Anatomical, physiological and biochemical studies of the cerebellum from mutant mice. I. Electrophysiological analysis of cerebellar cortical neurons in the staggerer mouse

Brain Research, 98 (1975) 135-147 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

135

ANATOMICAL, P H Y S I O L O G I C A L A N D B I O C H E M I C A L STUDIES OF T H E C E R E B E L L U M F R O M M U T A N T MICE. I. E L E C T R O P H Y S I O L O G I C A L ANALYSIS OF C E R E B E L L A R CORTICAL N E U R O N S IN T H E S T A G G E R E R MOUSE

FRANCIS CREPEL AND JEAN MARIANI* Laboratoire de Physiologie Comparde, Universit~ Pierre et Marie Curie, Paris and Unit~ de Neurobiologie Moldculaire, Ddpartement de Biologie Mol~culaire, Institut Pasteur, Paris (France}

(Accepted April 28th, 1975)

SUMMARY An electrophysiological analysis of cerebellar cortical neurons was performed in staggerer mutant mice on postnatal days 18 to 22, and data were compared with cerebellar activities in normal mice of the same age. Bioelectrical activities were elicited through local (LOC) and juxtafastigial (JF) stimulation. In staggerer mice, parallel fibers (PF) exhibited no major alterations, i.e. they conducted impulses and they activated most of the cerebellar cortical units encountered, but some minor differences existed in comparison with normal mice, i.e. their excitability was reduced, as tested by their refractory period and conduction velocity. On the contrary, Purkinje cell (PC) responses presented marked abnormalities. (1) Depth profiles during antidromic invasion of PC and unitary antidromic responses revealed a lower safety factor than normal in the invasion of the somatodendritic region of PC. In particular, a marked IS-SD delay or block occurred in about 60 of unitary antidromic responses of the mutant mouse. (2) Spontaneous or evoked typical climbing fiber responses (CFR) were never recorded in staggerer mice, despite functional synapses between climbing fibers (CF) and PC disclosed with harmaline injections. Activation of PC via CF under harmaline consisted of rhythmical trains of simple spikes. Finally, most of the PC studied in the staggerer mice were certainly activated via PF, but with a low efficacy. These results suggest that PC are directly affected by the mutation.

* Present address: Laboratoire de Physiologie Compar6e, Universit6 Pierre et Marie Curie, Paris, France.

136 INTRODUCTION

The development of synapses in the nervous system of vertebrates 13,3° as well as invertebrates x,z2 is subject to a stringent genetic control. Nevertheless, in a recent theory of synaptic epigenesis, Changeux and co-workers have proposed that the final connectivity of the neuronal network is modulated within a genetically encoded envelope by its own bioelectrical activity at critical stages of development 3,4. Despite its complex structure the cerebellum of mammals would be suitable for analysis of the respective role of such 'genetic' and 'epigenetic" factors acting throughout maturation: (I) its morphology and physiology are well understood in the adult (refs. in 10, 28); (2) it contains only a few classes of cells but these are repeated a large number of times, thus allowing direct biochemical analysis23,24; (3) in rodents most of its development occurs postnatally and therefore has been extensively studied through histological and electrophysiological methods (refs. in 5); (4) in the mouse several mutations affect the organization of the cerebellar cortex. In this respect, the recessive 'staggerer' (sg) mutation 32 is of especial interest because one of its major phenotypical expressions in homozygous sg/sg animals is the selective and almost complete absence of synapses between cerebellar parallel fibers (PF) and Purkinje cells (PC) 18,3°,34. This failure occurs despite the presence of attachment plates between these two elements at early developmental stages 33,34. The understanding of this striking abnormality requires a precise knowledge of the phenotypical expressions of the mutation in the pre- and postsynaptic structures involved in this defect. On the basis of anatomical observations it has been shown that PC are much more affected than PF at least during the first three postnatal weeks, and it has been proposed that PC would be the primary target of the mutation 3°,34. The progressive disappearance of granule cells and PF would in turn be attributed to a transsynaptic retrograde degeneration 3~. The aim of the present study was to reinvestigate this question by other methods, i.e. electrophysiologically, with particular emphasis on the properties of PF and PC. It will be shown that PC are much more affected than PF. Preliminary results of this work have been already reported 7,2,~'. METHODS

C57B1/6J mice originating from the Jackson Laboratory (Bar Harbor, Maine, U.S.A.) were raised at the Institut Pasteur. Homozygous mutants sg/sg were obtained by intercrossing heterozygous + +sg/dse ~-, d ('dilute') and se ('short ear') being two genetic markers located near the sg ('staggerer') locus. The mutant mice were distinguished from their littermates by clinical features as reported elsewhere 3°,:~l. Normal mice were recognized on the basis of their pate color. Normal and staggerer mice were used for electrophysiological studies on postnatal days 18 to 22, i.e. at the time of weaning, because staggerer mice generally did not survive after the end of the first month. Staggerer mice body weights ranged from 4 to 7 g, compared with 6-9.5 g in normals.

