HETEROSENSORY AND HETEROCORTICAL

ACTIVATION OF THE PURKINJE NEURON

Brain .Iesearch I nst i tute University of California, Los hgeles

Los hgeiles, California

C. Batini

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Running T i t l e : Act ivat ion o f the Purkinje Neuron

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D r . C . Ba t in i Space Biology Laboratory, Brain Kesearch I n s t i t u t e Center for the Health Sciences University of Cal i fornia , Los Angeles tos Engeles, Ca l i fo rn ia y3324

1 Heterosensory and Heterocortical Activation of the Purkinje Neuron

by

C. Batini

Brain Research Institute, University of California, Los Angeles

Summary. Convergence of heterogeneous afferents has been described

in the cerebellsr surface with recording of gross evoked potentials. An

hypothesis is presented here that this phenomenon should appear at the

single neuron level in the cerebellar cortex. Steel microelectrodes have

been used for recording the unitary potential in the extracellular field

of the Purkinje neuron as well as of the granular cell.

obtained can be summarized as follows:

The results

1. Phasic activation of peripheral receptors, visua1,acoustic and

somesthetic, as well as electrical stimulation of primary and extraprimary

cortical areas, elicit responses in single granular cells and in single

Purkinje neurons,

of spikes appearing at an appropriate delay and was followed by long-

lasting inhibition. This implies that the three sensory afferent pathways,

as well as the cortico-ponto-cerebellar system, may converge on the single

Purkinje neuron through the polysynaptic intracerebellar circuit activated

The response consisted in both cases of a rapid burst

by the W;SY f:bei afferents.

2. In the Purkinje neuron records, a second spike response shortly

delayed from the mossy fiber'sresponse was observed.

of a single spike appearing on a slow positive wave, with a higher threshold

for activation. This second response in the Purkinje neuron is attributed

to the climbing fiber afferents producing a monosynaptic activation.

It consisted usually

The

2.

a .

-. convergence phenomenon of heterogeneous afferents has also been observed

in this climbing fiber response.

3 . Paired cortical and peripheral stimuli delivered at varying

intervals have shown the existence of two different patterns of interac-

tion at the Purkinje neurons. First, a long inhibitory interaction

lasting for 100-150 msec which has been demonstrated by others, using

direct stimulation of cerebellar system. This study shows that intra-

cerebellar polysynaptic inhibitory system may be activated by more

peripheral stimulations. Second, a facilitatory interaction appeared

at very short interstimulus intervals, and was easily obtained at inten-

sities which elicited no responses when unpaired.

tatory interaction (short interval) was observed only on the climbing

f 1 ber response.

This form of facili-

The results have been interpreted and discussed in accordance with

the available anatomical data.

that cortical and peripheral cerebellar afferents studied here may possibly

feed into precerebellar stations of Convergence, which would be the

sources of the mossy and climbing fibers.

The present work has led to the hypothesis

Key Words: Cerebellum -- Convergence of afferents -- Mossy fiber afferent -- Climbing fiber afferent

The sensory input to the cerebellar cortex, as well as cortico-

cerebellar projections,have been widely investigated by use of gross evoked

potentials (see Dow and krutzi, 1958). Initial studies attempted

a functional classification of the entire cortical surface in terms of

regional localization of afferents. Later studies correlated peripheral

and cortical projections to specific cerebellar regions for particular

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sensory modalities (see 3ow and Woruzzi, 1958).

This concept of regional specificity seems questionable, since

overlap has been found between sensorial fields (Dow, 1342; Fadiga,

Pup1111 and Berger, 1956; Jansen, JF., 1957; Buser and Franchel, 1960;

Batini, Castellanos and Buser, 1964, 1wa) as well as interaction between

heterogeneous afferents (Bremer and Bonnet, 135 1 ; Albe-Fessard and

Szabo, 1954; Levy, Loeser and Koella, 1961; Batini, 1965). More recent

findings have emphasized that,

face, sensory and cortical inputs appear to be largely convergent

(Batini , Caste1 lanos and Buser, 1966a) .

over almost the whole cerebellar sur-

The present study has tested the hypothesis that this "convergence"

first appears at the unitary level.

by recording single units from a particular area of the cerebellar

cortex, while varying the applied afferent stimulus.

strated that heterosensory as well as heterocortical afferents can in-

fluence the firing pattern of the same neuron. This "convergence"

phenomenon will be shown to act on the Purkinje neuron through two

parallel systems, one of which i s postulated to be transmitted indirectly

by the mossy fibers, the other monosynaptically by the climbing fibers.

