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Responses of the granule cells of the selachian cerebellum (Mustelus canis)
87
BRAIN RESEARCH
RESPONSES OF THE G R A N U L E CELLS OF THE SELACHIAN C E R E B E L L U M ( M U S T E L U S CANIS)
J. C. ECCLES, H. T~BORfKOV/~ AND N. T S U K A H A R A
Department of Physiology, State University of New York at Buffalo, Buffalo, N.Y., and .~4arine Biological Laboratory, Woods Hole, Mass. (U.S.A.) (Accepted June 15th, 1969)
INTRODUCTION
In their structural details, somata, dendrites and axons, the granule cells of the selachian cerebellum very closely resemble the granule cells of the mammalian cerebellum 17,18,z1,z4. In their general morphological arrangement they offer peculiar advantages for physiological investigation. Instead of the widely dispersed arrangement in which the granule cell layer is coextensive with both the molecular layer and the intervening layer of Purkyn~ cell somata, the selachian granule cells are concentrated in two cords lying adjacently on each side of the midline, and following parasagittally all the cortical folding, as is illustrated in Fig. 1B of the preceding paper 12. In the dogfish used in our experiments these cords were 2-3 mm in diameter, so it was easy to insert a microelectrode into a granule cell cord and record responses of granule cells virtually uncontaminated by the responses of other nervous elements of the cerebellum. It was found that, as a consequence, the responses of granule cells could be investigated more effectively than in the mammalian cerebellum. The granule cords also have a relatively sparse population of so-called Golgi cells, but these cells differ from the mammalian Golgi cells in that their dendrites are restricted to the granule cords (cf. ref. 12, Fig. 2), whereas in the mammal the dendrites widely ramify in the molecular layer. It was of interest therefore to discover if the selachian Golgi ceils resembled mammalian Golgi cells in having an inhibitory action on granule cells. A preliminary account of these investigations has been publishedZL METHOD
The general experimental procedures have been described in the preceding paper r*. The arrangements of the stimulating and recording electrodes are shown diagrammatically in Fig. I B, which is a drawing of a transverse section of the posterior lobe of the dogfish cerebellum along a plane indicated in Figs. IA and B of the preceding paperlL Usually the microelectrode was inserted perpendicularly to the dor-
Brain Research, 17 (1970) 87-102
88
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Fig. 1. Transverse sections of the cerebellum. A, Drawing of the transverse section of the cerebellum of Scyllium at the level of the acoustico-lateral commissure. The dorsal (era. gr.) and ventral granule cell cords are seen. The pathways of the fibers entering the cerebellum from the peduncle are well shown and form the stratum fibrosum (str. fibr.) deep to the Purkyn~ cell layer and the molecular layer (str. tool.). Also shown is the cerebellar ventricle (v. cb.) the fourth ventricle (V. 4) and the spino-cerebellar (tr. sp. cb.) and olivo-cerebellar (ol. cb.) tracts (modified from Larsell)lL B, Diagrammatic representation of the transverse section of the posterior lobe of the cerebellum showing the recording microelectrode (ME) in the dorsal granule cord. Mossy fibers (M.F.) are drawn in the stratum fibrosum on the route to the granule cell cords and ending synaptically on granule cells (GrC). The axons of the granule cells bifurcate to form the parallel fibers (PF) that are transversely directed in the molecular layer (M.L.). Also shown is the layer of Purkyn?: cells (PC) and the end of the concentric stimulating electrode is drawn symbolically in the stratum fibrosum (Stim. El.). sum o f the cerebellum strictly in a p a r a s a g i t t a l plane. F o r o p t i m a l r e c o r d i n g from the g r a n u l e cell c o r d it was usually located a b o u t 1 m m from the midline. A t depths up to a b o u t 4 m m it w o u l d be r e c o r d i n g f r o m the d o r s a l cord, b u t on further insertion across the ventricular cavity the ventral c o r d was entered. M o s t o f our investigation was on this c o r d because it gave responses that were well m a i n t a i n e d d u r i n g experiments o f m a n y h o u r s ' d u r a t i o n . The afferent fibers to the granule cells have not been recognized to have the characteristic histological features o f m o s s y fibers 17,24; nevertheless it has been f o u n d t h a t their p h y s i o l o g i c a l action is strictly c o m p a r a b l e to m a m m a l i a n mossy fibers; hence it is c o n v e n i e n t to recognize this h o m o l o g y b y alluding to them as mossy fibers. These fibers enter the cerebellum via the p e d u n c l e a n d course in the s t r a t u m fibrosum between the Purkyn~ cells a n d the ventricular cavity as indicated in Fig. I A. In their f u r t h e r p a s s a g e to the granule c o r d s they are seen to continue j u s t deep to the Purkyn6 cell layer a n d then to t u r n into the g r a n u l e cords where they end by ramifying a m o n g s t the granule cells. A s i n d i c a t e d in Fig. IB, the m o s s y fibers have been excited by applic a t i o n o f c u r r e n t pulses t h r o u g h a concentric electrode t h a t has been inserted into the Brain Research, 17 (1970) 87-102
G R A N U L E CELLS OF SELACHIAN CEREBELLUM
89
stratum fibrosum at a site usually close to its origin from the peduncle. Fig. 1B also shows that the axons of granule cells pass into the molecular layer. By the characteristic T-shaped dichotomy they form the parallel fibers that run transversely in the molecular layer and even cross the midline. RESU LTS
Synaptic excitation of granule cells In Fig. 2A, with progressive increase in the peduncular stimulation, from 9 to 36 V, there is paripassu an increase in size of a characteristic potential wave form - an initial diphasic (positive-negative) spike and a later slow negative potential with a relatively fast rising phase and a slower decay. With stronger stimulation (45-90 V) there is further increase in both the initial diphasic spike and the slow negative potential, but a negative spike potential is superimposed on its rising phase. There is a close similarity to the simplest potentials produced in a mammalian granular layer by a mossy fiber volley TM. ]-he initial diphasic spike can be attributed in part to the mossy fiber volley, and its synaptic excitatory action on the granule cells would give the slow negative wave, the spike being dt~e to the generation of impulse discharges. Fig. 2A was recorded at a depth of 9.1 mm from the dorsum of the posterior lobe and so would be deep in the ventral cord (cJi Fig. 1). Fig. 2B illustrates part of a depth profile of potentials evoked by a 63 V stimulus and recorded along the microelectrode track that included the recording site for Fig. 2A. More superficially than 9.1 mm thele was a progressive decline of the response, which otherwise had approximately the same time course. More deeply, at 9.5 mm, there is the sign of a reverse polarity for the spike discharge, which would be expected for the concentration of impulse discharges propagating in the granule cell axons on t h e way to the deep molecular layer (U~ Fig. 1B). Finally at 9.7 mm there was a large positive potential in each of the three superimposed traces. Presumably this later spike is generated by a Purkyn6 cell excited by the parallel fiber volley. At 0.4 mm deeper still all potentials were greatly reduced, as would be expected in the deep molecular layer. Fig. 2C illustrates for this same experiment a series of potentials generated at a depth of 4.35 mm in the dorsal granule cord by double peduncular stimulation. It is seen that the second stimulus produced quite a large negative wave at the shortest interval (3 msec) and there was virtually a full-sized wave at 4 msec. The only significant difference was the shorter time course of the second response, and this can be seen to advantage at testing intervals of 12, 16 and 32 msec. Fig. 3 gives an example in another experiment of a depth profile of the granule cell potentials recorded in the ventral cord. As in Fig. 2B both the slow negative wave and the superimposed spike reached a maximum at about the same depth, 8.9-9. I ram. At 9.5 mm the initial negative wave appeared to be terminated suddenly by a reversed potential, which can be attributed to the propagation of impulses more deeply along the granule cell axons. At still deeper recording sites, the potentials were virtually reversals of the negative wave recorded at the optimal sites. Evidently the field potential
Brain Research, 17 (1970) 87-102
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Fig. 2. Synaptically evoked potentials of granule cells. Stimuli are applied to the fibers in the stratum fibrosum by the concentric electrode shown in Fig. 1B, and potentials are recorded in A at a depth of 8.4 mm deep in the ventral granule cord. Responses evoked by the indicated strengths of stimuti of O.2 msec duration are shown in A. Series B is taken at the indicated depths along the same microelectrode track, the stimuli being 54 V for 0.2 msec. Series C is taken in the dorsal cord depth (4.9 ram) along the same microelectrode track, there being double stimuli at various intervals, both stimuli being 54 V for 0.2 msec. Note change in sweep speed. Same voltage scale for A and B, and a separate scale for C, all with DC recording. indicates t h a t the axons o f the granule cells d o w n into the deep m o l e c u l a r layer were acting as sources to the active sinks generated in the g r a n u l a r cord by the synaptic e x c i t a t o r y action o f the mossy fiber volley. The p l o t t e d p o i n t s o f Fig. 3B illustrate the relation o f this d e e p e r source to the active sinks a n d o f the progressive deciine o f the negative wave m o r e superficially. The small field p o t e n t i a l s in the u p p e r p a r t o f the p l o t t e d curve were p r o b a b l y n o t actively generated there, b u t merely represent the decline o f the general p o t e n t i a l field m o r e superficially than the deeper sink-source generating zone. Brain Research, 17 (1970) 87-102