137

lOmsec

4 msec

Fig. 1. Responses evoked by local (LOC) stimulations on beam in the cerebellar cortex of the mouse. All traces were recorded in the molecular layer except B1, B~, El, E2, and F, picked up in the Purkinje cell (PC) layer. Left column: normal mice. A illustrates the parallel fiber (PF) volley, followed bya slow negative wave (arrow). BI: unitary response of a PC driven by a threshold stimulation. B2: same PC as in B1 with a stronger stimulus that now evokes a burst of 3 simple spikes. Middle and right columns: staggerer mice. C illustrates the same type of experiment as A. Note that the PF response and the slow negative wave (arrow) are conserved in staggerer mice. The broken horizontal line emphasizes the slow negative component following the PF volley. D~ : fast speed record of the PF volley in another experiment, showing the triphasic (positive-negative-positive) shape of the response. D2: same response as in D~ but it was preceded by a conditioning PF volley of the same magnitude, revealing the refractoriness of these fibers. E~: superimposed unitary responses of a PC to LOC stimulation on beam. The PC was identified by its antidromic invasion after JF stimulation (E~). F: maximum response of an unidentified cortical neuron through LOC stimulation on beam. Horizontal calibration bar below the right column is 2 msec in E~, 1 msec in E~ and 10 msec in F. Negativity is upward for field potentials and downward for unitary responses.

A f t e r initial ether anesthesia, a n i m a l s were p a r a l y z e d with gallamine triethiodide (Flaxedil 60 mg/kg) injected i.p., a n d were artificially respired. In a few cases s o d i u m p e n t o b a r b i t o n e was a d d i t i o n a l l y used, as indicated at the a p p r o p r i a t e places in the results. T h e physiological c o n d i t i o n s o f the a n i m a l s during the experiments were c o n t r o l l e d b y m o n i t o r i n g the e l e c t r o c a r d i o g r a m , a n d b o d y t e m p e r a t u r e was k e p t at 37 °C. The vermis was e x p o s e d , the d u r a removed, a n d a b i p o l a r stimulating electrode was inserted in the region o f the fastigial nucleus ( J F stimulation). T h e electrode consisted o f two silver wires (each 50 # m in diameter) insulated except at the tip a n d j o i n e d side b y side with Epon. T h e p o s i t i o n o f the stimulating electrode was confirmed at the end o f experiments by an electrolytic lesion (300/~A; 40 sec). A n o t h e r , identical stimulating electrode was a p p l i e d on the surface o f the vermis ( L O C stimulation), 270 or 500 # m laterally to the microelectrode, d e p e n d i n g on the e x p e r i m e n t (see Results). Extracellular field potentials and u n i t a r y responses were r e c o r d e d with glass m i c r o p i p e t t e s filled with 1.5 M NaC1 a n d methyl blue ( D C resistance 1.5-5 Mf~). T h e p o s i t i o n o f the tip o f the m i c r o e l e c t r o d e was d e t e r m i n e d by i o n t o p h o r e t i c injection o f methyl blue 8~. M o v e m e n t s o f the tissues were minimized b y covering the structures with 4 ~ a g a r in 1 0 ~ saccharose solution.