The interaction patterns between heterogeneous afferents will show

functional differences of the two convergent systems.

The problem has been investigated

It will be demon-

Methods

Thirtyltwo adult cats were used in this experiment: I9 were deeply

anesthetized with chlorolose (70 mg/Kg) , 8 were decerebrated at the

precollicular level under ether anesthesia, and 5 were left intact and

maintained under local anesthesia. All wound edges and pressure points

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were i n f i l t r a t e d w i th procaine ch lor ide solut ion. A l l i n t a c t animals

were immobilized w i t h F laxed i l and maintained by a r t i f i c i a l respi ra-

t ion. An in f ra red CO monitor (Capnograph Godart type CG/58003) was

used to susta in a constant expired C O A large anter io r

craniectomy was performed i n order t o expose the c o r t i c a l surface for

the e l e c t r i c a l st imulat ion.

fo l ium and tuber vermis f o r microelectrode recording and was covered

w i t h zgar.

2 a t 3 to 4%. 2

A small pos te r io r craniectony exposed the

Single e l e c t r i c shocks (0.2 msec, 1 t o 5 vo l t s ) were del ivered t o

surface electrodes 2.0 m apart, t o var ious c o r t i c a l areas, inc lud ing

motor cortex, and primary v isua l and acoust ic areas. Sensory st imola-

t i o n involved shor t f lashes {a f te r a t rop in iza t ion of the pupi I ) , c l i c k s

and subcutaneous e l e c t r i c shock t o the forepaws. The v isua l system i n

the decerebrate animals was act ivated by e l e c t r i c s t imu la t ion of the

an te r io r c o l l i c u l i ( 6a t i n i , Castellanos and Buser, 1966b).

U n i t a c t i v i t y was recorded w i t h s ta in less s tee l microelectrodes,

prepared according t o the method of Green (1958).

d isp lay systems consisted o f a ~6 Grazs preampl i f ier , a 502A Tektron ix

osci l loscope. Traces were photographed w i t h a Grass camera. Spike

a c t i v i t y was recorded on one channel o f a tape recorder (2 channel

Tanberg Model 6) and square waves on the second channel, for st imulus

marking.

Mnemotron computer of average t rans ients (C.A.T. Model 4008).

were prepared o f the spike d i s t r i b u t i o n versus summed per iods of time

fo l low ing or j u s t preceding the stimulus presentation, the stimulus being

del ivered monotonously w i t h per iods between 4-10 sec. A pu lse height

Pmpl i fy ing and

A fu r the r analysis of the evoked f i r i n g was performed using a

Histograms

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I 5 . I - . ~ se lector was interposed between the tape recorder and the Mnemotrom

computer, i n order t o suppress minor spikes (d isc r im ina t ion l eve l ad just -

ab le from 200 mV to 5 V) , t o select e i t h e r p o s i t i v e o r negative spikes,

and t o t ransform the spike i n t o a constant square pulse (adjustable a t

1-3 msec and between 250 mV and 10 V) . One hundred and twenty-seven u n i t s were recorded from the cerebe l la r

cortex, 77 i n anesthetized animals, 23 i n decerebrated preparat ions and

21 i n the intact F l a x e d i l i t e d subjects.

KesQlts

A. Evoked a c t i v i t y i n qranular and molecular layers.

When the microelectrode was inserted i n t o the cerebel lar cortex,

la rge pos i t ive-negat ive or p o s i t i v e spikes were encountered a t a depth

of 0.3 to 0.4 mn corresponding t o the Purk in je c e l l layer. Th is a c t i v i t y ,

which has been shown to der ive from Purk in je neurons (Grani t and P h i l l i p s ,

i956, 1957; Andersen, Eccles and Voorhoeve, 1964), was o f t e n found a t

deeper leve ls depending upon the angle of the v e r t i c a l l y penetrat ing

e lect rode on the varying cerebel lar surface. Using the method f i r s t

described by Grani t and P h i l l i p s (1956), the Purk in je neurons were also

recognized by t h e i r antidromic response to an e l e c t r i c a l shock appl ied i n

the subjacent cerebel lar whi te matter. A t greater c o r t i c a l depths only

small spikes were encountered, usual ly negative i n p o l a r i t y and of shor t

duration. These were in te rpre ted as the a c t i v i t y o f the granular ce l l s .