GRANULE CELLS OF SELACHIANCEREBELLUM
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Fig. 3. Depth profile of synaptically evoked potentials of granule cells. A shows a series of specimen records (DC recording) at the indicated depths along a microelectrode track in the ventral granular cord of the posterior lobe in response to a stimulus of 63 V for 0.2 msec. The potentials measured at a fixed interval of 2.5 msec after the stimulus are plotted in the depth profile of B. Fig. 2A gives an illustration o f spatial summation in the excitatory action of mossy fibers on granule cells. Convergence of two or more impulses was required to generate a spike discharge such as is seen with stimulus strengths o f 45 V or more. Fig. 4 gives an example o f temporal summation. A single mossy fiber volley evoked a slow negative wave with the superposition o f only a minimal spike discharge. This spike was greatly facilitated by superposition o f a second mossy fiber volley, though the added slow potential was not potentiated, which would correspond to the series o f Fig. 2C in which the slow negative waves were not appreciably distorted by spike discharge. The plotted measurements (Fig. 4B) from the series partly illustrated in Fig. 4A show that this facilitation could be observed out to a volley interval of 70 msec. It can be postulated that this temporal facilitation has the standard explanation, Brain Research, 17 (1970) 87-102
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Fig. 4. Facilitation of synaptic excitation of granule cells. The specimen records of A are evoked by double stimuli of 63 V for 0.2 msec at a depth of 8.95 mm alongthe same microelectrode track asin Fig. 3 and at the indicated intervals. Note the three different sweep speeds, but there is the same voltage scale with DC recording. In B the size of the response added by the second stimulus is calculated as a percentage of the control response and plotted against the stimulus interval to give a facilitation curve. namely that it is due to the superposition of the EPSP produced by the second volley on the residual EPSP of the first (cJ~ ref. 3). This value of 70 msec is certainly an underestimate of the duration of the granule cell EPSP, because, in accord with intracellutar recording from other species of neuron (ef. ref. 4), accommodation would be expected to raise the threshold depolarization at which the second EPSP would ge:aerate an impulse discharge. it was often observed that the potentials evoked by a mossy fiber volley in the granular cord had irregular trains of small spikes superimposed on the slow negative potential. For example in Fig. 4A two or three small spikes can be seen at the arrows after the initial facilitated spike. When microelectrodes with a finer tip were employed, it was possible to record quite large unitary spike potential~ as in Fig. 5. In Fig. 5A, with progressive increase in the peduncular stimulation, there was an increased number of unitary spike potentials, it being possible to discern two distinct populations: a large spike that failed at 27 V, was single at 36 and 45 V and double at stronger stimulation; and a smaller spike that was single at 27 V, double at 36-54 V and apparently numbering three or four with stronger stimuli. Since these responses were recorded in the ventral granule cord, they are likely to be produced by granule cells. This identification is supported by the still smaller spike potentials that can be seen in the responses of Fig. 5A that were evoked by stimuli of 45-90 V. Evidently there were many ceils in fairly close relationship to the tip of the recording microelectrode, and one must have been in close proximity. It might be contended that granule cells are so small and so tightly packed that the extraceltular Brain Research, 17 (1970) 87-102
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Fig. 5. Synaptically evoked responses of single granule cells. Mossy fiber volleys were evoked as usual by the concentric stimulating electrode in the stratum fibrosum (of. Fig. IB) and the recording microelectrode was carefully adjusted so as to record selectively from a single unit deep in the ventral granule cord at a depth of 10.13 ram. In A are the responses evoked by stimuli ofO.2 msec duration and the indicated voltages. Each record of B resembles the fellow record of A except that there were two stimuli of the same voltage as in A, and 7 msec apart. In C there were two stimuli of strength 90 V and at various intervals as indicated. In D the response to a single stimulus of 72 V was tested after slight movements of the microelectrode as described in the text. Same voltage scale throughout, but the sweep speed was changed as indicated by the time scales.
spike potentials of one could not be recorded to give such a large spike both absolutely (almost 1 mV) and relatively to the other cells. However, such a selective recording required very careful positioning of the microelectrode, and was secured only in the stable mechanical conditions provided by deep recording in the ventral granule cord. Moreover very small movements of the microelectrode greatly reduced the size of the spike potential, as may be seen in Fig. 5D. Withdrawal of the microelectrode from the depth of 10.13 mm in two successive steps of 10 #m each resulted in a response with a series of small spikes, none of which could be identified with the large spike. With reversal of the movement the spike returned at I0.12 and 10.13 mm but it was greatly depressed at 10.14 ram, I0/~m below the original recording site. Fig. 5A shows that the increased size of the mossy fiber volley provided by the stronger peduncular stimulus resulted in an increased response of the two granule cells Brain Research, 17 (1970) 87-102
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Fig. 6. Antidromic activation of granule cells. A is a diagrammatic illustration of the experimental arrangement with a surface stimulation of the parallel fibers (P.F.) laterally on the dorsum by a concentric electrode (Stim. El.). The microelectrode is seen recording from the granule cells in the dorsal granule cord (Gr. Cord), M.F. and P.A. are mossy fibers and Purkyn6 cell (P.C.) axons. In B are the responses evoked by the stimuli shown in volts and of 0,2 msec duration and recorded in the dorsal granule cord at 3.25 mm depth. The microelectrode track was about 1 mm from the midline and the surface stimulating electrode was about 2 mm laterally thereto. There was DC recording, stars and dots signalling respectively Purkyn~ cell and granule cell spike potentials.
under observation, i.e. spatial summation of two or more impulses gave a n increased synaptic excitatory action on these granule cells. Fig. 5B illustrates the summational effect of two mossy fiber volleys at 7 msec apart, and of the same size as for the corresponding single stimulation in A. There was always an increased response to the double stimulation, but with 90 V strength the effect was more than double, both for the large spike and the small spike. The specimen records of Fig. 5A and B were chosen from a larger number - - 3 or 4 each for the single and double at each strength. The mean number of spikes shows that at several strengths the double stimulation was more than twice as effective as the single, the ratios of double/single being respectively: 1.3/0.8; 2.0/1.0; 3.0/1.2; 3.0/1.7; 4.0/1.8; 4.0/2. l; 6.0/2.0 for stimulus strengths of 27, 36, 45, 54, 63, 72 and 90 V. The high ratios provide further evidence :for temporal facilitation of the excitatory synaptic action of mossy fibers on granule cells (of. Fig. 4). Since with 90 V stimulation there was a large temporal facilitation of spike discharges at 7 msec interval in B, it was of interest to investigate the time course of this facilitation. In C with double stimulation there was an increased response (3 spikes) to the second volley even at 72 msec test interval, and at briefer intervals the test volley evoked even four or five spike discharges. This evidence for temporal facilitation is thus in good agreement with Fig. 4 in showing facilitation of granule cell discharge at test intervals up to 70 msec.
Brain Research,17 (1970) 87-102
GRANULE CELLS OF SELACHIAN CEREBELLUM
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Fig. 7. Recovery curves for Purkyn6 and granule cells antidromically activated. A shows specimen records of potentials evoked as in Fig. 6B, but for two stimuli at various intervals as shown. The recording site was identical with that of Fig. 6B and the stimulus strength was 90 V for 0.2 msec. DC recording. In B and C are respectively the recovery curves for the antidromic spikes of the granule cells and the Purkyn6 cells as measured from records of which a few are shown in A, where stars indicate Purkyn6 spikes a ad dots granule spikes of the testing responses. The spike potentials are measured as negativities from the base line and are plotted as percentages of the mean controls.