138 RESULTS

I. Electrophysiological properties of parallel fibers In normal mice LOC stimulation applied laterally to the recording site (stimulation 'on beam') evoked in the molecular layer a triphasic positive - negative - positive field potential (Fig. I A) which was identical to that described in the cat under the same experimental conditions10; it was therefore interpreted as being due to the activation of parallel fibers (PF). This response was followed by a negative slow wave (Fig. 1A) timed with unitary responses recorded in the Purkinje cell (PC) layer (Fig. IB1 and 1B2). As in other mammals lo, the slow component was attributed to the synaptic activity of cerebellar neurons excited via PF. In staggerer mice LOC stimulations on beam evoked in the molecular layer a response similar to that in normal mice (Fig. 1C). However, the absolute refractory period of PF, measured by double stimulations applied through the same electrode (Fig. 1D1 and 1D2) was significantly longer (P = 0.05) in mutant mice (mean value m = 2,81 msec, n ~- 27, S.E.M. ~- 0.13) than in normal mice (m ~ 1.61 msec, n ~- 22, S.E.M. ~- 0.08). Conduction velocities of PF were determined by successively applying along the axis of the folium two identical stimulation pulses at 270 and 500 #m from the recording microelectrode. These velocities differed significantly (P = 0.05) mean value being 0.258 m/sec in mutant mice (n -= 16, S.E.M. = 0.016) and 0.361 m/sec in normal mice (n = 14, S.E.M. = 0.018). I1. Electrophysiological properties of Purkinje cells (1) Antidromic activation (a) Field potentials. In normal mice JF stimulations evoked at the depth of the PC layer a complex field potential. The initial component of the response exhibited a biphasic positive-negative shape (Fig. 2A) and had a short latency of about 0.5 msec. This biphasic wave followed high frequency repetitive stimulations, up to 500/sec, and was therefore identified with the PIN1 complex due to PC antidromic activation 1°. In staggerer mice JF stimulations also evoked the P1N1 response (Fig. 2B) but the absolute refractory period of PC, measured with two consecutive shocks, was longer (P = 0.02) in mutant mice (mean value m = 1.88 msec, n = 16, S.E.M. -- 0.05) than in normal mice (m = i.25 msec, n == 26, S.E.M. = 0.04). In addition the evolution of the antidromic field response with depth was also disturbed in staggerer mice. For each track, field potentials were recorded in the superficial and in the deep (i.e. sheltered) cortex corresponding to the same folium. The axis of the microelectrode was carefully adjusted to be orthogonal to the surface of the folium. During each penetration the depth of PC layers were determined in the superficial and in the deep cortex by electrophysiological criteria (maximum size of unitary antidromic responses upon the N1 wave) and a deposit of methyl blue (Fig. 2C and 2D) was made in the superficial or in the sheltered cortex just at the level at which the N1 wave reversed into a positive wave (see below). After iontophoretic

139

A

B

C

O. . V % . . , . ~

106

"-~ 200. Q_ bd 306

D

400,

"v

I!

2 msec

Fig. 2. Evolution with depth of the antidromic field responses evoked in the cerebellar cortex through juxtafastigial (JF) stimulation. A: normal mice. B: staggerer mice. In each column the intensity of stimulation was kept constant from top to bottom and superimposed traces were recorded at indicated depths from the surface in both the superficial and the sheltered cortex (full explanation in text). In normal mice the negative component of the antidromic response (N1 wave) was present at all depths except 600 #m (PC layers at 230/tm and 410/~m; arrows). In staggerer mice the N1 wave reversed into a positive wave in both superficial and sheltered cortex at the level of PC somas (located respectively at 120/~m and at 310/tm; arrows). C and D: examples of methyl blue deposition at the tip of the microelectrode when the Nt wave reversed into a positive wave in the sheltered cortex of a normal (C) and a staggerer (D) mouse. Magnifications are × 42 in C and x 77 in D. Arrows in C and D emphasize the presumed axis of the track of the microelectrode during penetration (see text). injection, a n o t h e r recording was m a d e before m o v i n g the m i c r o e l e c t r o d e to check t h a t the field response was unchanged, i.e. always at the reversal p o i n t o f the N1 wave. O n the basis o f the methyl blue m a r k a n d o f the o r t h o g o n a l direction to the surface o f the folium in histological slides (Fig. 2C a n d 2D), the tracks were r e c o n s t r u c t e d a n d they were only r e t a i n e d for analysis when their axis deviated by less t h a n 20 ° f r o m a direction o r t h o g o n a l to the plane o f each P C layer (15 t r a c k s in n o r m a l a n d 12 in m u t a n t mice); real deviations d u r i n g p e n e t r a t i o n s could be slightly undere s t i m a t e d o r o v e r e s t i m a t e d by this m e t h o d . In n o r m a l mice (Fig. 2A), the N1 wave was always p r e s e n t at all depths o f the superficial cortex. Its a m p l i t u d e progressively increased f r o m the u p p e r d o m a i n o f the m o l e c u l a r layer (Fig. 2A, 0/~m) to deeper regions, reaching a m a x i m u m located at the level o f P C s o m a o r P C axons (Fig. 2A, 300 #m). W h e n the m i c r o p i p e t t e entered the deep cortex, i.e. the inverted face o f the folium, the a m p l i t u d e o f the N1 wave did n o t change significantly in the g r a n u l a r layer. Then, in 20 % o f the t r a c k s the N1 wave reversed into a positive wave at the level o f P C somas, the positivity persisting t h r o u g h the whole m o l e c u l a r layer o f the sheltered cortex. In all o t h e r cases, the reversal o f

140

Number,

AI

÷

4

lmsec

of

ceils

1

~°~6 °'

