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Vagal and somatic representation by the climbing fiber system in lobule V of the cat cerebellum
Brain Research, 552 (1991) 58-66 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS (100689939116689K
58
BRES 16689
Vagal and somatic representation by the climbing fiber system in lobule V of the cat cerebellum Gang Tong, Lee T. Robertson and John Brons Oregon Health Sciences University, Department of Anatomy, School of Dentistry, Portland, OR 97201 (U.S.A.)
(Accepted 8 January 1991) Key words: Cerebellum; Climbing fiber; Vagus; Autonomic; Somatosensory; Viscerosensory
The organization of the climbing fiber representation of the vagal afferents and the body surface in the vermal and intermediate zones of lobule V was examined in cats anesthetized with a-chloralose. Extracellular single-unit recordings were made from 428 Purkinje cells. Electrical stimulation of the vagus nerve elicited climbing fiber responses in 40% of the cells, most of which had convergent somatic input, Activation of A~ vagal afferent fibers accounted for 65% of the responses, whereas the Ap fibers involved 27% and the C fibers included 8% of the responses. The responses driven by vagal nerve stimulation were encountered throughout the lobule, although a significantly increased representation of the vagus was identified for 3 longitudinal 0.5 mm wide sectors (two in the vermis and one in the intermediate region). In the vermis, the fine-grain organization consisted of a mixture of representations of the various parts of the body surface with and without convergent vagal input, although there was little convergence in the medial vermis where many of the responses were elicited by only vagal nerve stimulation. In the intermediate cortex, most of the vagal climbing fiber representation was convergent with forelimb input. These results suggest that vagal input into the cerebellum could have important modulatory effects on the cerebellar somatosensory input. INTRODUCTION The cerebellum has traditionally been considered important in the regulation and coordination of somatic motor activity, but substantial evidence also exists for a modulation of autonomic functions. Moruzzi 28 first showed that stimulation of the anterior lobe of the cerebellum could inhibit blood pressure and respiratory responses. Since Moruzzi's work, numerous studies have demonstrated that stimulation or ablation of the cerebellar cortex and nuclei can induce a variety of cardiovascular changes, including alterations in blood pressure, heart rate and blood flow 3"5'10'29'30. The cerebellum also receives information from the cardiovascular system via vagal nerve afferents 6'18"24'25'33. However, there are conflicting conclusions on the arrangement of the vagal projection to the cerebellum. Field potentials elicited by stimulation of the vagal nerve have been identified in the medial vermis of the anterior lobe 6'24'25'33, throughout the vermis of sublobule V c and lobule V133, and as a longitudinal strip in the intermediate zone of lobule V TM. H e n n e m a n and Rubia TM suggested that the responses in the intermediate zone represent climbing fiber input whereas the responses in the vermis may be due to far-field potentials originating
in the brainstem. Recently, Perrin and Crousillat 33 made single unit recordings of 23 Purkinje cells in the vermis and identified both climbing and mossy fiber vagal input, although little information was given on the magnitude of the vagal climbing fiber representation or how the climbing fiber vagal representation was distributed in the lobule. Several physiological studies of the climbing fiber input in the anterior lobe have demonstrated an extensive representation of the body surface 15'23'27'34'36.Therefore, it is likely that the vagal climbing fiber input to the anterior lobe will converge with the somatic input. It is necessary to know the extent and distribution of the possible convergence before we can appreciate the functional implications of the vagal input. The examination of the receptive fields of cells with convergent somatic and vagal input may provide some insight on the cerebellum's role in cardiovascular function. The purpose of this study was to re-examine the organization of the climbing fiber representation of the vagus nerve in the vermal and intermediate regions of lobule V by using fine-grain, single'unit mapping procedures. By mapping a large n u m b e r of individual Purkinje cells, it was possible to calculate the regional proportions of the Purkinje cells receiving vagal climbing fiber inputs,
Correspondence: L. Robertson, Department of Anatomy, School of Dentistry, Oregon Health Sciences University, 611 S.W. Campus Drive, Portland, OR 97201, U.S.A.
59 tO d e t e r m i n e
the
spatial
organization
of
the
vagal
r e p r e s e n t a t i o n t h r o u g h o u t l o b u l e V, and to establish the spatial r e l a t i o n s h i p b e t w e e n the c l i m b i n g fiber r e p r e s e n t a t i o n of the v a g u s n e r v e and b o d y surface. MATERIALS AND METHODS The experiments were carried out on 12 cats of either sex, weighing 2.5-4.0 kg. The animals were anesthetized with intravenous injections of a-chloralose (100 mg/kg), followed by supplementary doses (30 mg/kg). Chloralose anesthesia was used instead of barbiturates because small doses of barbiturates can influence normal cardiovascular activity 17a9. Catheters were placed in the femoral vein and artery for administering fluids and drugs and for monitoring the blood pressure, respectively. The rectal temperature was maintained between 37 and 38 °C. The animal was placed in a stereotaxic apparatus and the dorsal surface of Iobule V of the cerebellar cortex was surgically exposed. The dura over the anterior cerebellum was removed and the cerebellum was covered with warm mineral oil or Ringer's agar. Tungsten microelectrodes (8-10 MI2 and 1000 Hz) and conventional electrophysiological equipment were used to extracellularly record single Purkinje cells, which were distinguished by spontaneous climbing fiber responses TM.The microelectrode placement was controlled stereotaxically and the penetrations were made in the parasagittal plane at a 15-20 ° angle from the vertical with an average interpretation distance of 400 gm. Each electrode penetration traversed through several Purkinje cell layers and, at each layer, an attempt was made to isolate a Purkinje cell and elicit a climbing fiber response by vagal or somatic stimulation. A Purkinje cell was considered to be responsive if a climbing fiber response was elicited in 70% of the trials. Typically, responsive units exceeded the 90% level of discharge probability. The vagus nerve was isolated, bathed in a pool of warm mineral oil, cut caudally, and stimulated at the level of the nodose ganglion with a pair of Ag-AgCl electrodes via a constant current stimulus isolator. In each experiment, compound action potentials (CAPs) were recorded from the vagal nerve with electrodes placed 26-30 mm proximal to the stimulating electrodes. The CAPs were checked several times during each experiment, although the threshold levels were consistently stable. The stimulus level was expressed as multiples of the lowest threshold required to produce a nerve volley. Stimulation consisted of either a single, 0.3 ms rectangular pulse or a train of 3 pulses at 200 Hz, every 4-5 s. The somatic stimulation included taps to the skin surface with hand-held probes, including calibrated yon Frey nylon filaments. Climbing fiber responses elicited by punctate stimuli of less than 2.0 g were considered cutaneous; those requiring more than 2.0 g were defined as deep. The response latencies were obtained by electrically stimulating the cutaneous receptive field with small needle electrodes.