Antidromic excitation of granule cells The large pure c o n c e n t r a t i o n of granule cells in the granule cords gives opportunity for investigating the events occurring when impulses propagate antidromically in the axons of granule cells, i.e. from parallel fibers and down the stem of the T - j u n c t i o n to the granule ceils. With the m a m m a l i a n cerebellum there was only a single presumed example of the a n t i d r o m i c invasion of a granule cell 7. With the much more favorable a r r a n g e m e n t in the selachian cerebellum, massive a n t i d r o m i c invasion of granule cells was regularly observed. The experimental a r r a n g e m e n t is shown in Fig. 6A, where a concentric electrode either on the surface or very superficially in the molecular layer
Brain Research, 17 (1970) 87-102
96
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was used for exciting parallel fibers and the microelectrode was inserted into the dorsal granule cord. Strong stimuli (up to 90 V and 0.2 msec duration) had to be applied in order to excite the very thin non-medullated parallel fibers, and as a consequence the deeper-lying axons of Purkyn6 cells were also excited. In Fig. 6B weak stimuli evoked an initial spike and then a slower and larger spike (starred) which may be identified as due to antidromic invasion of Purkyn6 cells (cf. ref. 12). Stimuli of 54-90 V evoked in addition a much later spike (dotted) with a negative summit at a latency of about 10 msec. This spike response was much later than the spike response of granule cells produced by orthodromic excitation via mossy fibers, which had a latency of less than 3 msec to the summit in Figs. 2A, B. 3 and 4 despite the longer conduction pathway. However, this long latency would be expected for the spike potential produced by the antidromic invasion of granule cells because the conduction velocity in their axons, the parallel fibers, is only about 0.2 m/sec 22, es, which is in agreement with the value of 0.3 m/sec lbr the mammalian parallel fiberse, 6. When double stimuli were applied at various intervals, it was fbund that, alter antidromic invasion, the granule cells recovered fast¢r than the Purkyne cells. This can be seen in the specimen records of Fig. 7A, where there was already a large second granule spike (indicated by dot) at the briefest testing interval (2 reset) and at 12 msec the recovery was almost complete, whereas at that test interval the second Purkyn6 response was merely the very small spike indicated by the star. The time course of the recovery of the Purkyne cell response is plotted in Fig. 7C. there being full recovery in 40 msec, which is in precise accord with the recovery illustrated in the previous paper re. The much earlier recovery of the antidromic invasion of granule cells is seen in Fig. 7B, where it was virtually complete in 15 msec. Fig. 7C shows another feature that would be expected when the conditioning stimulus very effectively excited parallel fibers, namely a slight facilitation at 40-80 msec followed by a slight depression. "l-hese effects have been attributed to an admixture of two opposing synaptic actions on the Purkyne cells, namely a direct excitation by the parallel fiber impulses and an indirect inhibition via the pathway: parallel fiber impulses to stellate cells that in turn give inhibitory synapses to Purkyn6 cells le. Antidromic invasion of granule cells is indicated in Fig. 8 for two other experiments. Since the recording microelectrode was much more medially placed in tile dorsal granule cord, the Purkyn6 spike potential was small in the series o f A ~',ld B, and undetectable in C and D, where the recording was by an electrode accurately placed in the midline, so as to be remote from Purkyn~ cells (cJ~ Fig. I B). The depth profile in A shows a diphasic (positive-negative) spike potential when the electrode was relatively superficial (2.0-3.2 mm), while more deeply the negative spike potential was earlier. A possible explanation is that the deeper granule cells have thicker axons and hence there is a briefer antidromic conduction time despite the longer conduction distance. This explanation is in line with the histological observation in the mammalian cerebellum that the deeper granule cells have larger axons that give the larger parallel fibers deep in the molecular layer 13,~5,23. The double stimulation series (B) reveals a recovery curve of the antidromic spike potential of the granule cells corresponding to that plotted in Fig. 7B. Brain Research, 17 (1970) 87-102
97
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Fig. 8. Antidromic activation of granule cells. There is in A a depth profile of DC recorded responses evoked by antidromic activation of Purkyn6 cells and granule cells, indicated by stars and dots respectively. The recordings were in the dorsal granule cord about 1 mm from the midline and at the indicated depths, and the responses were evoked by stimuli of 72 V for 0.2 msec. In B there was double stimulation, each stimulus being 72 V for 0.2 msec and the recording at a depth of 3.4 mm in the track of A. C and D resemble A and B, but are from a different experiment, with recording in the midline so as to elimiaate the field potentials of Purkyn~ cell spikes. In D there are responses to double stimuli 54 V for 0.2 msec recorded at a depth of 2.8 ram, all stimuli being indicated by arrows. There was DC recording throughout Fig. 8. In the depth profile o f C, there is the surprising observation that the a n t i d r o m i c spike potential reversed to a large positive wave at the deepest recording sites o f the dorsal granule cord. The most p r o b a b l e e x p l a n a t i o n is that the m i c r o e l e c t r o d e had injured some granule cells in that region, with the consequence that their spikes were being recorded as positive potentials. Again in D there is seen to be the typical fast recovery o f D anule cell a n t i d r o m i c response, which was already considerable at the briefest testing interval (5 msec), and a l m o s t fully recovered by 20 msec. DISCUSSION in general the granule cell responses described in this p a p e r c o n f o r m e d with Brain Research, 17 (1970) 87-102
98
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those described for the mammalian cerebellum7, lo, but in several respects the present investigation was more illuminating because of the peculiar advantages deriving from the concentration of the granule cells in the parasagittal cords. For example, spatial and temporal facilitation are well illustrated in Figs. 2A, 4 and 5A-C and antidromic invasion in Figs. 6-8. With double antidromic activation granule cells display a more rapid recovery lhan Purkyn6 cells. A second response was evoked at only 2 msec interval (Fig. 7) and full recovery occurred by 20 msec. It seems that refractoriness after the initial spike response provides an adequate explanation of a depression with such a relatively fast recovery. By contrast after Purkyn~ cell activation, the recovery time of about 40 msec indicates that, superimposed on the initial refractoriness, there is a more prolonged depression such as would result from after-hyperpolarization. The rapid recovery of granule cells also is displayed by the very short intervals between successive spike discharges, which were as brief as 3 msec in Fig. 5 for example. With powerful synaptic stimulation or double antidromic stimulation Purkyn~ cells have discharged at intervals as brief as 5 msec (cfi ref. 12, Figs. 5 and 6A). A most interesting discovery with respect to the granule cell responses has not yet been referred to because it is a negative finding, namely that there has been no trace of a synaptic inhibitory action on granule cells. Such an inhibitory action was a very prominent feature of the mammalian cerebellum, and with some confidence has been attributed to the Golgi cells of the granular layera,7,1°, H. Favorable conditions for detection of inhibitory action on granule cells would be provided by double mossy fiber stimulation, but, as shown in Figs. 4 and 5, even when the stimulus interval was only a few milliseconds, there was no trace of a depression of the granule cell spike discharge evoked by the second volley. Favorable conditions would also be provided by double parallel fiber stimulation, because the so-called Golgi cell of the granular layer conceivably could be synaptically excited by impulses antidromically propagated in the axons of granule cells. However. as shown in Figs. 7B and 8B and D, after the initial recovery from refractoriness there was no inhibition of the antidromic response of the granule cells. In contrast to the mammalian cerebellum the so-called Golgi cells of the selachian cerebellum have their dendritic branches restricted to the granular layerlV,2t,24; hence it would be expected that the main excitatory input would be from the mossy fibers, which are known to provide a subsidiary excitation in lhe mammal 5,1°,16. Nevertheless, conditioning by a mossy fiber volley failed to produce any depression of the granule cells. It could of course be suggested that the conditioning mossy fiber volley had an inhibitory action indirectly via Golgi cells as well as a direct excitatory action, and that the latter was more powerful and just as prolonged, hence the absence of any overt inhibition in such tests as are illustrated in Figs. 4 and 5. It is certainly important to have electronmicroscopic examination of the mossy fiber-granule cell synapses in order to see if there are superimposed synapses homologous with the Golgi cell inhibitory synapses in the mammaP 4,16. Fig. 9 shows diagrammatically the neuronal connectivities and actions that we postulate for the cortex of the selachian cerebellum. The features of this diagram are Brain Research, 17 (1970) 87-102
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P
99
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Fig. 9. Diagram of neuronal connections. Diagrammatic representation of the dorsum of a transverse section in order to show the neuronal connections that are postulated on the basis of the known anatomical structures and in order to explain the responses reported in this paper and the preceding paper. Synapses are drawn in conventional manner, and presumed inhibitory cells and their inhibitory synapses are shown in black. M.F., mossy fiber; C.F., climbing fiber, note the branching remote from their terminals; Gr.C., granule cell; P.F., parallel fiber with synapses on Purkyn~ cells, P.C., and stellate cells, S.C. Further description is given in the text.