D i f f e r e n t a f fe ren t s t i m u l i were applied t o a l l u n i t s recorded from e i t h e r

the molecular or granular layer. The most f requent ly e l i c i t e d response

consisted o f a s ing le spike or a short burs t appearing w i t h a f i x e d delay

a f te r the stimulus, fo l lowed by a per iod of suppression of spontaneous

6 . - .

f i r i n g for 100-150 msec. Since a l l a f fe ren t f i b e r s act ivated i n our

experiments have been shown t o end i n the cerebel lar cor tex as mossy

f i b e r s (see Discussion), we would expect t h a t the onset o f response of

the u n i t s i n the granular layer ( f i r s t re lay i n the cerebel lar cor tex

f o r the mossy f i be rs ) to a given stimulus would be shorter than the delay

i n t h e response of a Purk in je neuron (which i s secondari ly act ivated by

the granular c e l l s ef ferents). In f ac t , latency of the responses e l i c i t e d

i n the granular c e l l s was always found t o be a t least 4-5 msec shorter than

the latency o f the responses i n the Purk in je neurons t o the same stimulus.

- - - - - - - - - - - - - - - - - - Place Figure 1 about here

- - - - - - - - - - - - - - - - - - Although i t was more d i f f i c u l t to record from small granular c e l l s

and they generally survived f o r a shorter period, we were able i n some

instances t o t e s t a l l desired s t imul i , and t o observe evoked f i r i n g f o r

eadh st imulus (Fig. 1).

A greater number o f Purk in je neurons has been tested, almost a l l

responding t o the applied s t imu l i . Here again, as shown i n Fig. 2, i t

was poss ib le to e l i c i t a response i n the same u n i t by using e i t h e r

per iphera l or c o r t i c a l s t imu l i .

- - - - - - - - - - - - - - - - - - Place Figure 2 about here

- - - - - - - - - - - - - - - - - - These r e s u l t s were found i n both the anesthetized and non-anesthetized

preparations (Fig. 3 ) .

Purk in je c e l l can be inf luenced by the s p e c i f i c sensory systems as w e l l

They demonstrate t h a t a s i n g l e granular and

.. 7.

as by the cortico-ponto-cerebellar system.

Place Figure 3 about here

A simple hypothesis would be t o a t t r i b u t e transmission of these

heterogeneous impulses to the mossy f i b e r s through the granular c e l l s to

the Purk in je ce l l s . Anatomical substrates f o r such m u l t i p l e connections

between mossy f i b e r s and granular c e l l s have already been emphasized

(Fox and Barnard, 1957; Gray, 1961). However, accurate analysis o f

la tenc ies and evoked f i r i n g patterns of Purk in je neurons i n t h i s study,

has shown more complex evoked a c t i v i t y , f o r which an add i t iona l afferent

system must be postulated.

8. Purk in ie c e l l evoked a c t i v i t y .

i n these uni ts , spontaneous f i r i n g appeared to be s l i g h t l y d i f f e r e n t

i n the three preparat ions studies. The h igh frequency discharges f i r s t

described by Brookhart, Moruzzi and Snider (1950, 1951) were more o f t e n

observed i n both types o f non-anesthetized preparations, although t o a

lesser extent i n the i n tac t F laxed i l i zed preparation. Under ch loro lose

anesthesia, the most frequent pa t te rn o f discharge was a more regular l o w

rate. However, epochs o f randm rapid f i r i n g , fo l lowed by per iods of

slow f i r i n g , or even s i lence appearing i n a very random fashion (see

a lso Grani t and P h i l l i p s , 1956), was seen i n the record o f an i nd i v idua l

c e l l i n a l l three preparations. The superimposed t ime histogram of the

spikes c l e a r l y ex t rac ts the evoked spikes from the background a c t i v i t y

even f o r very h igh frequencies o f spontaneous f i r i n g (Fig. 3 ) .