RESULTS
Population studied T h e c l i m b i n g f i b e r r e s p o n s e s o f 428 P u r k i n j e cells w e r e isolated throughout the vermal and intermediate regions of s u b l o b u l e s Vb_ f. Vagal s t i m u l a t i o n successfully elicited c l i m b i n g fiber r e s p o n s e s in 4 0 % (171/428) of t h e i s o l a t e d P u r k i n j e cells. In this g r o u p , 3 2 % o f t h e c l i m b i n g fiber responses
were
by b o t h
vagal
and
tactile
tion. T h e 6 0 % o f t h e cells w i t h o u t v a g a l i n p u t i n c l u d e d 2 0 % (86/428) with c l i m b i n g f i b e r r e s p o n s e s d r i v e n o n l y by tactile s t i m u l a t i o n a n d 4 0 % t h a t w e r e u n r e s p o n s i v e to any s t i m u l a t i o n e m p l o y e d . T h e d i f f e r e n t types o f v a g a l a f f e r e n t fibers w e r e not e v e n l y r e p r e s e n t e d . T h e classification of a f f e r e n t fiber types
that
elicited t h e
climbing fiber responses
was
d e t e r m i n e d by the s t i m u l u s t h r e s h o l d and c o n d u c t i o n v e l o c i t y o f t h e C A P s 31 (Fig. 1). A/~ v a g a l a f f e r e n t s h a d an a v e r a g e t h r e s h o l d o f 70.5 /~A a n d a m e a n c o n d u c t i o n v e l o c i t y of 38.8 m/s. A~ v a g a l a f f e r e n t s r e q u i r e d stimulus
A~!3(27%)
I)
~tl
activated
s t i m u l a t i o n and 8 % w e r e e l i c i t e d o n l y by v a g a l stimula-
B
An
t
In an attempt to identify possible longitudinal zones, the vagal and the somatic elicited responses were segregated into 0.5mm-wide longitudinal sectors. Grouping the data into 0.5-mm-wide sectors permitted the statistical comparison between populations of responses, although a narrow spatial organization or an organization that spans multiple sectors may not be discerned. Differences between the sectors for the number of responses elicited by either vagal or somatic stimulation were analyzed using a xE-test. Latency differences between the sectors for vagal and somatic stimulation were statistically assessed by a one-way analysis of variance, followed by the Tukey method for a post hoc comparison of the means 12. At the conclusion of each experiment, an electrolytic lesion was made in the last electrode tract, which provided histological verification of the recording site. Each cat was killed with a lethal dose of sodium pentobarbital, the brain removed, and the cerebellum processed with standard histological procedures. The boundaries of the folia and the electrode tract locations were traced on camera lucida projections of 40 /~m parasagittai sections. The interelectrode tract distance provided a calibration for shrinkage due to histological processing. The locations of the isolated Purkinje cells were obtained from the adjusted depth measurements from the pial surface.
c
ill A, ,
,,j
A8(65%) (n=171)
13(8%)
Fig. 1. A: compound action potential of the cervical vagus nerve to electrical stimulation (arrow) at 50 times threshold. The distance between stimulating electrodes and recording electrodes was 28 mm. B: the proportion of the climbing fiber responses elicited by stimulation of the three fiber types of the vagus nerve.
60
VERMAL ZONE
i Sample size:
58
51
67
61
1, 31
A
INTERMEDIATE ZONE 39
16
21
36
10
70
38
80-
Vaclal Afferents
6O
60-
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13_
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~ 30
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20
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10 0
0.0
0.5
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4.0
4.5
B
5.0
Distance from midline (mm) [] •
Vagat+somatic vagal
[] []
50
Somatic Unresponsive
0<.001
Fig. 2. The proportion of climbing fiber responses in 0.5 mm wide sectors of lobule V that were elicited by vagal, vagal and somatic, somatic stimulation or were unresponsive to the stimulation. The climbing fiber responses elicited by only vagal nerve stimulation were encountered in the medial vermis and the sector 4.0 mm lateral from the midline. The number of climbing fiber responses elicited by vagal stimulation were significantly greater (*P < 0.001) in the sectors of 0.5, 1.5 and 4.0 mm lateral to midline than in the remaining sectors.
intensities of 4-12 times threshold and had an average conduction velocity of 12.5 m/s. The C vagal afferents required a stimulus intensity of 35-55 times threshold and had a conduction velocity of 1.1 m/s. A b o u t 65% (111/171 cells) of the vagal climbing fiber responses were elicited by A6 fibers, 27% of the responses were elicited by vagal At~ fibers, and only 8% of the responses were elicited by vagal C-fiber.
Longitudinal organization Dividing the vermal and intermediate regions of lobule V into a series of 0.5-mm-wide longitudinal sectors revealed regional differences in the proportion of responses elicited by stimulation of only the vagus nerve, stimulation of both the vagus nerve and the body surface (somatic stimulation), and only somatic stimulation (Fig. 2). Climbing fiber responses elicited by only vagal stimulation were encountered mainly in the medial vermis and in one sector of the intermediate cortex. Responses elicited by both vagal and somatic stimulation were encountered in all sectors, except in the most medial sector where only 5% of the cells had convergent input. Three longitudinal sectors (0.5, 1.5 and 4.0) had significantly more responses elicited by vagal stimulation (including the responses with convergent somatic input) than the remaining sectors (Pearson ;(2, F1 = 36.13; P < 0.001).