based both upon anatomical investigations 1,17,1s,21,24 and upon our present electrophysiological study 12, there being one exception in that the Purkyn~ cells are shown as being inhibitory solely on analogy with their function in the m a m m a l 5. The input pathway by the climbing fiber is branched in accord with the findings in the previous paper, and there is, as in the mammal, applied to each Purkyn~ cell only one climbing fiber with its extensive synaptic contacts. The input pathway via the mossy fibers gives converging synaptic excitation on the granule cells, and the granule axons bifurcate to form parallel fibers that excite Purkyn~ cells via spine synapses and also stellate cells that in turn give inhibitory synapses on the dendrites o f the Purkyn6 cells. After extensive branching this mossy fiber pathway exercises a limited convergent action on each granule cell, but there would be an enormously greater convergence in the next stage, namely parallel fibers to the spines o f the Purkyn6 cell dendrites. It should be noted that, as in the mammal, the Purkyn6 dendrites branch extensively to form a kind o f leaflet orthogonal to the direction o f the parallel fibers, so giving maximal opportunity for convergent excitation, but this cannot be depicted in the plane o f the diagram Brain Research, 17 (1970) 87-102
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which cuts across the dendritic leaflets. The histological evidence is that the axon of a stellate cell does not extend fal beyond the region of its soma and dendrites; hence it would be expected that the Purkyn6 cells excited by a beam of parallel fibers would also be inhibited by those stellate cells excited by that same beam. There would not be the extensive lateral inhibition that is such a feature in the mammalian cerebellum, where basket cells exert an inhibitory action up to 1 mm transversely from the beam of excited parallel fibersS, 9. We have as yet made no attempt to test for this presumed absence of lateral inhibition. In summary, Fig. 9 discloses that already in its evolutionary development the cerebellum had achieved most of the characteristic features of the neuronal machinery in the mammalian cerebellum 5. There appear to be only three missing features: the powerful feedback inhibition on granule cells that is mediated by the parallel fiber excitation of Golgi cell dendrites in the molecular layer; the powerful lateral inhibition that is exerted on Purkyn6 cell somata by the basket cells through their extensive transversely directed axons; the inhibition by axon collaterals of Purkyn~ cells 5, s These investigations on the connections and functions of the neurons of the selachian cerebellum are of interest in relation to the evolutionary development of the cerebellum. At this early stage there are already almost all of the neuronal elements of the highly developed cerebella of mammalia. The mossy fiber-granule celt-parallel fiber-Purkyn6 cell and the climbing fiber-Purkyn6 cell pathways are fully developed except that there probably is less convergence of parallel fibers on Purkyn~ cells. In contrast to these excitatory pathways the inhibitory pathways are less developed, but there is good evidence for an inhibitory action on Purkyn~ cells by the mossy fibergranule cell-parallel fiber-stellate cell pathway 12. Though there appears to be the homologue of the Golgi cell in the granular layer, no inhibitory action by it on the mossy fiber-granule cell synapses has yet been demonstrated. Another inhibitory deficiency arises from the absence of Purkyn5 axon collaterals. It can be postulated that all the component cells of the mammalian cerebellum are represented at least primordially in the selachian cerebellum, evolution merely resulting in the development of synaptic connections, so enhancing their functional design and exploiting potentialities latent in this neuronal machine. Some of the stellate cells developed into basket cells by growing long axons transversely across the folium and so achieving a very effective functional contact (the baskets) with the somata of the Purkyn~ cells. The dendrites of the Golgi cells very largely grew up into the molecular layer where they were able to secure very effective synaptic excitation by the parallel fibers, and p a r i passu their axons branched extensively in the granular layer so that the Golgi cells became very effective inhibitol s of the granule cells. The Purkyn6 cells grew axon collaterals that developed extensive inhibitory synaptic connections on several types of cell in the cerebellar cortex. We therefore postulate that the essential neuronal elements of the cerebetlar cortex were present in the primordial ancestors of the selachians, and that evolution resulted in the realization of the latent potentialities. This concept of evolution contrasts with the postulate of Llin~ts et al. 19 that inhibition first developed in the reptilian cerebellum because it was not demonstrated in the cerebellum of anura. Recently Nicholson and Llin~is'-'° report Brain Research, 17 (,1970) 87-102
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101
the existence of Purkyn~ cell inhibition in the elasmobranch czreb:!lum, and attempt to develop an evolutionary explanation by which this may be accommodated to the absence of inhibition in the anuran cerebellum. However, as stated in the preceding paper t'-), the question of inhibition in the anuran cetebzllum is still opzn. SUMMARY
The granule ceils of the selachian cerebel um are concentl ated in two parasagittal cords closely adjacent on either side of the midline, which is an arrangement that gives very favorable conditions for selective recording from granule cells by an extracellular microelectrode. A mossy fiber volley generates a negative potential wave wilh the time course and depth profile expected for the synaptic excitatory l esponses of granule cells. With large volleys, tile spike responses of granule cells are superimposed thereon. With two mossy fiber volleys at intervals up to 70 lnsec, there is facilitation of the spike discharge, and this has been studied in unitary recordings from granule cells which are found to respond by brief repetitive discharges. When the axons of granule cells (the parallel fibers) are excited by a surface electrode, the field potentials in the granule cord show that there is antidromic invasion of the granule cells. After this invasion the recovery is faster than after the antidromic invasion of Purkyn~ cells, being complete within 20 msec. There has been no evidence for an inhibitory action of the so-called Golgi cells on the granule cells. The physiological findings on the neurons of the dogfish cerebellar cortex have been correlated with the histological data, and this correlation has been displayed diagrammatically. ACKNOWLEDGEMENT Our grateful thanks are supported this project that was Lectureship by one of us (J. C. from the National Institute of N0822101.
extended to the Grass Foundation who generously carried out during the tenure of an Alexander Forbes Eccles). This work was partially supported by a grant Neurological Diseases and Stroke, Grant No. R01-
REFERENCES 1 ARIENSKAPPERS,C. U., HUBER, G. C., AND CROSBY, E. C., The Comparative Anatomy of the Nervous System of Vertebrates Including Man, Vol. 2, Macmillan, New York, 1936, pp. 696-1239. 2 Dow, R. S., Action potentials of cerebellar cortex in response to local electrical stimulation, J. Neurophysiol., 12 (1949) 245-256. 3 ECCLES,J. C., The Physiology of Nerve Cells, Johns Hopkins Press, Baltimore, 1957, 270 pp. 4 ECCLES,J. C., The Physiology of Synapses, Springer, Heidelberg, Berlin, G6ttingen, 1964, 316 pp. 5 ECCLES,J. C., ITO, M., AND SZENTfiIGOTHAI, J., The Cerebellum as a Neuronal Machine, Springer, Heidelberg, Berlin, G6ttingen, New York, 1967, 335 pp. Brain Research, 17 (1970) 87-102
102
,~. ~'
~3 ~ 1.1~ C," '~i,'
6 ECCLES, J. C., LLINAS, R., AND SASAKI, K., Parallel fiber stimulation and the resp~)asc~ induced thereby in the Purkinje cells of the cerebellum, Exp. Brain Res., I (1966) 17 39. 7 ECCLES, J. C., LLINAS, R., AND SASAKI,K., The mossy fiber-granule cell relay in the ~.:ereb~'lium a n d its i n h i b i t i o n by G o l g i cells, Exp. Brain Res., 1 (1966) 82-101. 8 ECCLES, J. C., LLINAS, R., AND SASAKI, K., The action of antidromic impulses o~ the ccrebeilar Purkinje cells, J. Physiol. (LonJ.), 182 (1966) 316-345. 9 ECCLES, J, C., SASAKI, K., AND STRATA, P., The profiles of physiological events produ,ced by a parallel fiber volley in the cerebellar cortex, Exp. Brain Res., 2 (1966) 18-34. 10 ECCLES, J. C., SASAKI,K., AND STRATA,P., The potential fields generated in the cerebzllar cortex by a mossy fiber volley, Exp. Brain Res., 3 (1967) 58-80. 11 ECCLES, J. C., SASAKI, K., AND STRATA, P., A comparison of the inhibitory actions of Golgi ceils and of basket cells, Exp. Brain Res., 3 (1967b) 81-94. 12 ECCLES, J. C., T.~BORIKOV.~, H., AND TSUKAHARA, N., Responses of the Purkyn~ ceils of a selachian cerebellum ( Mustelus canis), Brain Research, 17 (1970) 57--86. 13 Fox, C. A., AND BARNARD, J. W., A quantitative study of the Purkinje cell dendritic branch lets and their relationship to afferent fibers, J. Anat. (Lond), 91 (1957) 299-313. 14 Fox, C. A., HILLMAN, D. E., SIEGESMUND, K. A., ANO DtJTTA, C. R., The primate cerebellar cortex: a Golgi and electron microscopic study. In C. A. Fox AND R. S. SN1OER (Eds. h The Cerebellum, Progress in Brain Research, Vol. 25, Elsevier, Amsterdam, 1967, pp. 174-225. 15 Fox, C. A., SIEGESMUND, K. A., AND DUTTA, C. R., The Purkinje cell dendritic branchlets and their relation with the parallel fibers: Light a,ld electron microscopic observations. In M. M. COHEN AND R. S. SNIDER (Eds.), Morphological and Biochemical Correlates of Neural Activity, Harper and Row, New York, 1964, pp. 112-141. 16 HAMORI, J., AND SZENT/~GOTH'AI,J., Participation of Golgi neurone processes in the cerebellar glomeruli: An electron microscope study, Exp. Brain Res., 2 (1966) 35-48. 17 HOUSER, G. L., The neurons and supporting elements of the brain of a selachian, J. comp. Neurol., 11 (1901) 65-175. 18 LARSELL,O., [J. JANSEN (Ed.)], The Comparative Anatomy and Histology of the Cerebellum from Myxinoids through Birds, Univ. Minnesota Press, Minneapolis, 1967, 291 pp. 19 LLIN.g,S, R., NICHOLSON, C., FREEMAN, J. A., AND HILLMAN, D, E., Dendritic spikes and their inhibition in alligator Purkinje cells, Science, 160 (1968) 1132-1135. 20 NICHOLSON, C., AND LLINAS, R., Inhibition of Purkinje cells in the cerebellum of etasmobranch fishes, Brain Research, 12 (1969) 477-481. 21 NIEUWENHUYS,R., Comparative anatomy of the cerebellum. In C. A. Fox AND R. S. SNIPER (Eds.), The Cerebellum, Progress in Brain Research, Vol. 25, Elsevier, Amsterdam, 1967, pp. 1-93. 22 PAUL, D. [-I., Parallel fibre response in the elasmobranch cerebellum, J. Physiol. {Lond.), 202 (1969) 64P-65P. 23 RAM6N Y CAJAL, S., Histoh~gie du Syst~me Nerveu,t de l'Homme et des Vertdbrds, Vol. 2, Maloine, Paris, 1911, 993 pp. 24 SCHAPER, A., The finer structure of the selachian cerebellum (Mustelus vulgaris) as shown by chrome-silver preparations, J. comp. Neurol., 8 (1898) 1-20. 25 TSUKAHARA, N., TAao~iKovX, J., AND ECCLES, J. C., The responses of granule cells in the cerebellum of Mustelus canis, Biol. Bull., 135 (1968) 440.
