suppression; but in an extensive study of CN-evoked Purkinje cell responses (Gresty, 1970), none gave an evoked discharge long enough to account for a spike suppression in the FN exceeding 50 msec. Neither an unexpectedly long inhibitory action of Purkinje cells nor an intranuclear mechanism seems a likely explanation for these observations because JF stimulation, which might excite both Purkinje cell axons and FN neurones directly, failed to suppress spike discharge for more than 25 msec. An alternative hypothesis is that spike suppression is due in part to disfacilitation of FN cells, if the assumption is made that their main excitatory drive derives from CF collaterals. This agrees with the observation
M. A. GRESTY AND D. H. PAUL 664 that afferent collateral actions on FN neurones were principally identified with CF inputs, but implies extensive convergence of CF collaterals to maintain the spontaneous excitation of FN cells, because of the low spontaneous discharge rate of individual CFs (about 1/sec, Oscarsson, 1967; Bell & Grimm, 1969; Latham & Paul, 1971). Given such a convergence and bearing in mind that CFs themselves cannot normally be driven at rates much above 10 Hz (Eccles, Provini, Strata & Taborikov&, 1968; Gresty & Paul, 1970; Latham & Paul, 1971), the nearly simultaneous excitation of large numbers of CFs by the CN stimulus could result in disfacilitation of FN cells, thereby prolonging the suppression of spike discharge. The main objection to this hypothesis is that the 'gating' on the CF path is probably at the inferior olivary neurone level (Latham & Paul, 1971) and the 'gate' is activated by the olivary neurones discharging. Thus any FN cells which were disfacilitated should also have exhibited a preceding CF excitation (via a collateral), but this was not confirmed during the experiments. Difficulties are also encountered when trying to explain the facilitation which sometimes followed spike suppression. The obvious mechanism would be a cessation to Purkinje activity producing an enhanced (disinhibitory) discharge of the FN cell. However, in many cases the facilitation did not begin until 50-100 msec after the stimulus and sometimes persisted for more than 150 msec. The necessary reciprocal behaviour of Purkinje cells has not been observed in experiments on the caudatocerebellar path, although such a relationship between FN and anterior lobe cortex has been demonstrated following cutaneous stimulation (Latham et al. 1970), and Armstrong et al. (1973) found evidence for a similar reciprocal relationship between cerebellar Purkinje cells and interpositus neurones in chloralosed cats subjected to peripheral afferent stimulation. We have to conclude that although known cerebellar cortical and nuclear mechanisms are adequate in principle to explain the effects of CN stimulation on responses of FN cells, the patterns of activity in cortex and nucleus do not show the same apparent reciprocity when the CN is the source of afferent input as they do when peripheral afferents provide the input. Furthermore, with repetitive CN stimulation the late facilitatory effect was progressively enhanced in CL units and suppressed in IL units. Whilst this latter result might arise from some reciprocal inhibitory mechanism operating either on the projection paths or on the FN cells themselves, we know of no evidence to support such a suggestion. We are indebted for expert technical assistance to Mr P. Cheetham and Mr E. Balog. During the course of this work, M.A.G. held an M.R.C. studentship for training in research.
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