0.0 0.5 1.0 1,5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Forelimb
40 t E
~ 30. ®
~ 20. 10. 0"
0.0 0.5 1.0 1.5 2.0 2.$ 3.0 3.5 4~0 4.5 5.0 Distance from mldline (mm)
Fig. 3. The latencies (mean + S.E.M.) of the climbing fiber
responses elicited by stimulation of the vagus nerve (A) or the forelimb receptive field (B) in the 0.5-mm-wide parasagittal sectors. The number of responses within each sector is shown on the bottom part of each histogram. The latencies for vagal nerve stimulation were not significantly different between sectors, whereas the latencies for forelimb stimulation were significantly (*P < 0.001) longer in sectors 1.0 and 1.5 than in the remaining sectors. The distribution of climbing fiber responses elicited by somatic stimulation was similar to previous observations 34"36. The most medial sector contained few Purkinje cells activated by tactile stimulation with 81% of the cells unresponsive to any of our stimulation. In the rest of the vermis and the intermediate regions, 3 7 - 8 5 % of the ceils had responses driven by tactile stimulation (including cells with convergent vagal input). The number of cells elicited by somatic or vagal and somatic stimulation was significantly different (Pearson ;(2, Fl ° = 85.6; P < 0.001) than the n u m b e r of unresponsive units and cells driven by only vagal stimulation for various sectors. The sectors at 0.0 and 0.5 mm from the midline had significantly fewer somatic responses than unresponsive units or vagal elicited responses. The sector at 1.5 mm had more somatic elicited responses than the other types of units. The response latencies for vagal stimulation ranged from 25 to 43 ms and were fairly consistent between sectors, except for sectors 2.5 to 3.5, w h e r e the latencies were slightly longer than the other sectors (Fig. 3A).
61
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1
[ ] AI$ Fiber A A8 Fiber •
Forelimb
•
Hindlimb
•
Face
o Unresponsive
2,at A N # 5 -4.0 m m
50
"rim
Fig. 5. A-C: representative parasagittal sections of the intermediate cortex showing the distribution of isolated Purkinje cells. Distance from midline in millimeters is indicated below each section. Abbreviations same as in Fig. 4.
H o w e v e r , no significant differences (F3,58 = 0.270, P < 0.947) were f o u n d a m o n g the sectors. A scatter diagram of the latencies also did not reveal any distinct longitudinal zones, which m a y have been m a s k e d by combining d a t a into 0.5 m m wide sectors. T h e response latencies for forelimb stimulation were significantly different (F8,114 ~- 8.68; P < 0.001) among longitudinal sectors. T h e average response latency to stimulation of the forelimb at 1.5 m m was 32.7 ms, which was significantly longer than those of any o t h e r sector, except in the sector at 1.0 mm. T h e latency in the sector at 1.0 m m was also significantly longer than the lateneies in sectors 2 . 5 - 5 . 0 (Fig. 3B). The response latencies of stimulation of the face ranged from 24.6 to 28.9 ms, but no significant differences (F4,47 = 0.384; P < 0.819) were found b e t w e e n sectors where the face was represented. The 18 climbing fiber responses representing the hindlimb were all e n c o u n t e r e d in the lateral vermis. The hindlimb response latencies ranged from 21.5 to 53.1 ms,
Split (n=20) Face (n=,46) Hindlimb (n=lS) Forelimb (n=138) 0
20
40
60
80
100
Percent k-~ vagal+somatic
[ ] Somatic
Fig. 6. The proportion of the climbing responses elicited by stimulation of the vagus nerve and different general body areas. All the responses with split receptive fields (non-contiguous body area) and most of the responses with representations of the face had convergent vagal input. About half of the responses representing either ipsilateral extremity had convergent vagal input.
63
A Vagal + somatic
40-
(n=137)
•
Cutaneous
Deep 30O O 0-
20.
10"
0
Face
.
~
Forelimb
Hindlimb
Split
Forelimb
Hindlimb
Split
B Somatic (n=85)
4C
~
30
O 132O
10
0
Face
Receptive field
Fig. 7. The proportion of climbing fiber responses of various body surfaces that were elicited by cutaneous or deep tactile stimulation. In responses with convergent input (A), the face representation was mainly elicited by cutaneous stimulation and the forelimb representation was elicited predominantly by deep stimulation. In responses elicited by only somatic input (B), the face and forelimb representation were elicited equally with cutaneous and deep stimulation.
but the small sample size prevented any statistical analysis of possible differences between sectors.
Topological organization The examination of fine-grain organization within each section revealed that the responses elicited by vagal nerve stimulation were fairly evenly distributed throughout, from the surface to the depths of the folia (Figs. 4 and 5). In the vermis, a considerable mixture of response types was evident. For example, in the most medial sections (Fig. 4A), the cells isolated in the dorsal sublobule included responses elicited by only vagal nerve, both vagal and somatic, and only somatic stimulation, as well as unresponsive units. The responses elicited by vagal nerve stimulation included a mixture of A#, A 6 and C fiber types. Responses elicited by stimulation of the vagal C fibers predominated in the medial vemis (Fig. 4A), with only an occasional response encountered in the lateral vermis (Fig. 4C) or intermediate regions.
In the lateral vermis, climbing fiber responses representing various body surfaces were frequently encountered in various sized patches representing the same general body area. In Fig. 4B, representation of the hindlimb, forelimb, and face were encountered in different patches as the electrode traversed from the surface to the deeper regions. Most patches contained a mixture of cells with and without vagal convergent input. The vagal input usually represented both A 6 and A# fiber types. For example, in Fig. 4B, only some of the responses representing the hindlimb included convergent vagal input whereas the responses representing either the forelimb or the face included convergent vagal input from both A 6 and A# afferent fibers. The intermediate region consisted mainly of cells with climbing fiber responses representing the forelimb (Fig. 5) and an occasional response representing the face, as well as scattering of unresponsive units. In this region, most of the vagal representation was convergent with somatic input, although an occasional response elicited only by vagal nerve stimulation was encountered. In some regions of particular animals, all the responses that were elicited by vagal nerve stimulation represented either A 6 or A# fibers. For example, in Fig. 5B and C, all the vagal input was mediated by A 6 at 4.0 mm from the midline and by Aa fibers at 5.0 mm. Thus, in this animal, there was a separation of the A 6 representation from the A# input. However, in other animals, the approximate same regions contained the opposite representation or a mixture of vagal nerve fiber types.
