Axonal branching in the climbing fiber pathway to the cerebellum

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22 VERHAART,W. J. C., The pyramidal tract of 7bpaia, compared to that in t)thcr prmmtcs, ,I, comp. Neurol., 126 (1966) 43--50. 23 WlSLOCKI, G. B., Observations on the gross and microscopic anatomy of the sloths ¢Bradvpus griseus griseus Gray and Choloepus hoffmanni Petersj, Ji Morph. Physh~l., 46 (1928) 317-377. (Accepted June 16th, 1969)

Brain Research, 15 (1969) 259-262

Axonal branching in the climbing fiber pathway to the cerebellum Cerebellar afferents fiom the inferior olivary nuclei arise from approximately one million nerve cells in the human ~5 whereas the number of Purkinje cells is estimated at 15 million 13. If it be assumed that the inferior olivary nucleus is the only source of climbing fibers (CF) in the cerebellum (ref. 19: but cfi refs. 2, 5. t8), and that each Purkinje cell is innervated by one CFL the observed numerical discrepancy of 1-15 would suggest that the CFs must branch somewhere along their couise. It appears likely that a qualitatively similal ratio exists in the cat 3,a0. in this context it is notable that, although branching of CFs within a single folium in the cerebellar cortex has been observed in several species using the Golgi technique 17, the branching of a single C F in the parasagittal plane with terminations in other folia or lobules has never been reported in anatomical studies (ref. 6, pp. 36-42). In the present study, experiments were designed to test the hypothesis that CFs do indeed branch so that one parent fiber innervates Purkinje cells in more than one lobule within the cerebellum. The presence of branching CFs would provide a mechanism whereby information fi om one cell in the inferior olive might be dispersed to more than one lobule of the cerebellar cortex. Such branchings were looked for primarily in the parasagittal plane because of the observations by Oscarsson 16 that inputs from individual limb nerves to the cerebellar cortex are distributed in part along narrow parasagittal strips in the anterior lobe. Experiments were performed on adult cats lightly anesthetized with Pentothal. The cerebellar cortex was exposed by a dorsal craniotomy and covered with a mineral oil pool. In initial experiments responses produced by surface stimulation with concentric stimulating electrodes were recorded from various portions of the cerebellar cortex by means of a lightly sprung silver ball electrode. It is known that surface stimulation activates CFs, as is shown by the antidromic propagation of impulses back to the inferior olivO. Stimulation was usually applied on the surface of the fifth lobule 14 just anterior to the fissura prima and just medial to the paravermal vein: at other times stimulation was applied to the third or fourth lobules. In these experimental situations, prominent surface responses were recorded in the anterior lobe at discrete points along a parasagittal strip just medial to the paravermai vein (Fig. 1C, D). This strip corresponds to the region in which Oscarsson has described a somatotopic organization of afferents from ipsilateral hindlimb and Brain Research, t 5 (1969) 262-267