012 0',¢

0'2

04

08

08

m sec

I

~2

"12

Fig. 3. Unitary antidromic responses of Purkinje cells (PC) evoked by juxtafastigial (JF) stimulation. A1 and A2: normal mice. In A1 several sweeps were superimposed to illustrate the fixed latency of the response with slightly suprathreshold stimulation at I/sec. Arrow: presumed IS-SD inflexion. A2: same response as in A1 but with repetitive stimulations at 120/sec. The latency of the IS component remained constant but the amplitude of the response gradually decreased. B and C: staggerer mice. Unitary antidromic responses in the superficial cortex. B: same parameters of stimulation as in A1 to show the constant latency of the IS component and the variable delay or even the block of the SD component (arrow). C: unitary antidromic response of another PC driven by a repetitive stimulation at 40/sec. Histograms: distributions of the latencies of the PC antidromic responses in normal (upper histogram) and in staggerer mice (lower histogram). the N1 wave occurred in the sheltered m o l e c u l a r layer, generally in its outer half, as revealed by the methyl blue m a r k m a d e at this level (Fig. 2C). In the superficial cortex o f staggerer mice a d e p t h profile o f the N1 wave resembling that o f n o r m a l mice was encountered in only 50~o o f p e n e t r a t i o n s ; in all other cases the N1 wave was mainly restricted to the g r a n u l a r layer and reversed into a positive wave at the level o f P C somas (Fig. 2B, 120 #m), or just above. In the sheltered cortex, the Na wave always reversed into a positive wave at the level o f P C s o m a s (Fig. 2B and 2D), thus confirming their position as electrophysiologically determined. (b) Unitary antidromic responses. In normal mice J F stimulation evoked extracellular unitary responses upon the N1 wave, and their a n t i d r o m i c nature was established on the basis o f their short and fixed latency even with repetitive stimulation at m o r e than 100/sec a n d up to 300/sec (mean value o f latencies m - - 0.67 msec, n .... 56, S.E.M. = 0.03 (Fig. 3A1 and 3A2). The a n t i d r o m i c spike usually exhibited a discrete inflexion on its rising phase (Fig. 3 A 0 , p r e s u m a b l y due to the I S - S D delay s,9. This inflexion became more a p p a r e n t with repetitive high frequency stimulation (Fig. 3A2). However, the S D c o m p o n e n t followed stimulation rates up to 100300/sec. A m o n g a total n u m b e r o f 103 neurons, 56 were a n t i d r o m i c a l l y excited and consequently identified as PC (Fig. 3, upper histogram). In mutant mice unitary antid r o m i c responses were also recorded with J F stimulation (Fig. 3B a n d 3C) from 46 o f 181 neurons tested. These 46 cells were subsequently identified as PC. Latencies o f the a n t i d r o m i c responses were slightly longer t h a n those in n o r m a l mice (Fig. 3, lower histogram), despite the reduced size o f the cerebellum in staggerer mice. P r o b a bly because o f the l o c a t i o n o f the microelectrode with regard to PC soma, an I S - S D delay was seen on unitary a n t i d r o m i c responses for only 31 o f the 46 identified PC.

141 In about 60 % of these 31 PC the duration of the I S - S D delay was greatly lengthened and the SD component shifted in latency or failed to occur at low frequency stimulation (Fig. 3B). This was markedly strengthened during repetitive stimulation with a frequency as low as 30-50/sec (Fig. 3C). The antidromic responses of the other PC appeared much more similar to those in normal mice as regards their I S - S D delay, and the ability of the SD component to follow high frequency stimulation.

(2) Orthodromic activation of PC (a) JF stimulation. In normal mice JF stimulation evoked in PC two classes of unitary orthodromic responses. The first one (Fig. 4A) consisted of one or two simple spikes with a latency range of 3.4-5.6 msec (mean value = 4.67 msec, n = 1 I, S.E.M. = 0.24) and it was considered as being due to PC activation via the M F granule cells - PF pathway 10. Of 67 PC tested, 11 exhibited this type of response. The second class of activity consisted of typical all-or-none climbing fiber responses (CFRs) 1° (Fig. 4B and 4C); their latency ranged from 1.6 to 7.6 msec (mean value m ~- 4.7 msec, n = 59, S.E.M. = 0.2) (Fig. 6); 59 of the 67 PC tested presented such CFRs. In staggerer mice JF stimulation also evoked extracellular unitary orthodromic responses in PC but they exhibited a striking abnormality: we were unable to identify any evoked or spontaneous CFRs. Accordingly, when recording extracellularly close to PC soma we never saw in staggerer mice the classical 'inactivation response '1~

A.f B

C _

10 m s e c

Ill

6 m sec

Fig. 4. Orthodromic responses evoked through juxtafastigial (JF) stimulation in the cerebellar cortex of the mouse. Superimposed sweeps in all traces except Ex, E2, E8 and E4. Left column: normal mice. In A the antidromic spike was followed by a simple spike due to PC activation via the mossy fiber (MF) pathway. In B the antidromic spike was followed by an all-or-none climbing fiber response (CFR). C: another example of antidromic response followed by a C F R with a typical inactivation response (see text). Middle and right columns: staggerer mice. Dx, D2 and Da: orthodromic activation of a PC through progressively increasing stimulations from top to bottom. In D1 the stimulation was at threshold and the response consisted of two single spikes. In D2 and D3 the interspike interval within the response decreased and the inactivation of the second spike increased (dot) when the intensity of stimulation became greater. An antidromic response was evoked in Ds with a marked IS-SD delay (arrow). El, E2, E3 and E4: same experiment with another PC. The stimulation was also progressively increased from top to bottom. Note the increase of the number of spikes within the response.