Vagal and somatic afferent convergence The proportion of cells with somatic and vagal convergence differed for various receptive fields (Fig. 6). All of the 20 climbing fiber responses with split receptive fields and 91% (42/46) of the responses representing the face had converging vagal input, but only half of the responses representing the ipsilateral extremities had converging input. Approximately 87% of the responses representing the face and the vagus nerve were encountered in the lateral vermis, whereas the responses with split receptive fields or representing the extremities were encountered throughout the lobule. The modality of cutaneous versus deep stimulation was different between responses having convergent vagal and face versus vagal and forelimb input (Fig. 7A). The responses with face convergent input were twice as likely to be elicited by cutaneous than deep stimulation, whereas the reverse was true for responses with forelimb convergent input, where there was a 1:3 cutaneous versus deep relationship. This is in contrast to the responses elicited only by somatic stimulation, where cutaneous and deep stimulation drove an equal number of responses
64 representing the face and forelimb (Fig. 7B). DISCUSSION The fine-grain mapping of the vagal climbing fiber representation in lobule V of this study revealed a larger representation of the vagus, a more complex spatial organization, and a more extensive convergence of somatic and vagal input than has been observed previously6'1s'24"25'33. This study demonstrated that approximately 40% of the climbing fiber responses isolated in lobule V were elicited by electrical stimulation of mainly the vagal A6 and A/~ afferent fibers. These fibers probably convey information from the cardiac and pulmonary organs via several brainstem nuclei to the vago-olivocerebellar projection. The vago-olivocerebellar input could substantially influence cerebellar activity through its distinct distribution and by the convergence of vagal and somatic representations.
Origin of vago-olivocerebellar fibers How information from the vagal afferents reaches the inferior olive, the origin of the climbing fibers, is not completely known. The range of the response latencies (18.5-68.8 ms) of the vagal climbing fiber responses suggest that several synaptic connections are possible. Anatomical studies demonstrate that vagal afferents project to the nucleus of the solitary tract (NTS), area postrema, and the dorsal motor nucleus of the vagus nerve in the cat 4'21'22. Only the NTS has a direct projection to the inferior olive 26, whereas the connections of other brainstem nuclei may provide vagal signals as an indirect pathway to the inferior olive. Many of the vagal afferents to the cerebellar cortex likely originate from the cardiac and pulmonary organs, since these organs provide the majority of myelinated axons of the vagal afferent system, whereas the C fibers mainly originate from the visceral organs 31. However, it has been shown previously that stimulation of abdominal vagal afferents at the level of the lower esophageal sphincter did not induce any cerebellar responses in the vermis 33, which was similar to the low proportion of responses elicited by C fiber stimulation in this study.
Distribution of the vagal representation Previous studies indicated that the vagal climbing fiber responses in the vermis are distributed to the medial parts 6'33 or throughout the caudal part of Iobule V and the rostral part of lobule V133. However, the mapping techniques of this study showed that the vagal climbing fiber representation was not only distributed throughout the whole vermis but was significantly increased in two longitudinal sectors (Fig. 2). These longitudinal sectors
may coincide with some of the spino-olivocerebetlar longitudinal zones in the vermis, which have been previously defined by the latencies of field potentials and the body areas represented 1'1<3s'39. The sectors at 1.0 and 1.5 mm lateral from the midline, in this study, may correspond to the medial part of the b zone, since this region contained long-latency responses to stimulation of ipsilateral forelimb receptive fields and contained mixed representation of the forelimb and hindlimb, which was similar to previously reported representations 1;39. If the 1.0 and 1.5 mm sectors correspond to the medial b zone, then the 0.5 mm sector, which contained a significantly increased proportion of vagal climbing fiber input, would correspond to zone x. Although the 0.5 sector is close to the midline, there appears to be considerable variability in the reported width of the most medial zone (e.g. compare the width of the zones of Figs. 1F and 213 of ref. 39). The most medial sector presumably corresponds to zone a. The medial sector mainly contained a distinct population of responses elicited by just the vagus nerve. The low incidence of convergent vagal and somatic input in this region is partly due to the limited representation of the body surface that is characteristic of this zone 15"34. However, vagal convergence may occur for proprioceptive 7, vestibular 37, or visual 9 input that also project to this region. The concentration of climbing fiber input from vagal afferents and from the various receptors related to postures suggests the possibility that the vagal afferents may participate in regulating the cardiovascular activity during the orthostatic reflex or may be associated with visceral activity during motion sickness. Vagal climbing fiber responses were also encountered throughout the intermediate zone of lobule V, although a significantly increased proportion of responses occurred in the sector at 4.0 mm lateral from the midline. The occurrence of a vagal representation throughout the intermediate cortex rather than a single longitudinal zone may be due to the chloralose anesthesia used in this study and by Perrin and Crousillat 33 instead of the sodium pentobarbital used by Hennemann and Rubia 18. The barbiturate anesthesia may have interfered with vagal afferent input ~9. The increased vagal representation in the sector at 4.0 mm of this study probably corresponds to the 500-800-/,m-wide longitudinal zone of vagal representation identified by Hennemann and Rubia TM in the intermediate cortex. It was not possible to identify any spatial correspondence of the vagal representation with various spino-olivocerebellar longitudinal zones in the intermediate cortex 16'3s, since no significant differences in the response latencies to vagal or somatic stimulation were identified and the representation of body surface did not match the zonal somatic represen-
65 tations of E k e r o t and L a r s o n 16. H o w e v e r , the absence of clearly defined longitudinal zones in the intermediate cortex for the somatic r e p r e s e n t a t i o n , in this study, is similar to previous studies that have used natural stimulation to elicit the responses 15'27'34'36.