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263

forelimb 16. The responses consisted of short latency (1-6 msec) positive waves (Fig. l A), similar to the surface positivity produced by climbing fiber input 16. Corresponding surface responses were evoked by surface stimuli applied at other points along the parasagittal strip. The postulated pathway for the transmission of impulses which produce these responses is schematically represented in Fig. 1E. These surface responses have been interpretedg, TM as representing passive sources to the sinks produced by CF activation of the Purkinje cell dendritic tree. Such responses could not be distinguished when stimulating on the surface on the same folium, perhaps due to in part to the presence of large potentials produced by excitation of the parallel fibers in this situation (Fig. IB). Using these surface recordings as a guide, multiple microelectrode tracks were made in the vermis and pars intermedia of the anterior and posterior lobes of the cerebellum to depths of up to 7 mm beneath the surface. Extracellular unit and field potentials were monitored with glass micropipettes filled with 4 M NaC1 and having resistances of 2-4 Mr2. lpsilateral forelimb (superficial and deep radial) and hindlimb (quadriceps, sural, plantar cutaneous, and superficial peroneal) nerves were dissected and placed in a warm mineral oil pool. Potentials evoked by these inputs were used to determine the layer of the cerebellar cortex in which the microelectrode tip was located 9. Recording sites wele confirmed by histological identification. When typical Purkinje cell responses to CF activation were elicited by stimulation of the peripheral nerves, surface stimulation with the concentric electrode was then employed to see if similar responses could be produced. On those occasions when surface stimulation was successful in evoking responses thought to be due to CF activation, the field potentials were characterized by a very brief latency, a short rise-time, and a depth profile similar to that of the CF responses evoked by peripheral nerve stimulation (Fig. 2A). This profile consisted of a gradual change from negative to positive polarity as the electrode was advanced from the molecular layer through the Purkinje cell layer and into the granular layer. Stimulation of the cerebellar cortex may excite Purkinje cell axon collaterals which are located in the molecular layer4, n. At no time in the present experiment did we observe responses which could be interpreted on the basis of latency, form, or duration as representing antidromic activation of Pmkinje cells6,12 (cf. Fig. 2B, C). In addition when the probing microelectrode penetrated Purkinje cells, intracellular excitatory PSPs were recorded up to 2 lobules distant from the surface stimulation electrodes (Fig. 2E) thus confirming an orthodromic pathway to the Purkinje cells. The responses to climbing fiber activation could be distinguished from mossy fiber responses by the all-or-none firing characteristics of the former (Fig. 2B-D; also of. ref. 7) as well as by the multiple spike firing in response to a single stimulus (Fig. 2B, C). When utilizing higher resistance electrodes (about 10 Mf~) large unitary, all-ornothing negative potentials are often recorded in the lower one-third of the molecular layer in response to all CF inputs to the cerebellum (unpublished observations). Such responses are recorded without a preceding negative shift in DC potential and are presumed to occur when the microelectrode tip is located in intimate apposition Brain Research, 15 (1969) 262-267

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Fig. 1. Distribution of surface potentials evoked by stimulation of the surface of nearby lobules. A, Typical positive fields recorded on the 4th (upper) and 3rd (lower) lobules with stimulation of the 5th lobule by a parasagittally oriented concentric electrode surface, stimulating current being 0.2 mA. B, Examples of potentials produced by activation of a beam of parallel fibers with stimulation o n the same folium. In both A and B arrows indicate time of stimulation. The lower trace in B shows 5 superimposed responses; the remaining traces show the Fabritek 1062 computer output produced by 8 summated responses. C, D, Surface maps from 2 different experiments showing distribution of surface positive responses ( + ) found when stimulating the surface of the 5th (C) or 3rd (D) lobules. Regions in which response was looked for but not found are labeled with minus signs (--). P.V. : paravermal vein; P.I. : pars intermedia; F.P. : fissura prima; M L : midline; R o m a n numerals refer to lobules of cerebeltar cortex 14. E. Three-dimensional drawing o f anterior lobe in parasagittat plane portraying postulated pathway (arrows) f o r response to climbing fiber (cf) activation as described in this study. I.O.: inferior olive; PC: Purkinje cells; other labeling as above,

Brain Research, 15 (1969) 262-267

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Fig. 2. A, Depth profile of responses elicited by activation of climbing fibers as produced by translobule surface stimulation and by stimulation of the ipsilateral plantar cutaneous (PI. Cut.) nerve. The responses were recorded from the superficial molecular layer (1), the molecular layer just above the level of the Purkinje cell bodies (2), and the granular layer (3), as depicted in the photomicrograph. Vertical calibration represents 0.5 mV and is the same for all traces except upper trace of surface column where 0.5 mV is indicated by smaller vertical bar. Time bars represent 10 msec. B G, Examples from different experiments of Purkinje cell responses recorded in the depths of the cortex to surface stimulation of different lobules in the parasagittal plane. B and C illustrate multiple firing of a Purkinje cell due to climbing fiber input stimulation at threshold intensity so that on each series of superimposed traces there was failure in one. D, Giant extracellular unitary responses due to CF activation produced by threshold trans-lobule surface stimulation illustrating all-or-nothing property. E, lntracellular recording from a deteriorating Purkinje cell showing computer averaged excitatory PSP (8 responses averaged). F, Effects of a conditioning trans-lobule surface stimulation on the unitary CF response evoked by inferior olive (1.O.) stimulation at the intervals indicated in msec. Note the virtually complete block at 7 msec and partial recovery at 12 msec intervals. In each interaction record the response to the conditioning stimulus alone is superimposed on the interaction responses. G, The converse of F. Again note almost complete block at 14 msec. In B-G arrows are used to indicate onset of stimulation artifact where necessary.