142

.,i ! i_/il_J i i W

-W ~ "

Ii" ~ll-'lit

11-W

llt l.i i/lil li.ljl lrl

111111II117

] i._ t1_I t] li !. J_JJ l lr [r 11!-111 F llir lrrf,

i+

1 sec.

Fig. 5. Activity of cerebellar cortical neurons after harmaline injection. A1-B2: normal mice. Al: rhythmical bursting activity of a Purkinje cell (PC) at 5/sec. A2: superimposed successive fast speed records of the same PC as in A1 showing that each burst was composed of a typical climbing fiber response. Each sweep was triggered by the first spike of the response. BI : rhythmical activity of an unidentified cortical neuron (see text). The frequency was the same as in At and each burst consisted of simple spikes as shown by fast speed records in B2 (one sweep, triggered by the first spike). C1-D: staggerer mice. In C1 a clear-cut rhythmical effect of harmaline is apparent in a PC. The corresponding fast speed record in C2 (one sweep) shows that the bursts were composed of simple spikes. D illustrates the more irregular firing of another PC under harmaline. Horizontal calibration bar below fast speed records: 2 msec in A2 and 10 msec in B~ and C2. Vertical calibration bar: 2 mV in all records. which a p p e a r s in n o r m a l cerebellum as a large positive wave (Fig. 4C). All PC tested r e s p o n d e d with only simple spikes with latencies ranging from 1.7 to 10 msec. In 16 o f the 33 PC studied the o r t h o d r o m i c response consisted o f one, or sometimes two, single spikes at all stimulus intensities used. In 17 other PC, the simple spike response was graded, i.e. the n u m b e r o f spikes (Fig. 4E) a n d / o r the firing frequency within the response (Fig. 4D) increased with intensity o f stimulus. (b) Activation by harmaline. As m e n t i o n e d above, the study o f o r t h o d r o m i c activation o f PC t h r o u g h J F stimulation had failed to d e m o n s t r a t e an excitatory inp u t o f climbing fibers (CF) onto PC in the mutant. Studies o f M o n t i g n y and Lamarre 27 a n d Llinas a n d V o l k i n d 21 have shown that h a r m a l i n e induces a r h y t h m i c discharge in C F originating from the inferior olive. W e have therefore a t t e m p t e d to reveal a C F input onto P C with h a r m a l i n e injection (50 mg/kg i.p.), followed by b a r b i t u r a t e anesthesia ( N e m b u t a l 30 m g / k g i.p.) in o r d e r to depress PC activation via the M F - P F p a t h w a y 14. PC were recorded in the sagittal region o f the vermis. In normal mice a steady rhythmical discharge o f C F R s a p p e a r e d in every PC recorded 4-5 min after h a r m a l i n e injection, with a frequency o f 4--8/sec d e p e n d i n g on the e x p e r i m e n t (Fig. 5 A1 a n d 5A2). R h y t h m i c a l bursts o f simple spikes at the same frequency were also recorded from some unidentified cerebellar cortical neurons (Fig. 5B1 and 5B2), i.e. cells which showed t h r o u g h J F stimulations neither a n t i d r o m i c responses n o r evoked or s p o n t a n e o u s C F R . These cells might well be inhibitory interneurons2L In staggerer mice a rhythmical activation o f identified P C was also o b t a i n e d by

143 harmaline injection, but it consisted of bursts of 2-8 simple spikes at 3-7/sec (Fig. 5C1) instead of typical CFRs, the interspike interval within the burst being 4-5 msec (Fig. 5C2). Only 40 ~ of the PC studied had a rhythmical discharge as steady as in normal mice under harmaline, strongly suggesting they were innervated by CF. The firing of the others was rather irregular (Fig. 5D), although more synchronized than before harmaline injection. (c) L O C stimulation. In normal mice LOC stimulation on beam evoked unitary responses in PC. They were timed with the slow negative wave following the PF response in the molecular layer (see Fig. 1A, 1B1 and 1B~). The number of spikes varied gradually with the intensity of stimulus (Fig. 1B1 and 1Bz) from one spike at threshold to 3 or 4 spikes in the maximum response. As in the cat ~0, this response was interpreted as being due to activation of PC via PF, the repetitive firing of PC in our experimental situation being similar to that previously described in unanesthetized preparationsL In staggerer mice a slow negative wave also followed the PF volley evoked in the molecular layer by a LOC stimulation 'on beam' (Fig. 1C). In addition, 12 of the 15 identified PC studied exhibited a unitary response in this situation with the same latency as that of the slow negative wave recorded in the molecular layer. At all intensities of stimulation tested it only consisted of one, or sometimes two, single spikes (Fig. 1El). These responses were never obtained with LOC stimulation 'off beam'. It must be emphazised that LOC stimulation 'on beam' also routinely evoked similar responses from unidentified cortical units. Only on rare occasions (Fig. IF) did they consist of more than one or two spikes. A number of them are probably unidentified PC, but others might be inhibitory interneurons since histological data 3°, as,34 have demonstrated that their synapses with PF are grossly unaffected by the mutation. As a whole, these results strongly suggest that PF are still able to activate PC in the mutant. DISCUSSION