Convergence o f vagal and somatic representations T h e large p r o p o r t i o n of climbing fiber responses with convergent vagal and somatic input from the extremities suggests an interaction b e t w e e n autonomic and somatom o t o r activity. Various postural adjustments or increases in m o t o r activity, such as running, require changes in the c a r d i o p u l m o n a r y and o t h e r visceral systems that may be partly r e g u l a t e d by the vagal afferents to the cerebellum 29'32. H o w e v e r , it is not clear what the significance is for the strong association between the vagal input and the somatic r e p r e s e n t a t i o n s of the face. Possibly, the cutaneous stimulation of the cat's face m a y effectively trigger orienting responses that are a c c o m p a n i e d by a u t o n o m i c changes. T h e consequences of vagal nerve and somatic interactions on the discharge p r o p e r t i e s of the Purkinje cell have yet to be elucidated. Climbing fiber responses elicited by vagal afferents m a y have a short-term influence on REFERENCES 1 Andersson, G. and Eriksson, L., Spinal, trigeminal, and cortical climbing fiber paths to the lateral vermis of the cerebellar anterior lobe in the cat, Exp. Brain Res., 41 (1981) 71-81. 2 Armstrong, D.M., Functional significance of connections of the inferior olive. Physiol. Rev., 54 (1974) 358-417. 3 Bradley, D.J., Ghelarducci, B., Paton, J.F.R. and Spyer, K.M., The cardiovascular responses elicited from the posterior cerebellar cortex in the anaesthetized and decerebrate rabbit, J. Physiol. (Lond.), 383 (1987) 537-550. 4 Cottle, M.K.W., Degeneration studies of primary afferents of the IXth and Xth cranial nerves in the cat, J. Comp. Neurol., 122 (1964) 329-345. 5 Del Bo, A., Sved, A.F. and Reis, D.J., Fastigial stimulation releases vasopressin in amounts that elevate arterial pressure, Am. J. Physiol., 224 (1983) H687-H694. 6 Dell, D. and Olson, R., Projections thalamiques, corticales et cerebeleuses des afferences viscerales vagales, C.R. Soc. Biol. (Paris), 45 (1951) 1084-1088. 7 Denoth, E, Magherini, P.C., Pompeiano, O. and Stanojevic, M., Responses of Purkinje cells of cerebellar vermis to sinosoidai rotation of the neck, J. Neurophysiol., 43 (1980) 46-59. 8 Dietrichs, E. and Haines, D.E., Interconnections between hypothalamus and cerebellum, Anat. Embryol., 179 (1989) 207-220. 9 Donaldson, I.M.L. and Hawthorne, M.E., Coding of visual information by units in the cat cerebellar vermis, Exp. Brain Res., 34 (1979) 27-48. 10 Dormer, K.J., Andrezik, J.A., Person, R.J., Braggio, J.T. and Foreman, R.D., Fastigial nucleus cardiovascular response and brainstem lesions in the beagle, Am. J. Physiol., 250 (1986) 231-239. 11 Dow, R.S., Kramer, R.E. and Robertson, L.T., Disorders of the cerebellum. In R.J. Joynt (Ed.), Clinical Neurology, Lippincott, Philadelphia, in press. 12 Dowdy, S. and Wearden, S., Statistics for Research, Wiley, New
reception of convergent somatic input, since there is an a p p r o x i m a t e l y 100 ms refractory p e r i o d following a climbing fiber response 2'35. Thus, a climbing fiber response elicited by vagal input could effectively block subsequent somatic climbing fiber input or, conversely, a somatic elicited response could i m p e d e the vagal input. Vagal activation of a climbing fiber response also could m o d u l a t e simple spike activity of the Purkinje cell by increasing the gain of the mossy fiber input 13. In addition to the short-term effects, activation of the vagus nerve by a d e c a p e p t i d e can selectively induce long-term changes (up to 30 h) in climbing fiber activity2°. Overwhelming e x p e r i m e n t a l and clinical evidence indicates the cerebellum participates in the control of skeletal muscles during posture and m o v e m e n t . However, there is an accumulation of evidence suggesting that the cerebellum is associated with vegetative functions, e m o t i o n a l behavior, and even cognitive functions 8'11. The extent of the vago-olivocerebellar p r o j e c t i o n to lobule V, as o b s e r v e d in this study, supports a reappraisal of cerebellar function.
Acknowledgements. We would like to thank Mary Dennis for her technical assistance. This work was supported by an NIH Grant NS18242. York, 1983, 537 pp. 13 Ebner, T.J. and Bloedel, J.R., Climbing fiber afferent system: intrinsic properties and role in cerebeUar information processing. In J.S. King (Ed.), New Concepts in Cerebellar Neurobiology, Liss, New York, 1987, pp. 371-386. 14 Eccles, J.C., Llin~is, R.R. and Sasaki, K., The excitatory synaptic action of climbing fibers on the Purkinje ceils of the cerebellum, J. Physiol. (Lond.), 182 (1966) 268-296. 15 Eccles, J.C., Provini, L., Strata, P. and T~bofi'kov~t, H., Topographical investigations on the climbing fiber inputs from forelimb and hindlimb afferents to the cerebellar anterior lobe, Exp. Brain Res., 6 (1968) 195-215. 16 Ekerot, C.-F. and Larson, B., The dorsal spino-olivocerebellar system in the cat. I. Functional organization and termination in the anterior lobe, Exp. Brain Res., 36 (1979) 201-217. 17 Gutman, J., Bergmann, F. and Chaimovitz, M., Blood pressure responses to electrical stimulation of peripheral nerves and their modifications by nembutal, Arch. Int. Physiol. Biochern., 69 (1961) 509-520. 18 Hennemann, H.E. and Rubia, EJ., Vagal representations in the cerebellum of the cat, Pfliigers Arch., 375 (1978) 119-123. 19 Hoffer, B., Ratcheson, R. and Snider, R.S., The effects of stimulation of the cerebellum on the circulatory system, Fed. Proc., 25 (1966) 701 (Abstract). 20 Kageyama, H. and Kurosawa, A., Long-lasting inhibitory action of caerulein on climbing fiber system in the cerebellum of the rat, Neuropharmacology, 28 (1989) 991-995. 21 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches, J. Comp. Neurol., 193 (1980) 467-508. 22 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. I. The cervical vagus and nodose ganglion, J. Comp. Neurol., 193 (1980) 435-465. 23 Leicht, R. and Schmidt, R.F., Somatotopic studies on the ventral cortex of the cerebellar anterior lobe of unanesthetized cats,
66