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to the C F synapse on the Pm kinje cell dendrite. All-or-nothing responses of this type could also be produced with surface trans-lobule stimulation (Fig. 2D, F, G). S t r o n g evidence that the responses produced by both surface and inferior olive stimulation were a result o f activation o f the same C F is provided by the depression of surface induced responses by a conditioning inferior olive stimulation (Fig. 2G), A similar depression was observed when surface stimulation preceded olive stimulation (Fig. 2FL just as occurs with any other double C F stimulationL At short intervals impulses produced in the C F axon by stimulation at the two sites collided with resultant blockage o f the Purkinje cell response (Fig. 2F, 7 msec; Fig. 2G, [4 msec), A comparison o f the timing o f the collisions in the two interaction experiments (Fig. 2F, G) suggests that the climbing fiber bifurcates in the cerebellum rather than near its exit point from the inferior olive. These experiments indicate that C F axons branch prior to reaching the cerebellar cortex so that a single fiber activates Purkinje cells o f different lobules ( Fig. I E). The extent o f this branching is indicated by the fact that stimulation on the fifth lobule near the fissura prima produced responses in t'olia of the fourth and third lobules (Figs. 1C, D; 2B, C). This widespread parasagittal distribution of axon branches arising from single inferior olive neurons 19 provides a means for distributing the impulses from these cells. Thus there appears to be a well ordered geometrical design in the arrangement o f the branches o f a single CF. Certainly this branching in the parasagittal plane must a c c o u n t in part for the parasagittal slrips of C F activation observed by Oscarsson IG. We thank Plof. J. C. Eccles for suggestions and criticisms extended in the course o f this study. Laboratory o f Neurobiology, State University o f New York at Buffalo. Amherst, N.Y. 14226 (U.S.A.)

DONALD S. FABER* JOHN T. MURPHY

1 ARMSTRONG.D. M., AND HARVEY, R. J.. Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat, J. Physiol. (Lond.), 187 (1966) 553-574. 2 BATINI, C., ANDPUMAIN.R., Activation of Purkinje neurons through climbing fibres alter chronic lesions of the olivo-cerebellar pathway, Experientia (Basel), 24 (1968) 914-916. 3 BRAITENBERG, V.. AND ATWOOD. R. P.. Morphological observations on the cerebellar cortex, J. comp. Neurol., 109 (1958) 1-33. 4 EAGER, R. M.. Cortical association pathways in the cerebellum of the cat. J. comp, Neurol.. 121 (1963) 381-394. 5 EAGER, R. P., The mode of termination and temporal course of degeneration of cortical association pathways in the cerebellum of the cat. J. comp. Neurol., 124 (1965) 243-258. 6 ECCLES,J. C., ITO, M., ANDSZENTAGOTHAI.J., The Cerebellum as a Neuronal Machine, Springer, New York. 1967. pp. 36-42, 71-80. 7 ECCLES,J. C., LLINAS,R., AND SASAKI,K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum, J. PhysioL (Lond.), 182 (1966) 268-296. 8 ECCLES, J. C.. LLIN~S, R., SASAKI. K., AND VOORHOEVE, P. E.. Interaction experiments on the

responses evoked in Purkinje cells by climbing fibers. J. PhysioL (Lond.), 182 (1966) 297-315,

*

Post-doctoral fellow NINDB (1 F2 NB 40, 544-01 NSRB).