Brain Research, 17 (1970) 87-102
BRAIN RESEARCH
RESPONSES OF THE G R A N U L E CELLS OF THE SELACHIAN C E R E B E L L U M ( M U S T E L U S CANIS)
J. C. ECCLES, H. T~BORfKOV/~ AND N. T S U K A H A R A
Department of Physiology, State University of New York at Buffalo, Buffalo, N.Y., and .~4arine Biological Laboratory, Woods Hole, Mass. (U.S.A.) (Accepted June 15th, 1969)
INTRODUCTION
In their structural details, somata, dendrites and axons, the granule cells of the selachian cerebellum very closely resemble the granule cells of the mammalian cerebellum 17,18,z1,z4. In their general morphological arrangement they offer peculiar advantages for physiological investigation. Instead of the widely dispersed arrangement in which the granule cell layer is coextensive with both the molecular layer and the intervening layer of Purkyn~ cell somata, the selachian granule cells are concentrated in two cords lying adjacently on each side of the midline, and following parasagittally all the cortical folding, as is illustrated in Fig. 1B of the preceding paper 12. In the dogfish used in our experiments these cords were 2-3 mm in diameter, so it was easy to insert a microelectrode into a granule cell cord and record responses of granule cells virtually uncontaminated by the responses of other nervous elements of the cerebellum. It was found that, as a consequence, the responses of granule cells could be investigated more effectively than in the mammalian cerebellum. The granule cords also have a relatively sparse population of so-called Golgi cells, but these cells differ from the mammalian Golgi cells in that their dendrites are restricted to the granule cords (cf. ref. 12, Fig. 2), whereas in the mammal the dendrites widely ramify in the molecular layer. It was of interest therefore to discover if the selachian Golgi ceils resembled mammalian Golgi cells in having an inhibitory action on granule cells. A preliminary account of these investigations has been publishedZL METHOD
The general experimental procedures have been described in the preceding paper r*. The arrangements of the stimulating and recording electrodes are shown diagrammatically in Fig. I B, which is a drawing of a transverse section of the posterior lobe of the dogfish cerebellum along a plane indicated in Figs. IA and B of the preceding paperlL Usually the microelectrode was inserted perpendicularly to the dor-
Brain Research, 17 (1970) 87-102
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Fig. 1. Transverse sections of the cerebellum. A, Drawing of the transverse section of the cerebellum of Scyllium at the level of the acoustico-lateral commissure. The dorsal (era. gr.) and ventral granule cell cords are seen. The pathways of the fibers entering the cerebellum from the peduncle are well shown and form the stratum fibrosum (str. fibr.) deep to the Purkyn~ cell layer and the molecular layer (str. tool.). Also shown is the cerebellar ventricle (v. cb.) the fourth ventricle (V. 4) and the spino-cerebellar (tr. sp. cb.) and olivo-cerebellar (ol. cb.) tracts (modified from Larsell)lL B, Diagrammatic representation of the transverse section of the posterior lobe of the cerebellum showing the recording microelectrode (ME) in the dorsal granule cord. Mossy fibers (M.F.) are drawn in the stratum fibrosum on the route to the granule cell cords and ending synaptically on granule cells (GrC). The axons of the granule cells bifurcate to form the parallel fibers (PF) that are transversely directed in the molecular layer (M.L.). Also shown is the layer of Purkyn?: cells (PC) and the end of the concentric stimulating electrode is drawn symbolically in the stratum fibrosum (Stim. El.). sum o f the cerebellum strictly in a p a r a s a g i t t a l plane. F o r o p t i m a l r e c o r d i n g from the g r a n u l e cell c o r d it was usually located a b o u t 1 m m from the midline. A t depths up to a b o u t 4 m m it w o u l d be r e c o r d i n g f r o m the d o r s a l cord, b u t on further insertion across the ventricular cavity the ventral c o r d was entered. M o s t o f our investigation was on this c o r d because it gave responses that were well m a i n t a i n e d d u r i n g experiments o f m a n y h o u r s ' d u r a t i o n . The afferent fibers to the granule cells have not been recognized to have the characteristic histological features o f m o s s y fibers 17,24; nevertheless it has been f o u n d t h a t their p h y s i o l o g i c a l action is strictly c o m p a r a b l e to m a m m a l i a n mossy fibers; hence it is c o n v e n i e n t to recognize this h o m o l o g y b y alluding to them as mossy fibers. These fibers enter the cerebellum via the p e d u n c l e a n d course in the s t r a t u m fibrosum between the Purkyn~ cells a n d the ventricular cavity as indicated in Fig. I A. In their f u r t h e r p a s s a g e to the granule c o r d s they are seen to continue j u s t deep to the Purkyn6 cell layer a n d then to t u r n into the g r a n u l e cords where they end by ramifying a m o n g s t the granule cells. A s i n d i c a t e d in Fig. IB, the m o s s y fibers have been excited by applic a t i o n o f c u r r e n t pulses t h r o u g h a concentric electrode t h a t has been inserted into the Brain Research, 17 (1970) 87-102
G R A N U L E CELLS OF SELACHIAN CEREBELLUM
89
stratum fibrosum at a site usually close to its origin from the peduncle. Fig. 1B also shows that the axons of granule cells pass into the molecular layer. By the characteristic T-shaped dichotomy they form the parallel fibers that run transversely in the molecular layer and even cross the midline. RESU LTS
Synaptic excitation of granule cells In Fig. 2A, with progressive increase in the peduncular stimulation, from 9 to 36 V, there is paripassu an increase in size of a characteristic potential wave form - an initial diphasic (positive-negative) spike and a later slow negative potential with a relatively fast rising phase and a slower decay. With stronger stimulation (45-90 V) there is further increase in both the initial diphasic spike and the slow negative potential, but a negative spike potential is superimposed on its rising phase. There is a close similarity to the simplest potentials produced in a mammalian granular layer by a mossy fiber volley TM. ]-he initial diphasic spike can be attributed in part to the mossy fiber volley, and its synaptic excitatory action on the granule cells would give the slow negative wave, the spike being dt~e to the generation of impulse discharges. Fig. 2A was recorded at a depth of 9.1 mm from the dorsum of the posterior lobe and so would be deep in the ventral cord (cJi Fig. 1). Fig. 2B illustrates part of a depth profile of potentials evoked by a 63 V stimulus and recorded along the microelectrode track that included the recording site for Fig. 2A. More superficially than 9.1 mm thele was a progressive decline of the response, which otherwise had approximately the same time course. More deeply, at 9.5 mm, there is the sign of a reverse polarity for the spike discharge, which would be expected for the concentration of impulse discharges propagating in the granule cell axons on t h e way to the deep molecular layer (U~ Fig. 1B). Finally at 9.7 mm there was a large positive potential in each of the three superimposed traces. Presumably this later spike is generated by a Purkyn6 cell excited by the parallel fiber volley. At 0.4 mm deeper still all potentials were greatly reduced, as would be expected in the deep molecular layer. Fig. 2C illustrates for this same experiment a series of potentials generated at a depth of 4.35 mm in the dorsal granule cord by double peduncular stimulation. It is seen that the second stimulus produced quite a large negative wave at the shortest interval (3 msec) and there was virtually a full-sized wave at 4 msec. The only significant difference was the shorter time course of the second response, and this can be seen to advantage at testing intervals of 12, 16 and 32 msec. Fig. 3 gives an example in another experiment of a depth profile of the granule cell potentials recorded in the ventral cord. As in Fig. 2B both the slow negative wave and the superimposed spike reached a maximum at about the same depth, 8.9-9. I ram. At 9.5 mm the initial negative wave appeared to be terminated suddenly by a reversed potential, which can be attributed to the propagation of impulses more deeply along the granule cell axons. At still deeper recording sites, the potentials were virtually reversals of the negative wave recorded at the optimal sites. Evidently the field potential
Brain Research, 17 (1970) 87-102
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Fig. 5. Synaptically evoked responses of single granule cells. Mossy fiber volleys were evoked as usual by the concentric stimulating electrode in the stratum fibrosum (of. Fig. IB) and the recording microelectrode was carefully adjusted so as to record selectively from a single unit deep in the ventral granule cord at a depth of 10.13 ram. In A are the responses evoked by stimuli ofO.2 msec duration and the indicated voltages. Each record of B resembles the fellow record of A except that there were two stimuli of the same voltage as in A, and 7 msec apart. In C there were two stimuli of strength 90 V and at various intervals as indicated. In D the response to a single stimulus of 72 V was tested after slight movements of the microelectrode as described in the text. Same voltage scale throughout, but the sweep speed was changed as indicated by the time scales.