For several decades mice have been used in neurobiological research, especially because of the presence of well-defined inbred strains and because of the discovery of numerous neuropathological mutants al. However, data were mainly obtained through histological and biochemical methods (see for example refs. 23, 30 and 33), the mouse seeming too small for refined electrophysiological studies. Obviously the lack of anatomofunctionai correlations at the cellular level constitutes a severe restriction for a greater use of this species. In a previous report in 19737, and in the present work, we demonstrate for the first time that such a detailed electrophysiological analysis is quite possible. As regards the staggerer mouse, one of the major expressions of the mutation is the important and selective diminution of the number of synapses between cerebellar PF and PC 30,33. Until now, the origin of this defect has been investigated by histological methodslS,~9,30,33, 34 which suggested that PC would be primarily affected by the mutation. The aim of the present study was to test this hypothesis with electrophysio-

144 logical data focussed upon the pre- and postsynaptic components of the synapse affected. For this reason we shall successively discuss abnormalities found in PF and PC responses.

I. Parallelfibers No major alterations in bioelectrical properties of PF appear in staggerer mice at the ages studied. In particular, our results demonstrate that PF are able to conduct impulses and to establish functional synapses with cortical units, including PC. A functional linkage between PF and PC in staggerer mice was unexpected on the basis of previous histological data18,29, 30. The activation of PC via PF disclosed in the present study was much weaker than normal and could be satisfactorily explained by the small number of atypical synapses between PF and PC recently identified in these animals 33. The reduced excitability of PF in the mutant, as manifested by changes of their refractory period and conduction velocity, might be due to the delayed maturation of the cerebellar cortex in the staggerer mouse 30 since similar features of PF responses were found at an earlier stage in the normal cerebellum 5. As a whole, these findings suggest that PF are not greatly affected by the mutation at the ages studied.

H. Purkinje cells Identifiable evoked or spontaneous CFRs were never observed in staggerer mice in our experimental conditions. However, functional connections between CF ~.nd PC exist, as revealed with harmaline, but they occur with certainty in only 40'~ of the PC tested. The responses consist of rhythmical trains of simple spikes instead of typical CFRs. The origin of this abnormal activation of PC by CF would lie either in the CF pathway or in PC. The normal rhythm of bursting induced by harmaline in staggerer mice, and the normal morphology of inferior olivary neurons in these animals 3'~ do not support the first hypothesis. On the other hand, the typical features of the CFRs in normal cerebellum are attributed to the mainly dendritic origin of this response12, 26. The defective activation of PC by CF could, therefore, be due to abnormalities of PC dendrites revealed by histological data 33. Anyway, the abnormal responses of PC to CF inputs do not seem to be due to the reduced number of P F - P C synapses since typical CFRs persist in the cerebellum rendered agranular during infancy 6,2°. The electrical excitability of PC as tested by their antidromic invasion appears modified in staggerer mice. Firstly, on the basis of previous interpretation of the depth profile of the N1 wave in mammals 19, our results reveal that the active antidromic invasion of PC tends to be restricted to the level of PC axons and somas in staggerer mice, whereas it reaches an important part of PC dendrites in normal mice. This suggests that the safety factor for the invasion from PC soma to dendrites 11 is lower than normal in the mutant. This could be related to the paucity of PF inputs onto these cells in staggerer