. - - - - - - - - - - - - - - - - - -

P 1 ace F i gute 4 about here

- - - - - - - - - - - - - - - - - - A simple p a t t e r n o f evoked a c t i v i t y i n a Purk in je c e l l i s seen i n

Fig. 4.

a b u r s t of spikes fo l lowed by a long per iod of suppression o f spontaneous

discharges.

Each stimulus, del ivered consis tent ly every 4 seconds, e l i c i t s

The delay w i t h which d i f f e r e n t u n i t s responded t o a given st imulus

var ied great ly. However, as shown i n Fig. 5, the latencies o f the f i r s t

evoked spike i n 25 neurons t o c o r t i c a l shocks (Fig. 5h) and t o f lashes

(Fig. re), have two p re fe ren t i a l latencies for e i t h e r stimulus.

- - - - - - - - - - - - - - - - - - Place Figure 5 about here

- - - - - - - - - - - - - - - - - - Furthermore, i n several records of a s ing le u n i t i t was possible

t o observe two responses fo l lowing a s ing le a f fe ren t vo l l ey (Fig. 6 and

Fig. 2). These two responses were found t o have the same latencies as

the p r e f e r e n t i a l values shown i n Fig. 5 . Thei r pat terns were o f t e n

d i f f e r e n t , the f i r s t cons is t ing of a very rap id bu rs t of spikes, the second

i n one or two spikes usual ly located i n the ascending p o r t i o n of a

large p o s i t i v e wave (see a lso the " i nac t i va t i on response" and the "D

po ten t ia l " described by Grani t and P h i l l i p s , 1956). A f u r t h e r d i s t i n c -

t i o n between these two responses was establ ished by using d i f f e r e n t

i n t e n s i t i e s of st imulat ion, the Itshort latency'' response having a lower

threshold than the "long latency".

9.

- - - - - - - - - - - - - - - - - - Place Figure 6 about here

- - - - - - - - - - - - - - - - - - That both the responses were generated in the same Purkinje neuron

However, if they is difficult to affirm from an extracellular record.

arose in two Purkinje neurons close together, they must then have

independent responses to a single afferent volley.

been pointed out that Purkinje neurons are activated by the granular

cells. The time of conduction through the granular efferents, the

parallel fibers, has been calculated to be 0.3-0.5 msec (Dow, 1939),

running for a maximal longitudinal length of 3-4 m (Fox and Garnard,

1957; Gray, 1961). The observed intervals between granular and Purkinje

neuron responses to the same stimulus i s consistent only for the first

response seen in the Purkinje neurons. Thus, another afferent system

has to be considered for the second response (see Discussion).

It has already

When tested by applying various stimuli, both responses were acti-

vated, with a comnon latency consistent with the site of stimulation.

More significantly the latency between the first and second responses

were unaltered by the site of stimulation, thus lending support to the

view that the anatomical convergence serves also as a converging

transmission system. However, using paired stimuli at varying inter-

vals, essential differences in the patterns o f interaction between hetero-

geneous afferences have been found.

C. Interaction patterns in Purkinie neurons between heteroqeneous afferents: inhibitory and facilitatory effects.

Paired stimuli with varying intervals were used in this experiment

!O, ..

to study the dynamic interaction between those cortical and peripheral

projections which have been shown to converge on a single Purkinje neuron

of the visuo-acoustic area in the cerebellar cortex. The paired stimuli

Were used in all the possible combinations provided that each of the pair

Place figure 7 about here

As shown in Fig. 7, the spike responses elicited by a cortical test

stimulus were suppressed when preceded by the flash conditioning stimulus.

On reversing the order of stimulation, the cortical shocks appeared to

have the same strong conditioning effect on the flash responses, suggesting

a reciprocal inhibitory interaction between cortical and peripheral

afferents.

broad time separation, with a maximum of 100-150 msec. An identical

inhibitory interaction was always obtained for any pair of stimuli, if

each stimulus elicited a response on the same Purkinje neuron. The long - lasting reciprocal inhibitory effect between the two heterogeneous afferent

volleys has been observed to be effective either on the units responding

at short latency (Fig. 7) or in those responding at long latency (Fig. 80

and 8F).

The mutually inhibitory effects are evident over a relatively

- - - - - - - - - - - - - - - - - - Place Figure 8 about here

- - - - - - - - - - - - - - - - - - I f we now combine the two heterogeneous stimuli at short intervals

a very strong facilitation is obtained. As shown in Fig. 9 the most

j *. I

11.

effective interval between the cortical and peripheral stimuli corres-

ponded to the almost simultaneous appearance of the two expected responses.