spike potentials of one could not be recorded to give such a large spike both absolutely (almost 1 mV) and relatively to the other cells. However, such a selective recording required very careful positioning of the microelectrode, and was secured only in the stable mechanical conditions provided by deep recording in the ventral granule cord. Moreover very small movements of the microelectrode greatly reduced the size of the spike potential, as may be seen in Fig. 5D. Withdrawal of the microelectrode from the depth of 10.13 mm in two successive steps of 10 #m each resulted in a response with a series of small spikes, none of which could be identified with the large spike. With reversal of the movement the spike returned at I0.12 and 10.13 mm but it was greatly depressed at 10.14 ram, I0/~m below the original recording site. Fig. 5A shows that the increased size of the mossy fiber volley provided by the stronger peduncular stimulus resulted in an increased response of the two granule cells Brain Research, 17 (1970) 87-102
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Fig. 6. Antidromic activation of granule cells. A is a diagrammatic illustration of the experimental arrangement with a surface stimulation of the parallel fibers (P.F.) laterally on the dorsum by a concentric electrode (Stim. El.). The microelectrode is seen recording from the granule cells in the dorsal granule cord (Gr. Cord), M.F. and P.A. are mossy fibers and Purkyn6 cell (P.C.) axons. In B are the responses evoked by the stimuli shown in volts and of 0,2 msec duration and recorded in the dorsal granule cord at 3.25 mm depth. The microelectrode track was about 1 mm from the midline and the surface stimulating electrode was about 2 mm laterally thereto. There was DC recording, stars and dots signalling respectively Purkyn~ cell and granule cell spike potentials.
under observation, i.e. spatial summation of two or more impulses gave a n increased synaptic excitatory action on these granule cells. Fig. 5B illustrates the summational effect of two mossy fiber volleys at 7 msec apart, and of the same size as for the corresponding single stimulation in A. There was always an increased response to the double stimulation, but with 90 V strength the effect was more than double, both for the large spike and the small spike. The specimen records of Fig. 5A and B were chosen from a larger number - - 3 or 4 each for the single and double at each strength. The mean number of spikes shows that at several strengths the double stimulation was more than twice as effective as the single, the ratios of double/single being respectively: 1.3/0.8; 2.0/1.0; 3.0/1.2; 3.0/1.7; 4.0/1.8; 4.0/2. l; 6.0/2.0 for stimulus strengths of 27, 36, 45, 54, 63, 72 and 90 V. The high ratios provide further evidence :for temporal facilitation of the excitatory synaptic action of mossy fibers on granule cells (of. Fig. 4). Since with 90 V stimulation there was a large temporal facilitation of spike discharges at 7 msec interval in B, it was of interest to investigate the time course of this facilitation. In C with double stimulation there was an increased response (3 spikes) to the second volley even at 72 msec test interval, and at briefer intervals the test volley evoked even four or five spike discharges. This evidence for temporal facilitation is thus in good agreement with Fig. 4 in showing facilitation of granule cell discharge at test intervals up to 70 msec.
Brain Research,17 (1970) 87-102
GRANULE CELLS OF SELACHIAN CEREBELLUM
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Antidromic excitation of granule cells The large pure c o n c e n t r a t i o n of granule cells in the granule cords gives opportunity for investigating the events occurring when impulses propagate antidromically in the axons of granule cells, i.e. from parallel fibers and down the stem of the T - j u n c t i o n to the granule ceils. With the m a m m a l i a n cerebellum there was only a single presumed example of the a n t i d r o m i c invasion of a granule cell 7. With the much more favorable a r r a n g e m e n t in the selachian cerebellum, massive a n t i d r o m i c invasion of granule cells was regularly observed. The experimental a r r a n g e m e n t is shown in Fig. 6A, where a concentric electrode either on the surface or very superficially in the molecular layer
Brain Research, 17 (1970) 87-102
96
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was used for exciting parallel fibers and the microelectrode was inserted into the dorsal granule cord. Strong stimuli (up to 90 V and 0.2 msec duration) had to be applied in order to excite the very thin non-medullated parallel fibers, and as a consequence the deeper-lying axons of Purkyn6 cells were also excited. In Fig. 6B weak stimuli evoked an initial spike and then a slower and larger spike (starred) which may be identified as due to antidromic invasion of Purkyn6 cells (cf. ref. 12). Stimuli of 54-90 V evoked in addition a much later spike (dotted) with a negative summit at a latency of about 10 msec. This spike response was much later than the spike response of granule cells produced by orthodromic excitation via mossy fibers, which had a latency of less than 3 msec to the summit in Figs. 2A, B. 3 and 4 despite the longer conduction pathway. However, this long latency would be expected for the spike potential produced by the antidromic invasion of granule cells because the conduction velocity in their axons, the parallel fibers, is only about 0.2 m/sec 22, es, which is in agreement with the value of 0.3 m/sec lbr the mammalian parallel fiberse, 6. When double stimuli were applied at various intervals, it was fbund that, alter antidromic invasion, the granule cells recovered fast¢r than the Purkyne cells. This can be seen in the specimen records of Fig. 7A, where there was already a large second granule spike (indicated by dot) at the briefest testing interval (2 reset) and at 12 msec the recovery was almost complete, whereas at that test interval the second Purkyn6 response was merely the very small spike indicated by the star. The time course of the recovery of the Purkyne cell response is plotted in Fig. 7C. there being full recovery in 40 msec, which is in precise accord with the recovery illustrated in the previous paper re. The much earlier recovery of the antidromic invasion of granule cells is seen in Fig. 7B, where it was virtually complete in 15 msec. Fig. 7C shows another feature that would be expected when the conditioning stimulus very effectively excited parallel fibers, namely a slight facilitation at 40-80 msec followed by a slight depression. "l-hese effects have been attributed to an admixture of two opposing synaptic actions on the Purkyne cells, namely a direct excitation by the parallel fiber impulses and an indirect inhibition via the pathway: parallel fiber impulses to stellate cells that in turn give inhibitory synapses to Purkyn6 cells le. Antidromic invasion of granule cells is indicated in Fig. 8 for two other experiments. Since the recording microelectrode was much more medially placed in tile dorsal granule cord, the Purkyn6 spike potential was small in the series o f A ~',ld B, and undetectable in C and D, where the recording was by an electrode accurately placed in the midline, so as to be remote from Purkyn~ cells (cJ~ Fig. I B). The depth profile in A shows a diphasic (positive-negative) spike potential when the electrode was relatively superficial (2.0-3.2 mm), while more deeply the negative spike potential was earlier. A possible explanation is that the deeper granule cells have thicker axons and hence there is a briefer antidromic conduction time despite the longer conduction distance. This explanation is in line with the histological observation in the mammalian cerebellum that the deeper granule cells have larger axons that give the larger parallel fibers deep in the molecular layer 13,~5,23. The double stimulation series (B) reveals a recovery curve of the antidromic spike potential of the granule cells corresponding to that plotted in Fig. 7B. Brain Research, 17 (1970) 87-102