145 mice 30, according to data obtained in cats 11. It must be emphasized that our interpretation of depth profiles is valid in staggerer mice since the required arrangement in space of cortical neurons16,17,19 is grossly retained at the age studied; PC somas are located in the superficial zone of the granular layer and most of their dendritic branches spread into the molecular layer along a vertical direction 33. Secondly, the important delay, or the block, in the IS-SD transmission which occurs in about 60 ~ of the unitary antidromic responses in staggerer mice demonstrates that the antidromic invasion of PC soma in the mutant also has frequently a lower safety factor than normal. Such a delay or a block in the IS-SD transmission is not seen either at early developmental stages in normal cerebellum when PF have established only a few connections with PC 5, or in X-irradiated agranular cerebellum where PF are missing 6. Similarly it is not encountered in the deafferented cat cerebellum 9. On the basis of these data, the reduced number of synapses between PF and PC in the mutant seems insufficient to explain this frequent, important IS-SD delay. To summarize, our results clearly establish that at the ages studied (1) PF have grossly normal responses and make enough synapses onto PC to activate them; (2) PC responses are markedly affected and some of these defects, such as the abnormal responses to CF inputs and the frequent low efficacy of the IS-SD transmission during their antidromic invasion, do not seem to be due to the reduced number of P F - P C synapses. Rather, they could represent a direct phenotypical expression of the mutation. This can be related to the pronounced decrease of one class of membrane proteins that have been found in PC of staggerer mice 24. Finally, our findings are consistent with the view that the mutation affects primarily PC. The early expression of the mutation on cell proliferation in the external granular layer described by Yoon z7 cannot be used to preclude this interpretation since affected PC could influence neighboring germinal cells at early developmental stages, and later on could transsynaptically 34 induce the degeneration of granule cells that has been observed in this animal 8o. ACKNOWLEDGEMENTS

We thank Drs J. P. Changeux, J. Mallet, C. Sotelo, and A. Zamora for useful comments during the experiments and preparation of the manuscript. We also thank Prof. P. Laget for his kind help with bistological controls. Mice were raised with the assistance of M. Huchet and J. L. Guenet at the Institut Pasteur (grants from the I.N.S.E.R.M., from the C.N.R.S. and from the D.G.R.S.T.). This work was supported by the I.N.S.E.R.M. (Grant 3878/01) and by the Fondation pour la Recherche M6dicale Frangaise.

REFERENCES 1 BENZER, S., Genetic dissection of behavior, Sci. Amer., 229 (1972) 24-37. 2 BLOEDEL,J. R., AND ROBERTS,W. J., Functional relationship among neurons of the cerebellar cortex in the absence of anesthesia, J. NeurophysioL, 32 (1969) 75-84.