The number of spikes elicited by the double stimulation was greater than

the sum of the spikes evoked by the two single stimuli. This effect was

easily obtained when the intensity of both stimuli was lowered to threshold,

and even more easily when the stimuli were presented at subliminal inten-

sities. As shown in Fig. 108 and lOC, either afferent stimulus delivered

separately was effective in producing a response, while their combined

effect elicited a very strong response (Fig. 10D). These findings

appeared to be independent of the anesthetic treatment of the animals

since they have been largely confirmed on the non-anesthetized

preparation (Fig. 10).

. . . . . . . . . . . . . . . . . . . . . Place Figures 9 and 10 about here

. . . . . . . . . . . . . . . . . . . . . Contrary to the inhfbitory effects obtained for both long and short

latency responses, facilitatory interaction was obtained only on the long

latency responses of the Purkinje neurcns. This difference is illustrated

in Fig. 1 1 , showing the histograms of a record in which two responses

(long and short latency) were obtained for the same afferent volley,

The histogram, as expected, shows a bimodal distributlon of the spikes

for a single stimulus (A).

the first response, but, at certain stimulation intervals, strongly en-

hanced the second response (B).

Combining the two stlmul6tions did not modify

- - - - - - - - - - - - - - - - - *

Place Figure 1 1 about here

- - - - _ - - - - - - - - - - - - -

12. . D i scuss ion

The u n i t po ten t i a l s recorded from the cerebel lar cor tex were

c l a s s i f i e d as granular c e l l and Purkinje c e l l a c t i v i t y according t o the

layer i n which they were found. The i d e n t i f i c a t i o n of the Purk in je

neurons as establ ished i n the present work has been discussed i n gre-

vious studies (Granit and Phi 11 ips, 1956; Andetsen, Eccles and Voorhoeve,

1364; Suds and Amand, 1964) and needs no f u r t h s r corrment. But :he iden-

t i f i c a t i o n o f the granular c e l l i s not supportcd by d i r e c t evidence.

The p o s s i b i l i t y ex i s t s for recording f r o m one o f the few s t e l l a t e

neurons w i t h i n the granular lsyer. However, the few d i r e c t a f f e r e r t

f i be rs to the s t e l l a t e c e l l s (Jontov, 1958) have beer. described as

a r r i v i n g from the medulla (Jontov, 1959).

experiments showed u n i t s responding w i t h very sho-t delay t o c o r t i c a l

as we l l as visuo-acoustic afferences i t would appear t h a t they were from

the granular c e l l s . I n the present invest igat ions, the a f fe ren t cerebel lar

pathways act ivated by e i t h e r c o r t i c a l s t imu la t ion or per iphera l v isua l

and acoustic, have been demonstrated t o run through the brachia pont is

(Jansen and Brodal, 1954) and end i n the granular layer as mossy f i b e r s

(Jelgersma, 1917; Lorente de Nd, 1335; Snider, 1936).

spino-cerebellar a f fe ren ts act ivated by somesthetic st imulat ion, end i n

the granular layer as mossy f i b e r s (Miskolczy, 1931; Brodal and Grant,

1962). Thus i n each case the act ivated granular c e l l s t ransmi t ted the

impulses monosynaptically t o the Purkinje neurons. However, the resu l t s

have shown t h a t a s ing le a f fe ren t vo l ley e l i c i t e d two responses i n the

Purk in je neurons. The fo l low ing reasons demonstrate tha t these two

responses are completely independent: (i) a s ing le modal i ty a f fe ren t

Since t h e r e s u l t s of our

S i m i l a r l y , the

13. .- vol ley can el icit the "firstti or the ''second1' separately; (i i) when

recorded simultaneously they may show different sensitivities to the

intensity of stimulation; ( i i i ) the facilitatory interaction between

heterogeneous afferents occured only on the "second responset'. According

to the conduction time of the efferent fibers from the granular cells,

only the first response can be attributed to the arrival of the impulses

through the mossy fibers.