97
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Fig. 8. Antidromic activation of granule cells. There is in A a depth profile of DC recorded responses evoked by antidromic activation of Purkyn6 cells and granule cells, indicated by stars and dots respectively. The recordings were in the dorsal granule cord about 1 mm from the midline and at the indicated depths, and the responses were evoked by stimuli of 72 V for 0.2 msec. In B there was double stimulation, each stimulus being 72 V for 0.2 msec and the recording at a depth of 3.4 mm in the track of A. C and D resemble A and B, but are from a different experiment, with recording in the midline so as to elimiaate the field potentials of Purkyn~ cell spikes. In D there are responses to double stimuli 54 V for 0.2 msec recorded at a depth of 2.8 ram, all stimuli being indicated by arrows. There was DC recording throughout Fig. 8. In the depth profile o f C, there is the surprising observation that the a n t i d r o m i c spike potential reversed to a large positive wave at the deepest recording sites o f the dorsal granule cord. The most p r o b a b l e e x p l a n a t i o n is that the m i c r o e l e c t r o d e had injured some granule cells in that region, with the consequence that their spikes were being recorded as positive potentials. Again in D there is seen to be the typical fast recovery o f D anule cell a n t i d r o m i c response, which was already considerable at the briefest testing interval (5 msec), and a l m o s t fully recovered by 20 msec. DISCUSSION in general the granule cell responses described in this p a p e r c o n f o r m e d with Brain Research, 17 (1970) 87-102
98
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those described for the mammalian cerebellum7, lo, but in several respects the present investigation was more illuminating because of the peculiar advantages deriving from the concentration of the granule cells in the parasagittal cords. For example, spatial and temporal facilitation are well illustrated in Figs. 2A, 4 and 5A-C and antidromic invasion in Figs. 6-8. With double antidromic activation granule cells display a more rapid recovery lhan Purkyn6 cells. A second response was evoked at only 2 msec interval (Fig. 7) and full recovery occurred by 20 msec. It seems that refractoriness after the initial spike response provides an adequate explanation of a depression with such a relatively fast recovery. By contrast after Purkyn~ cell activation, the recovery time of about 40 msec indicates that, superimposed on the initial refractoriness, there is a more prolonged depression such as would result from after-hyperpolarization. The rapid recovery of granule cells also is displayed by the very short intervals between successive spike discharges, which were as brief as 3 msec in Fig. 5 for example. With powerful synaptic stimulation or double antidromic stimulation Purkyn~ cells have discharged at intervals as brief as 5 msec (cfi ref. 12, Figs. 5 and 6A). A most interesting discovery with respect to the granule cell responses has not yet been referred to because it is a negative finding, namely that there has been no trace of a synaptic inhibitory action on granule cells. Such an inhibitory action was a very prominent feature of the mammalian cerebellum, and with some confidence has been attributed to the Golgi cells of the granular layera,7,1°, H. Favorable conditions for detection of inhibitory action on granule cells would be provided by double mossy fiber stimulation, but, as shown in Figs. 4 and 5, even when the stimulus interval was only a few milliseconds, there was no trace of a depression of the granule cell spike discharge evoked by the second volley. Favorable conditions would also be provided by double parallel fiber stimulation, because the so-called Golgi cell of the granular layer conceivably could be synaptically excited by impulses antidromically propagated in the axons of granule cells. However. as shown in Figs. 7B and 8B and D, after the initial recovery from refractoriness there was no inhibition of the antidromic response of the granule cells. In contrast to the mammalian cerebellum the so-called Golgi cells of the selachian cerebellum have their dendritic branches restricted to the granular layerlV,2t,24; hence it would be expected that the main excitatory input would be from the mossy fibers, which are known to provide a subsidiary excitation in lhe mammal 5,1°,16. Nevertheless, conditioning by a mossy fiber volley failed to produce any depression of the granule cells. It could of course be suggested that the conditioning mossy fiber volley had an inhibitory action indirectly via Golgi cells as well as a direct excitatory action, and that the latter was more powerful and just as prolonged, hence the absence of any overt inhibition in such tests as are illustrated in Figs. 4 and 5. It is certainly important to have electronmicroscopic examination of the mossy fiber-granule cell synapses in order to see if there are superimposed synapses homologous with the Golgi cell inhibitory synapses in the mammaP 4,16. Fig. 9 shows diagrammatically the neuronal connectivities and actions that we postulate for the cortex of the selachian cerebellum. The features of this diagram are Brain Research, 17 (1970) 87-102
GRANULE CELLS OF SELACHIANCEREBELLUM
P
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Fig. 9. Diagram of neuronal connections. Diagrammatic representation of the dorsum of a transverse section in order to show the neuronal connections that are postulated on the basis of the known anatomical structures and in order to explain the responses reported in this paper and the preceding paper. Synapses are drawn in conventional manner, and presumed inhibitory cells and their inhibitory synapses are shown in black. M.F., mossy fiber; C.F., climbing fiber, note the branching remote from their terminals; Gr.C., granule cell; P.F., parallel fiber with synapses on Purkyn~ cells, P.C., and stellate cells, S.C. Further description is given in the text.
based both upon anatomical investigations 1,17,1s,21,24 and upon our present electrophysiological study 12, there being one exception in that the Purkyn~ cells are shown as being inhibitory solely on analogy with their function in the m a m m a l 5. The input pathway by the climbing fiber is branched in accord with the findings in the previous paper, and there is, as in the mammal, applied to each Purkyn~ cell only one climbing fiber with its extensive synaptic contacts. The input pathway via the mossy fibers gives converging synaptic excitation on the granule cells, and the granule axons bifurcate to form parallel fibers that excite Purkyn~ cells via spine synapses and also stellate cells that in turn give inhibitory synapses on the dendrites o f the Purkyn6 cells. After extensive branching this mossy fiber pathway exercises a limited convergent action on each granule cell, but there would be an enormously greater convergence in the next stage, namely parallel fibers to the spines o f the Purkyn6 cell dendrites. It should be noted that, as in the mammal, the Purkyn6 dendrites branch extensively to form a kind o f leaflet orthogonal to the direction o f the parallel fibers, so giving maximal opportunity for convergent excitation, but this cannot be depicted in the plane o f the diagram Brain Research, 17 (1970) 87-102