146 3 CHANGEUX,J. P., COURREGE, PH., AND DANCHIN, A., A theory of the epigenesis of neuronal networks by selective stabilization of synapses, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2974-2978. 4 CHANGEUX, J. P., ET DANCHIN, A., Apprendre par stabilisation sSlective de synapses en cours de dSveloppement. In E. MORIN AND M. PIATTELLI - PALMARIN1(Eds.), L'unit~ de I'Homme, Seuil, Paris, 1974, pp. 320-350. 5 CREPEL, F., Excitatory and inhibitory processes acting upon cerebellar Purkinje cells during maturation in the rat. Influence of hypothyroidism, Exp. Brain Res., 20 (1974) 403-420. 6 CREPEL, F., DELHAYE-BOUCHAUD, N., AND LEGRAND, J., Electrophysiological analysis of the circuitry and of the corticonuclear relationships in the agranular cerebellum of irradiated rats, Arch. ital. Biol., in press. 7 CREPEL, F., MARIANI, J., KORN, H., ET CHANGEUX, J. P., Electrophysiologie du cortex cSrebelleux chez la souris mutante 'Staggerer'. C. R. Acad. Sei. (Paris'), 277 (1973) 2761-2763. 8 ECCLES, J. C., The Physiology of Synapses, Springer-Verlag, Berlin, 1964. 9 ECCLES, J. C., LLIN~,S, R., AND SASAKI, K., The action of antidromic impulses on the cerebella," Purkinje cells, J. Physiol. (Lond.), 182 (1966) 316-345. 10 ECCLES, J. C., ITO, M., AND SZENT~,GOTHAI,J., The Cerebellum as a Neuronal Machine, SpringerVerlag, Berlin, 1967. 11 ECCLES, J. C., FABER, D. S., AND T~,BOfdKOVA, H., The action of a parallel fiber volley on the antidromic invasion of Purkyn6 cells of cat cerebellum, Brain Research, 25 (1971) 335-356. 12 FUJITA, Y., Activity of dendrites of single Purkinje cells and its relationship to so-called inactivation response in rabbit cerebellum, J. Neurophysiol., 31 (1968) 131-141. 13 GLUECKSOHN-WAELSCH, S., Genetic factors and the development of the nervous system. In H. WAELSCH (Ed.), Biochemistry of the Developing Nervous System, Academic Press, New York, 1955, pp. 375-396. 14 GORDON, i . , RUBIA, J. F., AND STRATA, P., The effect of Pentothal on the activity evoked in the cerebellar cortex, Exp. Brain Res., 17 (1973) 50-62. 15 GRANIT, R., AND PHILLIPS, C.G., Excitatory and inhibitory processes acting upon individual Purkinje cells of the cerebellum in cat, J. Physiol. (Lond.), 133 (1956) 520-547. 16 KWAN, H. C., AND MURPHY, J. T., A basis for extracellular current density analysis in cerebellar cortex, J. NeurophysioL, 37 (1974) 170-180. 17 KWAN, H. C., AND MURPHY, J. T., Extracellular current density analysis of responses in cerebellar cortex to climbing fiber activation, J~ Neurophysiol., 37 (1974) 333-345. 18 LANDIS, D., Cerebellar cortical development in the staggerer mutant mouse, J. Cell Biol., 51 (1971) 159a. 19 LLIN/,S, R., Extracellular field analysis in the central nervous system. In J. I. HUBBARD, R. LLIN~,S, D. M. J. QUASTEL AND E. ARNOLD (Eds.), Eleetrophysiological Analysis" of" Synaptie Transmission, Monographs of the Physiological Society, London, 1969, pp. 265-294. 20 LLIN~.S, R., HILLMAN, D. E., AND PRECHT, W., Neuronal circuit reorganization in mammalian agranular cerebellar cortex, J. Neurobiol., 4 (1973) 69-94. 21 LLIN~,S, R., AND VOLKIND, R. A., The olivo-cerebellar system: functional properties as revealed by harmaline induced tremor, Exp. Brain Res., 18 (1973) 69-87. 22 LOPRESTI, V., MACAGNO, E. F., AND LEVINTHAL, C., Structure and development of neuronal connections in isogenic organisms: cellular interactions in the development of the optic lamina of Daphnia, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 433M.37. 23 MALLET, J., HUCHET, M., SHELANSKI, M., AND CHANGEUX, J. P., Protein differences associated with the absence of granule cells in the cerebella from the mutant weaver mouse and from X irradiated rat, FEBS Lett., 46 (1974) 243-246. 24 MALLET, J., HUCHET, M., POUGEOIS, R., AND CHANGEUX, J. P., A protein difference associated with defects of the Purkinje cell in staggerer and nervous mutant mice, FEBS Lett., 52 (1975) 216-220. 25 MARIANI, J., ET CREPEL, F., Mise en 6vidence d'anomalies bioelectriques de la r6gion somatodendritique des cellules de Purkinje du cortex c6r6belleux chez la souris mutante 'staggerer', In E. BOESIGER AND J. MEDIONI (Eds.), Colloque sur h's Mdcanis'mes Ethologiques de l'Evolution, Masson, Paris, 1975, in press. 26 MARTINEZ, F. E., CRILL, W. E., AND KENNEDY, T. T., Electrogenesis of cerebellar Purkinje cell responses in cats, J. NeurophysioL, 34 (1971) 348-356. 27 MONTIGNY,C. DE, AND LAMARRE,Y., Rhythmic activity induced by harmaline in the olivo-cerebello-bulbar system of the cat, Brahl Research, 53 (1973) 81-95.

147 28 PALAY,S., AND CHAN-PALAY,V., Cerebellar Cortex: Cytology and Organization, Springer-Verlag, Berlin, 1974. 29 SIDMAN,R. L., Development of interneuronal connections in brains of mutant mice. In F. D. CARLSON (Ed.), Physiological and Biochemical aspects of Nervous Integration, Prentice-Hall, Englewood Cliffs, N.J., 1968, pp. 163-193. 30 SIDMAN, R. L., Cell interactions in developing mammalian central nervous system. In L. G. SILVESTRI (Ed.), Cell Interactions, North-Holland Publ., Amsterdam, 1972, pp. 1-13. 31 SIDMAN, R. L., GREEN, M. C., AND APPLE, S. H., Catalog of the Neurological Mutants of the Mouse, Harvard University Press, Cambridge, Mass., 1965. 32 SIDMAN,R. L., LANE, P. W., AND DICKIE, M. M., Staggerer, a new mutation in the mouse affecting the cerebellum, Science, 137 (1962) 610-612. 33 SOTELO, C., Dendritic abnormalities of Purkinje cells in the cerebellum of neurological mutant mice (weaver and staggerer). In G. KREUTZBERG(Ed.), Proc. Syrup. Physiol. Path. of Dendrites (Advances in Neurology Series), New York, 1975, pp. 335-351. 34 SOTELO, C., AND CHANGEUX,J. P., Trans-synaptic degeneration 'en cascade' in the cerebellar cortex of staggerer mutant mice, Brain Research, 67 (1974) 519-526. 35 SOTELO,C., AND ZAMORA, A., in preparation. 36 THOMAS, R. C., AND WILSON, V. J., Precise localization of Renshaw cells with a new marking technique, Nature (Lond.), 206 (1965) 96-97. 37 YOON, C. H., Developmental mechanisms for changes in cerebellum of staggerer mouse, a neurological mutant of genetic origin, Neurology (Minncap.), 22 (1972) 743-754.