An explanation for the "second response" requires the existence of

another afferent system.

monosynaptically independent afferences over the climbing fibers.

de Nd (1924) and Mettler and Lubin (1942) claimed that all cerebellar

afferents end as mossy fibers, the climbing fibers belonging only to the

intracerebel lar systems (Carrea, Reissig ad resettler, 1~47; Scheibel and Scheibei,

1954) . However, SzentGothai and Rai kovi ts (1959) using the Nauta-Gyga method, recently demonstrated degenerating climbing fibers after various

extracerebellar lesions. The majority of these fibers seem to originate

in the inferior olive passing through the inferior peduncle, but some

degeneration was also found after lesiw of the brachium pontis and

brachium conjunctivum.

of the Purkinje neurons by afferent stimulations through the climbing

fibers cannot be excluded. This point of view is also supported in experi-

ments by Jansen and Fangel (1961), where two different components of evoked

activity were seen in the Purkinje layer after cortical stimulation: the

first was attributed to the arrival of impulses through the mossy fibers,

the second to those through the climbing fibers.

In fact the Purkinje cell dendrites receive

Lorente

Therefore, the possibility of a direct activation

. 14.

One of the main resu l ts from t h i s study is the suggestion tha t

a f fe ren t vo l leys coming from the ac t iva t ion o f d i f f e r e n t pathways may

converge a t the leve l o f a s ing le neuron i n the cerebel lar cortex.

Other invest igators (Bat in i , Castel lanos and Buser, 1966a) , using the

method o f gross evoked potent ia ls , were able to demonstrate w i t h macro-

electrodes, for a given point on the cerebel lar surface, the coexistence

o f three kinds of convergence: hetero-cort ical convergence (Dow, 1939;

Jansen, Jr. , 1957), hetero-sensory convergence (Bremer and Bonnet, 1951) , and convergence between c o r t i c a l and per ipheral vol leys (hibe-Fessard

and Szabo, 1954; Green, 1958). These resu l ts are here la rge ly confirmed

and extended t o the leve l of s ing le u n i t recordings i n the visuo-acoustic

region where independent systems seem able to converge on the Purk in je

neurons from each source studied.

The problem thus ar ises t o determine, for each of the two a f fe ren t

systems, the s i t e o f convergence of such heterogeneous sources, and

inversely, f o r each source, the locus o f divergence of the two a f fe ren t

cerebel lar systems.

already discussed i n previous work to which the reader i s re fer red

(Bat in i , Castel lanos and Buser, 1966a, 1966b). The hypotheses suggesting

Part o f t h i s complex hodological problem has been

that the pontine nuc!el possess the anatc%!cz? s.ubstrat2 for 8 -,re=tsre-

b e l l a r common relay, serving both per ipheral and c o r t i c a l afferences,

can be maintained as long as no other evidence i s added, This view i s

a r e s u l t o f the f a c t tha t pontine f l be rs have been demonstrated to end

i n the cerebel lar cortex exclusively as mossy f ibers . However, t h i s

system can be read i l y postulated only f o r the convergence of pa r t o f the

heterogeneous impulses a r r i v i n g v i a granular ce l ls ,

An analogous center fo r pre-cerebellar convergence f o r the d i r e c t

a f fe ren ts to Purk in je neurons i s suggested t o l i e i n the i n f e r i o r o l i ve .

It has been shown tha t the main source o f degenerating c l imbing f i b e r s

i n cerebe l la r cor tex ar ises from o l i v a r lesions (Szentagothai und

Rajkovits, 1959). Spinal a f ferents to the i n f e r i o r o l i v e have been

described (Brodal, Walberg and Blackstad, 1959) as we l l as c o r t i c a l

connections a r i s i n g from both primary and extraprimary areas (Mett ler ,

1935a, 1935b; Snider and Barnard, 1949; Walberg, 1956). The l o c a l i z a t i o n

of the af ferent f i b e r s w i t h i n the i n f e r i o r o l i v e i s poss ib ly compensated

by the d i f f u s e d i s t r i b u t i o n o f the o l i vo-cerebe l la r connections (Brodal,

1940a, lwb). Such a system recei.ving a f fe ren ts from spec i f i c pathways

and having m u l t i p l e a f fe ren ts from each subnucleus t o Purk in je neurons,

i s s t rong ly suggestive of a converging relay.