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9 ECCLES, J. C., PROVINI, L., STRATA, P., AND T,&BOP,IKov,~, H., Analysis of electrical potentials evoked on the cerebellar anterior lobe by stimulation of hindlimb and forelimb nerves, Exp. Brain Res., 6 (1968) 171-194. 10 ESCOBAR,A., SAMPEDRO,E. D., AND DOW, R. S., Quantitative data on the inferior olivary nucleus in man, cat and vampire bat, J. comp. Neurol., 132 (1968) 397-404. 11 FREZIK, J., Associative connections established by Purkinje axon collaterals between different parts of the cerebellar cortex, Acta morph. Acad. Sci. hung., 12 (1963) 9-14. 12 GRANIT, R., AND PHILLIPS, C. G., Excitatory and inhibitory processes acting upon individual Purkinje cells of the cerebellum in cats, J. Physiol. (Lond.), 133 (1956) 520-547. 13 KREUZFUCHS,A., Die Gr6sze der Oberfl~iche des Kleinhirns, Arb. Obersteinerlnst., (1902) 278 280. 14 LARSELL, O., The cerebellum of the cat and the monkey, J. comp. Neurol., 99 (1953) 135-199. 15 MOATAMED, F., Cell frequencies in the human inferior olivary nuclear complex, J. comp. Neurol., 128 (1966) 109-116. 16 OSCARSSON, O., Functional significance of information channels from the spinal cord to the cerebellum. In M. D. YAHR AND D. P. PURPURA (Eds.), Neurophysiological Basis o f Normal and Abnormal Motor Activities, Raven Press, Hewlett, N.Y., 1968, pp. 93-117. 17 SCHEIBEL, M. E., AYD SCHHBEL, A. B., Observations on the intracortical relations of the climbing fibers of the cerebellum, J. comp. Neurol., 101 (1954) 733-764. 18 SYIDER, R. S., Alterations which occur in mossy terminals of the cerebellum following transection of the brachium pontis, J. comp. Neurol., 64 (1936) 417 534. 19 SZENT,~GOTHAI,J., AND RAJKOVITS, K., Uber den Ursprung der Kletterfasern des Kleinhirns, Z. Anat. Entwickl.-Gesch., 121 (1959) 130-141. (Accepted June 17th, 1969) Brain Research, 15 (1969) 262-267

Cerebro-cerebellar connections mediated by fast and slow conducting pyramidal tract fibres of the cat Relationships between the cerebral and cerebellar cortices have invited many anatomical and physiological investigations. The basis for the present electrophysiological study, which concerns the cerebro-cerebellar connections mediated by the pyramidal tract, has been provided by recent microelectrode investigations independently performed on the respective cortices in the cat. Originally, pyramidal tract (PT) cells were classified into two main categories, fast and slow, according to whether their axons were fast or slowly conducting3,4,9,14,1s,20. Corresponding differences were found in the biophysical properties of the respective cell membranes13, is and in their synaptic organizationslg, 21-'~3. In the light of this evidence a study was made of pyramidally evoked responses of the cerebellum from the aspect of functional differentiation between fast and slow PT cells. Meanwhile it has been found that the stimulation of the sensorimotor area of the cerebral cortex induces two distinct types of field potential in the contralateral cerebellar cortex with short and long latencies respectivelyt°,11,1"L These potentials are similar to those elicited by stimulations of precerebellar nuclei and peripheral nerves, and are identified as the responses evoked by mossy (MF) and climbing fibres (CF)a, s. Different contributions of the fast and slow PT cells to the MF and CF responses will be demonstrated in this communication. Cats were anaesthetised with pentobarbitone sodium (35-40 mg/kg i.p.). The head of the animal was fixed in a stereotaxic metal frame (David K o p f InstruBrain Research, 15"(1969) 267-271