100
J. ¢:. E('( LES et al.
which cuts across the dendritic leaflets. The histological evidence is that the axon of a stellate cell does not extend fal beyond the region of its soma and dendrites; hence it would be expected that the Purkyn6 cells excited by a beam of parallel fibers would also be inhibited by those stellate cells excited by that same beam. There would not be the extensive lateral inhibition that is such a feature in the mammalian cerebellum, where basket cells exert an inhibitory action up to 1 mm transversely from the beam of excited parallel fibersS, 9. We have as yet made no attempt to test for this presumed absence of lateral inhibition. In summary, Fig. 9 discloses that already in its evolutionary development the cerebellum had achieved most of the characteristic features of the neuronal machinery in the mammalian cerebellum 5. There appear to be only three missing features: the powerful feedback inhibition on granule cells that is mediated by the parallel fiber excitation of Golgi cell dendrites in the molecular layer; the powerful lateral inhibition that is exerted on Purkyn6 cell somata by the basket cells through their extensive transversely directed axons; the inhibition by axon collaterals of Purkyn~ cells 5, s These investigations on the connections and functions of the neurons of the selachian cerebellum are of interest in relation to the evolutionary development of the cerebellum. At this early stage there are already almost all of the neuronal elements of the highly developed cerebella of mammalia. The mossy fiber-granule celt-parallel fiber-Purkyn6 cell and the climbing fiber-Purkyn6 cell pathways are fully developed except that there probably is less convergence of parallel fibers on Purkyn~ cells. In contrast to these excitatory pathways the inhibitory pathways are less developed, but there is good evidence for an inhibitory action on Purkyn~ cells by the mossy fibergranule cell-parallel fiber-stellate cell pathway 12. Though there appears to be the homologue of the Golgi cell in the granular layer, no inhibitory action by it on the mossy fiber-granule cell synapses has yet been demonstrated. Another inhibitory deficiency arises from the absence of Purkyn5 axon collaterals. It can be postulated that all the component cells of the mammalian cerebellum are represented at least primordially in the selachian cerebellum, evolution merely resulting in the development of synaptic connections, so enhancing their functional design and exploiting potentialities latent in this neuronal machine. Some of the stellate cells developed into basket cells by growing long axons transversely across the folium and so achieving a very effective functional contact (the baskets) with the somata of the Purkyn~ cells. The dendrites of the Golgi cells very largely grew up into the molecular layer where they were able to secure very effective synaptic excitation by the parallel fibers, and p a r i passu their axons branched extensively in the granular layer so that the Golgi cells became very effective inhibitol s of the granule cells. The Purkyn6 cells grew axon collaterals that developed extensive inhibitory synaptic connections on several types of cell in the cerebellar cortex. We therefore postulate that the essential neuronal elements of the cerebetlar cortex were present in the primordial ancestors of the selachians, and that evolution resulted in the realization of the latent potentialities. This concept of evolution contrasts with the postulate of Llin~ts et al. 19 that inhibition first developed in the reptilian cerebellum because it was not demonstrated in the cerebellum of anura. Recently Nicholson and Llin~is'-'° report Brain Research, 17 (,1970) 87-102
GRANULE CELLS OF SELACHIAN CEREBELLUM
101
the existence of Purkyn~ cell inhibition in the elasmobranch czreb:!lum, and attempt to develop an evolutionary explanation by which this may be accommodated to the absence of inhibition in the anuran cerebellum. However, as stated in the preceding paper t'-), the question of inhibition in the anuran cetebzllum is still opzn. SUMMARY
The granule ceils of the selachian cerebel um are concentl ated in two parasagittal cords closely adjacent on either side of the midline, which is an arrangement that gives very favorable conditions for selective recording from granule cells by an extracellular microelectrode. A mossy fiber volley generates a negative potential wave wilh the time course and depth profile expected for the synaptic excitatory l esponses of granule cells. With large volleys, tile spike responses of granule cells are superimposed thereon. With two mossy fiber volleys at intervals up to 70 lnsec, there is facilitation of the spike discharge, and this has been studied in unitary recordings from granule cells which are found to respond by brief repetitive discharges. When the axons of granule cells (the parallel fibers) are excited by a surface electrode, the field potentials in the granule cord show that there is antidromic invasion of the granule cells. After this invasion the recovery is faster than after the antidromic invasion of Purkyn~ cells, being complete within 20 msec. There has been no evidence for an inhibitory action of the so-called Golgi cells on the granule cells. The physiological findings on the neurons of the dogfish cerebellar cortex have been correlated with the histological data, and this correlation has been displayed diagrammatically. ACKNOWLEDGEMENT Our grateful thanks are supported this project that was Lectureship by one of us (J. C. from the National Institute of N0822101.
extended to the Grass Foundation who generously carried out during the tenure of an Alexander Forbes Eccles). This work was partially supported by a grant Neurological Diseases and Stroke, Grant No. R01-
REFERENCES 1 ARIENSKAPPERS,C. U., HUBER, G. C., AND CROSBY, E. C., The Comparative Anatomy of the Nervous System of Vertebrates Including Man, Vol. 2, Macmillan, New York, 1936, pp. 696-1239. 2 Dow, R. S., Action potentials of cerebellar cortex in response to local electrical stimulation, J. Neurophysiol., 12 (1949) 245-256. 3 ECCLES,J. C., The Physiology of Nerve Cells, Johns Hopkins Press, Baltimore, 1957, 270 pp. 4 ECCLES,J. C., The Physiology of Synapses, Springer, Heidelberg, Berlin, G6ttingen, 1964, 316 pp. 5 ECCLES,J. C., ITO, M., AND SZENTfiIGOTHAI, J., The Cerebellum as a Neuronal Machine, Springer, Heidelberg, Berlin, G6ttingen, New York, 1967, 335 pp. Brain Research, 17 (1970) 87-102
102
,~. ~'
~3 ~ 1.1~ C," '~i,'
6 ECCLES, J. C., LLINAS, R., AND SASAKI, K., Parallel fiber stimulation and the resp~)asc~ induced thereby in the Purkinje cells of the cerebellum, Exp. Brain Res., I (1966) 17 39. 7 ECCLES, J. C., LLINAS, R., AND SASAKI,K., The mossy fiber-granule cell relay in the ~.:ereb~'lium a n d its i n h i b i t i o n by G o l g i cells, Exp. Brain Res., 1 (1966) 82-101. 8 ECCLES, J. C., LLINAS, R., AND SASAKI, K., The action of antidromic impulses o~ the ccrebeilar Purkinje cells, J. Physiol. (LonJ.), 182 (1966) 316-345. 9 ECCLES, J, C., SASAKI, K., AND STRATA, P., The profiles of physiological events produ,ced by a parallel fiber volley in the cerebellar cortex, Exp. Brain Res., 2 (1966) 18-34. 10 ECCLES, J. C., SASAKI,K., AND STRATA,P., The potential fields generated in the cerebzllar cortex by a mossy fiber volley, Exp. Brain Res., 3 (1967) 58-80. 11 ECCLES, J. C., SASAKI, K., AND STRATA, P., A comparison of the inhibitory actions of Golgi ceils and of basket cells, Exp. Brain Res., 3 (1967b) 81-94. 12 ECCLES, J. C., T.~BORIKOV.~, H., AND TSUKAHARA, N., Responses of the Purkyn~ ceils of a selachian cerebellum ( Mustelus canis), Brain Research, 17 (1970) 57--86. 13 Fox, C. A., AND BARNARD, J. W., A quantitative study of the Purkinje cell dendritic branch lets and their relationship to afferent fibers, J. Anat. (Lond), 91 (1957) 299-313. 14 Fox, C. A., HILLMAN, D. E., SIEGESMUND, K. A., ANO DtJTTA, C. R., The primate cerebellar cortex: a Golgi and electron microscopic study. In C. A. Fox AND R. S. SN1OER (Eds. h The Cerebellum, Progress in Brain Research, Vol. 25, Elsevier, Amsterdam, 1967, pp. 174-225. 15 Fox, C. A., SIEGESMUND, K. A., AND DUTTA, C. R., The Purkinje cell dendritic branchlets and their relation with the parallel fibers: Light a,ld electron microscopic observations. In M. M. COHEN AND R. S. SNIDER (Eds.), Morphological and Biochemical Correlates of Neural Activity, Harper and Row, New York, 1964, pp. 112-141. 16 HAMORI, J., AND SZENT/~GOTH'AI,J., Participation of Golgi neurone processes in the cerebellar glomeruli: An electron microscope study, Exp. Brain Res., 2 (1966) 35-48. 17 HOUSER, G. L., The neurons and supporting elements of the brain of a selachian, J. comp. Neurol., 11 (1901) 65-175. 18 LARSELL,O., [J. JANSEN (Ed.)], The Comparative Anatomy and Histology of the Cerebellum from Myxinoids through Birds, Univ. Minnesota Press, Minneapolis, 1967, 291 pp. 19 LLIN.g,S, R., NICHOLSON, C., FREEMAN, J. A., AND HILLMAN, D, E., Dendritic spikes and their inhibition in alligator Purkinje cells, Science, 160 (1968) 1132-1135. 20 NICHOLSON, C., AND LLINAS, R., Inhibition of Purkinje cells in the cerebellum of etasmobranch fishes, Brain Research, 12 (1969) 477-481. 21 NIEUWENHUYS,R., Comparative anatomy of the cerebellum. In C. A. Fox AND R. S. SNIPER (Eds.), The Cerebellum, Progress in Brain Research, Vol. 25, Elsevier, Amsterdam, 1967, pp. 1-93. 22 PAUL, D. [-I., Parallel fibre response in the elasmobranch cerebellum, J. Physiol. {Lond.), 202 (1969) 64P-65P. 23 RAM6N Y CAJAL, S., Histoh~gie du Syst~me Nerveu,t de l'Homme et des Vertdbrds, Vol. 2, Maloine, Paris, 1911, 993 pp. 24 SCHAPER, A., The finer structure of the selachian cerebellum (Mustelus vulgaris) as shown by chrome-silver preparations, J. comp. Neurol., 8 (1898) 1-20. 25 TSUKAHARA, N., TAao~iKovX, J., AND ECCLES, J. C., The responses of granule cells in the cerebellum of Mustelus canis, Biol. Bull., 135 (1968) 440.
Brain Research, 17 (1970) 87-102