The funct ional s ign i f icance of the heterogeneous convergence a t the

cerebe l la r cor tex has already been pointed out (Bat in i , Castellanos

and Buser, 1366a, 1966b). Here the func t iona l s ign i f i cance o f the

double independent a f fe ren ts v i a Purk in je neurons can be discussed I n

the frame o f resu l t s obtained w i th pa i red s t imu l i .

The long- last ing i n h i b i t o r y e f f e c t described i n our experiments

annears +e 8 fundmental nrnnartv ef e!! afferents eiriyinn i n - “3 -rr--* #.. vr-. -, cerebe l la r cor tex and act ing a t the l eve l o f the Purk in je neurons.

A polysynapt ic i n h i b i t o r y system on the Purk in je neurons and the granular

c e l l s has been recent ly described (Andersen, Eccles and Voorhoeve, 1364;

Eccles, L l i ngs and Sasaki, 1966a, 1966b) t o be found i n the basket and

Golgi c e l l s which are interposed between granular and Purk in je neurons.

I5 . c

Inhibitory effects in this system are assumed to be activated by mossy

fibers. However, evidence exists for a second mechanism for the activa-

tion of this system through the collaterals of the climbing fibers

synapsing on Golgi cells of the granular layer (Scheibel and Scheibal,

Results of the present study have shown that a facilitatory response

may be obtained at the Purkinje neuron if the paired stimuli occur within

a very short period of time. The upper limit for this temporal relation

is probably determined by the transmission time of the polysynaptic

inhibitory system from the Golgi cell to the Purkinje neuron. Since a

collateral pathway i s shown between the climbing fiber and the Golgi

cell, it i s clear that a second ascending volley on the climbing fiber

may find the Purkinje neuron inhibited as a result of the first volley.

This assumption i s strengthened also by our earlier results obtained

by using the gross evoked responses: paired cortical and peripheral

stimuli delivered at short intervals and at low intensity elicited a

very strong facilitation of the late component of a complex cerebeiiar

evoked potential (Levy, loeser and Koella, 1961; Batini, 1965). The

same strong facilitatory effect on the early component of the evoked

p t e n t I a ! I s e!ir! 'ted hy B double shock delivered at very short inter-

vals on the inferior olive (3atini, in preparation).

17.

FOOTNOTES

1. The assistance and support of Dr. W.2. Adey and the facilities

of the Space Oiology Laboratory are gretefully acknowledged.

i s indebted to Mr. R.T. Kado for h i s assistance in the preparation of

th is manuscript. This study was supported in part by t h e AFOSR under

E649(638)-1387, and NASA under NsG 237-62.

The author

FIGURE LEGENDS

Fig. 1. Responses of a granular c e l l t o various s t imu l i i n an anesthetized

animal. A. Single e l e c t r i c shock t o the sigmoid gyrus; 8, to

the l a t e r a l gyrus; C. t o the ectosylv ian gyrus; D. f lash;

E. c l i ck . The s t imu l i are marked by a dot.

Responses t o various s t imu l i obtained i n a Purk in je neuron of

an anesthetized animal. A. Flash; B, c l i c k ; C. subcutaneous

e l e c t r i c shock i n the l e f t forepaw; D. c o r t i c a l st imulus o f the

v isua l cortex; E. of the acoustic cortex; F. of the motor cortex.

Note tha t the " f i r s t " and the "second response" are c l e a r l y

obtained i n a l l records.

Superimposed t i m e histograms o f the spike responses of a Purk in je

neuron i n an i n t a c t F laxedi l ized animal. I n t h i s and fo l lowing

histograms number of spikes i s shown on the ordinate, time i n

seconds on the abscissa, and N equals the number o f s t imu l i

presented. A. Acoustic st imulat ion; €3. somesthetic s t imulat ion

o f the r i g h t forepaw; C. e l e c t r i c shock o f the sigmoid gyrus.

The s t imu l i are del ivered j u s t before 0 time.

'If i rs t " and the "second response" are more c l e a r l y evident i n C.

Record of a Purk in je neuron i n an anesthetized preparation,

A. Responses t o flashes; E. t o e l e c t r i c shock o f the sigmoid

gyrus; C. c l i cks .

responds to each stimulus w i t h a burst o f spikes a t short

latency ( " f i r s t response"), followed by a long per iod o f inh ib i t ion .

Latency d i s t r i b u t i o n of 25 Purkinje neuron responses a f t e r

Fig. 2.

Fig. 3.