Exp. Brain Res., 27 (1977) 479-490. 24 Levchuk, O.V., Spontaneous activity and evoked responses of cerebellar cortical neurons to vagal and limb nerve stimulation in pigeons, Neurophysiology, 7 (1975) 294-299. 25 Levchuk, O.V., Bratus, N.V. and Grigor'yan, R.A., Unit responses of the cerebellar cortex to stimulation of the vagus nerve and nerves of the limb, Neurophysiology, 4 (1972) 360-366. 26 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. Comp. Neurol., 181 (1978) 421-450. 27 Miles, T.S. and Wisendanger, M., Organization of climbing fiber projections to the cerebellar cortex from trigeminal cutaneous afferents and from the SI face area of the cerebral cortex in the cat, J. Physiol. (Lond.), 245 (1975) 409-424. 28 Moruzzi, G., Paleocerebellum inhibition of vasomotor and respiratory carotid sinus reflexes, J. Neurophysiol., 3 (1940) 20-32. 29 Nakai, M., Iadecola, C., Ruggiero, D.A., Tucker, L.W. and Reis, D.J., Electrical stimulation of cerebellar fastigial nucleus increases cerebral cortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation, Brain Research, 260 (1983) 35-49. 30 Nisimaru, N. and Kawaguchi, Y., Excitatory effects on renal sympathetic nerve activity induced by stimulation at two distinctive sites in the fastigial nucleus of rabbits, Brain Research, 304 (1984) 372-376. 31 Paintai, A.S., Vagal afferent fibers, Ergebn. Physiol., 52 (1963)
74-156. 32 Paintal, A.S., Vagal sensory receptors and their reflex effects, Physiol. Rev., 43 (1973) 159-227. 33 Perrin, J. and Crousillat, J., The projection of vagal afferents on the cerebellar vermis of the cat, J. Auton. Nerv. Syst.. 13 (1985) 175-177. 34 Robertson, L.T., Laxer, K.D. and Rushmer, D.S.. Organization of climbing fiber input from mechanoreceptors to lobule V vermal cortex of the cat, Exp. Brain Res., 46 (1982) 281-291. 35 Rushmer, D.S., Roberts, W.J. and Augter, G.K., Climbing fiber responses of cerebellar Purkinje cells to passive movement of the cat forepaw, Brain Research, 106 (1976) 1-20. 36 Rushmer, D.S., WooUacott, M.H., Robertson, L.T. and Laxer, K.D., Somatopic organization of climbing fiber projections from low threshold cutaneous afferents to pars intermedia of cerebellar cortex in the cat, Brain Research, 181 (1980) 17-30. 37 Suzuki, D.A. and Keller, E.L., Vestibular signals in the posterior vermis of the alert monkey cerebellum, Exp. Brain Res., 47 (1982) 145-147. 38 Trott, J.R., Armstrong, D.M., The cerebellar corticonuclear projection from lobule Vb/c of the cat anterior lobe: a combined electrophysiological and autoradiographic study. I. Projections from the intermediate region, Exp. Brain Res., 66 (1987) 318-338. 39 Trott, J.R. and Armstrong, D.M., The cerebellar corticonuclear projection from lobule Vb/c of the cat anterior lobe: a combined electrophysiological and autoradiographic study. II. Projections from the vermis, Exp. Brain Res., 68 (1987) 339-354.
58
BRES 16689
Vagal and somatic representation by the climbing fiber system in lobule V of the cat cerebellum Gang Tong, Lee T. Robertson and John Brons Oregon Health Sciences University, Department of Anatomy, School of Dentistry, Portland, OR 97201 (U.S.A.)
(Accepted 8 January 1991) Key words: Cerebellum; Climbing fiber; Vagus; Autonomic; Somatosensory; Viscerosensory
The organization of the climbing fiber representation of the vagal afferents and the body surface in the vermal and intermediate zones of lobule V was examined in cats anesthetized with a-chloralose. Extracellular single-unit recordings were made from 428 Purkinje cells. Electrical stimulation of the vagus nerve elicited climbing fiber responses in 40% of the cells, most of which had convergent somatic input, Activation of A~ vagal afferent fibers accounted for 65% of the responses, whereas the Ap fibers involved 27% and the C fibers included 8% of the responses. The responses driven by vagal nerve stimulation were encountered throughout the lobule, although a significantly increased representation of the vagus was identified for 3 longitudinal 0.5 mm wide sectors (two in the vermis and one in the intermediate region). In the vermis, the fine-grain organization consisted of a mixture of representations of the various parts of the body surface with and without convergent vagal input, although there was little convergence in the medial vermis where many of the responses were elicited by only vagal nerve stimulation. In the intermediate cortex, most of the vagal climbing fiber representation was convergent with forelimb input. These results suggest that vagal input into the cerebellum could have important modulatory effects on the cerebellar somatosensory input. INTRODUCTION The cerebellum has traditionally been considered important in the regulation and coordination of somatic motor activity, but substantial evidence also exists for a modulation of autonomic functions. Moruzzi 28 first showed that stimulation of the anterior lobe of the cerebellum could inhibit blood pressure and respiratory responses. Since Moruzzi's work, numerous studies have demonstrated that stimulation or ablation of the cerebellar cortex and nuclei can induce a variety of cardiovascular changes, including alterations in blood pressure, heart rate and blood flow 3"5'10'29'30. The cerebellum also receives information from the cardiovascular system via vagal nerve afferents 6'18"24'25'33. However, there are conflicting conclusions on the arrangement of the vagal projection to the cerebellum. Field potentials elicited by stimulation of the vagal nerve have been identified in the medial vermis of the anterior lobe 6'24'25'33, throughout the vermis of sublobule V c and lobule V133, and as a longitudinal strip in the intermediate zone of lobule V TM. H e n n e m a n and Rubia TM suggested that the responses in the intermediate zone represent climbing fiber input whereas the responses in the vermis may be due to far-field potentials originating
in the brainstem. Recently, Perrin and Crousillat 33 made single unit recordings of 23 Purkinje cells in the vermis and identified both climbing and mossy fiber vagal input, although little information was given on the magnitude of the vagal climbing fiber representation or how the climbing fiber vagal representation was distributed in the lobule. Several physiological studies of the climbing fiber input in the anterior lobe have demonstrated an extensive representation of the body surface 15'23'27'34'36.Therefore, it is likely that the vagal climbing fiber input to the anterior lobe will converge with the somatic input. It is necessary to know the extent and distribution of the possible convergence before we can appreciate the functional implications of the vagal input. The examination of the receptive fields of cells with convergent somatic and vagal input may provide some insight on the cerebellum's role in cardiovascular function. The purpose of this study was to re-examine the organization of the climbing fiber representation of the vagus nerve in the vermal and intermediate regions of lobule V by using fine-grain, single'unit mapping procedures. By mapping a large n u m b e r of individual Purkinje cells, it was possible to calculate the regional proportions of the Purkinje cells receiving vagal climbing fiber inputs,
Correspondence: L. Robertson, Department of Anatomy, School of Dentistry, Oregon Health Sciences University, 611 S.W. Campus Drive, Portland, OR 97201, U.S.A.