Note t h a t the

Fig. 4.

The s t i m u l i are marked by a dot. The u n i t

Fig, 5.

t

s t imu la t ion o f the sigmoid gyrus (A) and f lashes (8).

of u n i t s i s shown on the ordinate, t ime i n 5 msec increments

i s p l o t t e d on the abscissa.

ca lcu lated as a mean of ten presentations of the stimulus,

See t e x t for f u r t h e r explanation,

Double response i n a Purk in je neuron t o f l ash s t imu la t ion ( the

st imulus a r t i f a c t i s marked by a dot) i n a decerebrated animal.

Note the d i f fe rence between the " f i r s t response", which consists

of a rap id burs t o f spikes, and the "second response", which

consis ts of two spikes appearing on the ascending p o r t i o n o f a

slow wave which has been shortened by the t ime constant. See

"D" p o t e n t i a l i n the t e x t for fu r the r descr ip t ion of the slow

poten t ia l , The double response i s followed by a long per iod of

i nhb i i t i on .

Number

The latency o f each u n i t has been

Fig. 6.

Fig. 7. I n h i b i t o r y i n te rac t i on by pai red s t i m u l i i n a Purk in je neuron

of an anesthetized animal. A. Control of the response to a

s ing le f l a s h stimulus ( l e f t side), and an e l e c t r i c shock t s

the acoustic cor tex ( r i g h t side).

response" only. Note the d i f fe rence i n the a r t i f a c t . 8 . L e f t

coiumn, the response to the test stiiittjtii~ (ce i t lcs! s k ~ ~ k ) i s

suppressed by the condi t ion ing ( f lash) st imulus presented a t

increasing in terva ls . I n the r i g h t column an iden t i ca l e f f e c t

i s obtained when the f l a s h i s the t e s t st imulus and the c o r t i c a l

shock i s the condi t ion ing stimulus. C. Control o f the response

t o a s ing le stimulus as i n A, a f t e r t he double s t imu la t ion period,

These e l i c i t the " f i r s t

20 , t

Fig. 8. Superimposed time h stograms showing i n h i b i t o r y i n te rac t i on by

pai red s t imul i . sigmoid gyrus s t imulat ion (2) i n an anesthetized animal.

A. Control for spontaneous a c t i v i t y ; B. responses t o flashes;

C. response to c o r t i c a l shocks; D. i n h i b i t i o n o f the f l a s h response.

The t e s t f l a s h s t imulat ion i s appl ied before the condi t ion ing

st imulus bu t the response t o the t e s t stimulus i s expected

a f te r the cond i t ion ing stimulus response. E. Summation of

the two responses which are expected t o appear simultaneously.

F. i n h i b i t i o n o f the c o r t i c a l t e s t response.

On the "second response", f 1 ashes (1) and

Fig. 9. Superimposed t i m e histograms o f the f a c i l i t a t o r y i n te rac t i on

by pai red s t imu l i i n Purkinje neuron of an anesthetized

animal. 1. Flashes; 2. motor cor tex s t imulat ion. Note the

long latency of the responses.

and t i m e i n msec on t h e abscissa.

Number of spikes on the ord inate

Fig. 10. Superimposed time histograms o f the f a c i l i t a t o r y i n te rac t i on

by pa i red s t i m u l i o f a P u r k i n j e neuron i n a decerebrated

preparation. The s t i m u l i are appl ied sho r t l y before 0 time.

A. Spontaneous a c t i v i t y ; B. subl iminal somesthetic s t imu la t ion

to t h e r i g h t forepaw; C. subi iminai clicks; G. pal ied sci ix i ! : a t

short i n te rva l s a t t h e same i n t e n s i t i e s as i n record C.

Fig. 11. Superimposed time histograms of the f a c i l i t a t o r y i n te rac t i on

by pa i red s t i m u l i on the Ilsecond response" o f a Purk in je

neuron i n an anesthetized animal, 1. Flashes; 2. e l e c t r i c

shock t o the sigmoid gyrus. A. Control before applying the

double st imulat ion. Note the bimodal d i s t r i b u t i o n o f the

spike response due to the appearance o f the "first" and the

"second response" on the unit record. 3 . Paired stimuli at

varying short intervals. The second response only is strongly

facilitated at certain intervals (see second line in B).

C. Control after applying the double stimulation.

22 ,

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