59 tO d e t e r m i n e
the
spatial
organization
of
the
vagal
r e p r e s e n t a t i o n t h r o u g h o u t l o b u l e V, and to establish the spatial r e l a t i o n s h i p b e t w e e n the c l i m b i n g fiber r e p r e s e n t a t i o n of the v a g u s n e r v e and b o d y surface. MATERIALS AND METHODS The experiments were carried out on 12 cats of either sex, weighing 2.5-4.0 kg. The animals were anesthetized with intravenous injections of a-chloralose (100 mg/kg), followed by supplementary doses (30 mg/kg). Chloralose anesthesia was used instead of barbiturates because small doses of barbiturates can influence normal cardiovascular activity 17a9. Catheters were placed in the femoral vein and artery for administering fluids and drugs and for monitoring the blood pressure, respectively. The rectal temperature was maintained between 37 and 38 °C. The animal was placed in a stereotaxic apparatus and the dorsal surface of Iobule V of the cerebellar cortex was surgically exposed. The dura over the anterior cerebellum was removed and the cerebellum was covered with warm mineral oil or Ringer's agar. Tungsten microelectrodes (8-10 MI2 and 1000 Hz) and conventional electrophysiological equipment were used to extracellularly record single Purkinje cells, which were distinguished by spontaneous climbing fiber responses TM.The microelectrode placement was controlled stereotaxically and the penetrations were made in the parasagittal plane at a 15-20 ° angle from the vertical with an average interpretation distance of 400 gm. Each electrode penetration traversed through several Purkinje cell layers and, at each layer, an attempt was made to isolate a Purkinje cell and elicit a climbing fiber response by vagal or somatic stimulation. A Purkinje cell was considered to be responsive if a climbing fiber response was elicited in 70% of the trials. Typically, responsive units exceeded the 90% level of discharge probability. The vagus nerve was isolated, bathed in a pool of warm mineral oil, cut caudally, and stimulated at the level of the nodose ganglion with a pair of Ag-AgCl electrodes via a constant current stimulus isolator. In each experiment, compound action potentials (CAPs) were recorded from the vagal nerve with electrodes placed 26-30 mm proximal to the stimulating electrodes. The CAPs were checked several times during each experiment, although the threshold levels were consistently stable. The stimulus level was expressed as multiples of the lowest threshold required to produce a nerve volley. Stimulation consisted of either a single, 0.3 ms rectangular pulse or a train of 3 pulses at 200 Hz, every 4-5 s. The somatic stimulation included taps to the skin surface with hand-held probes, including calibrated yon Frey nylon filaments. Climbing fiber responses elicited by punctate stimuli of less than 2.0 g were considered cutaneous; those requiring more than 2.0 g were defined as deep. The response latencies were obtained by electrically stimulating the cutaneous receptive field with small needle electrodes.
RESULTS
Population studied T h e c l i m b i n g f i b e r r e s p o n s e s o f 428 P u r k i n j e cells w e r e isolated throughout the vermal and intermediate regions of s u b l o b u l e s Vb_ f. Vagal s t i m u l a t i o n successfully elicited c l i m b i n g fiber r e s p o n s e s in 4 0 % (171/428) of t h e i s o l a t e d P u r k i n j e cells. In this g r o u p , 3 2 % o f t h e c l i m b i n g fiber responses
were
by b o t h
vagal
and
tactile
tion. T h e 6 0 % o f t h e cells w i t h o u t v a g a l i n p u t i n c l u d e d 2 0 % (86/428) with c l i m b i n g f i b e r r e s p o n s e s d r i v e n o n l y by tactile s t i m u l a t i o n a n d 4 0 % t h a t w e r e u n r e s p o n s i v e to any s t i m u l a t i o n e m p l o y e d . T h e d i f f e r e n t types o f v a g a l a f f e r e n t fibers w e r e not e v e n l y r e p r e s e n t e d . T h e classification of a f f e r e n t fiber types
that
elicited t h e
climbing fiber responses
was
d e t e r m i n e d by the s t i m u l u s t h r e s h o l d and c o n d u c t i o n v e l o c i t y o f t h e C A P s 31 (Fig. 1). A/~ v a g a l a f f e r e n t s h a d an a v e r a g e t h r e s h o l d o f 70.5 /~A a n d a m e a n c o n d u c t i o n v e l o c i t y of 38.8 m/s. A~ v a g a l a f f e r e n t s r e q u i r e d stimulus
A~!3(27%)
I)
~tl
activated
s t i m u l a t i o n and 8 % w e r e e l i c i t e d o n l y by v a g a l stimula-
B
An
t
In an attempt to identify possible longitudinal zones, the vagal and the somatic elicited responses were segregated into 0.5mm-wide longitudinal sectors. Grouping the data into 0.5-mm-wide sectors permitted the statistical comparison between populations of responses, although a narrow spatial organization or an organization that spans multiple sectors may not be discerned. Differences between the sectors for the number of responses elicited by either vagal or somatic stimulation were analyzed using a xE-test. Latency differences between the sectors for vagal and somatic stimulation were statistically assessed by a one-way analysis of variance, followed by the Tukey method for a post hoc comparison of the means 12. At the conclusion of each experiment, an electrolytic lesion was made in the last electrode tract, which provided histological verification of the recording site. Each cat was killed with a lethal dose of sodium pentobarbital, the brain removed, and the cerebellum processed with standard histological procedures. The boundaries of the folia and the electrode tract locations were traced on camera lucida projections of 40 /~m parasagittai sections. The interelectrode tract distance provided a calibration for shrinkage due to histological processing. The locations of the isolated Purkinje cells were obtained from the adjusted depth measurements from the pial surface.
c
ill A, ,
,,j
A8(65%) (n=171)
13(8%)
Fig. 1. A: compound action potential of the cervical vagus nerve to electrical stimulation (arrow) at 50 times threshold. The distance between stimulating electrodes and recording electrodes was 28 mm. B: the proportion of the climbing fiber responses elicited by stimulation of the three fiber types of the vagus nerve.
60
VERMAL ZONE
i Sample size:
58
51
67
61
1, 31
A
INTERMEDIATE ZONE 39
16
21
36
10
70
38
80-
Vaclal Afferents
6O
60-
~
, ~. 5o ~, 4o
13_
c
o-
~ 30
20- ~ U
20
40 :
10 0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
B
5.0
Distance from midline (mm) [] •
Vagat+somatic vagal
[] []
50
Somatic Unresponsive
0<.001
Fig. 2. The proportion of climbing fiber responses in 0.5 mm wide sectors of lobule V that were elicited by vagal, vagal and somatic, somatic stimulation or were unresponsive to the stimulation. The climbing fiber responses elicited by only vagal nerve stimulation were encountered in the medial vermis and the sector 4.0 mm lateral from the midline. The number of climbing fiber responses elicited by vagal stimulation were significantly greater (*P < 0.001) in the sectors of 0.5, 1.5 and 4.0 mm lateral to midline than in the remaining sectors.
intensities of 4-12 times threshold and had an average conduction velocity of 12.5 m/s. The C vagal afferents required a stimulus intensity of 35-55 times threshold and had a conduction velocity of 1.1 m/s. A b o u t 65% (111/171 cells) of the vagal climbing fiber responses were elicited by A6 fibers, 27% of the responses were elicited by vagal At~ fibers, and only 8% of the responses were elicited by vagal C-fiber.
Longitudinal organization Dividing the vermal and intermediate regions of lobule V into a series of 0.5-mm-wide longitudinal sectors revealed regional differences in the proportion of responses elicited by stimulation of only the vagus nerve, stimulation of both the vagus nerve and the body surface (somatic stimulation), and only somatic stimulation (Fig. 2). Climbing fiber responses elicited by only vagal stimulation were encountered mainly in the medial vermis and in one sector of the intermediate cortex. Responses elicited by both vagal and somatic stimulation were encountered in all sectors, except in the most medial sector where only 5% of the cells had convergent input. Three longitudinal sectors (0.5, 1.5 and 4.0) had significantly more responses elicited by vagal stimulation (including the responses with convergent somatic input) than the remaining sectors (Pearson ;(2, F1 = 36.13; P < 0.001).
0.0 0.5 1.0 1,5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Forelimb
40 t E
~ 30. ®
~ 20. 10. 0"
0.0 0.5 1.0 1.5 2.0 2.$ 3.0 3.5 4~0 4.5 5.0 Distance from mldline (mm)
Fig. 3. The latencies (mean + S.E.M.) of the climbing fiber
responses elicited by stimulation of the vagus nerve (A) or the forelimb receptive field (B) in the 0.5-mm-wide parasagittal sectors. The number of responses within each sector is shown on the bottom part of each histogram. The latencies for vagal nerve stimulation were not significantly different between sectors, whereas the latencies for forelimb stimulation were significantly (*P < 0.001) longer in sectors 1.0 and 1.5 than in the remaining sectors. The distribution of climbing fiber responses elicited by somatic stimulation was similar to previous observations 34"36. The most medial sector contained few Purkinje cells activated by tactile stimulation with 81% of the cells unresponsive to any of our stimulation. In the rest of the vermis and the intermediate regions, 3 7 - 8 5 % of the ceils had responses driven by tactile stimulation (including cells with convergent vagal input). The number of cells elicited by somatic or vagal and somatic stimulation was significantly different (Pearson ;(2, Fl ° = 85.6; P < 0.001) than the n u m b e r of unresponsive units and cells driven by only vagal stimulation for various sectors. The sectors at 0.0 and 0.5 mm from the midline had significantly fewer somatic responses than unresponsive units or vagal elicited responses. The sector at 1.5 mm had more somatic elicited responses than the other types of units. The response latencies for vagal stimulation ranged from 25 to 43 ms and were fairly consistent between sectors, except for sectors 2.5 to 3.5, w h e r e the latencies were slightly longer than the other sectors (Fig. 3A).
61
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_
_
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o
0_
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z
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o
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62 Tracks
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7
8
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10
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=2 i
1
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2
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,~'
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-/" ,,'
/
,
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,
'"
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,, //'
"/L''
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Cat AN #6 3.5 mm
1
[ ] AI$ Fiber A A8 Fiber •
Forelimb
•
Hindlimb
•
Face
o Unresponsive
2,at A N # 5 -4.0 m m
50
"rim
Fig. 5. A-C: representative parasagittal sections of the intermediate cortex showing the distribution of isolated Purkinje cells. Distance from midline in millimeters is indicated below each section. Abbreviations same as in Fig. 4.
H o w e v e r , no significant differences (F3,58 = 0.270, P < 0.947) were f o u n d a m o n g the sectors. A scatter diagram of the latencies also did not reveal any distinct longitudinal zones, which m a y have been m a s k e d by combining d a t a into 0.5 m m wide sectors. T h e response latencies for forelimb stimulation were significantly different (F8,114 ~- 8.68; P < 0.001) among longitudinal sectors. T h e average response latency to stimulation of the forelimb at 1.5 m m was 32.7 ms, which was significantly longer than those of any o t h e r sector, except in the sector at 1.0 mm. T h e latency in the sector at 1.0 m m was also significantly longer than the lateneies in sectors 2 . 5 - 5 . 0 (Fig. 3B). The response latencies of stimulation of the face ranged from 24.6 to 28.9 ms, but no significant differences (F4,47 = 0.384; P < 0.819) were found b e t w e e n sectors where the face was represented. The 18 climbing fiber responses representing the hindlimb were all e n c o u n t e r e d in the lateral vermis. The hindlimb response latencies ranged from 21.5 to 53.1 ms,
Split (n=20) Face (n=,46) Hindlimb (n=lS) Forelimb (n=138) 0
20
40
60
80
100
Percent k-~ vagal+somatic
[ ] Somatic
Fig. 6. The proportion of the climbing responses elicited by stimulation of the vagus nerve and different general body areas. All the responses with split receptive fields (non-contiguous body area) and most of the responses with representations of the face had convergent vagal input. About half of the responses representing either ipsilateral extremity had convergent vagal input.
63
A Vagal + somatic
40-
(n=137)
•
Cutaneous
Deep 30O O 0-
20.
10"
0
Face
.
~
Forelimb
Hindlimb
Split
Forelimb
Hindlimb
Split
B Somatic (n=85)
4C
~
30
O 132O
10
0
Face
Receptive field
Fig. 7. The proportion of climbing fiber responses of various body surfaces that were elicited by cutaneous or deep tactile stimulation. In responses with convergent input (A), the face representation was mainly elicited by cutaneous stimulation and the forelimb representation was elicited predominantly by deep stimulation. In responses elicited by only somatic input (B), the face and forelimb representation were elicited equally with cutaneous and deep stimulation.
but the small sample size prevented any statistical analysis of possible differences between sectors.
Topological organization The examination of fine-grain organization within each section revealed that the responses elicited by vagal nerve stimulation were fairly evenly distributed throughout, from the surface to the depths of the folia (Figs. 4 and 5). In the vermis, a considerable mixture of response types was evident. For example, in the most medial sections (Fig. 4A), the cells isolated in the dorsal sublobule included responses elicited by only vagal nerve, both vagal and somatic, and only somatic stimulation, as well as unresponsive units. The responses elicited by vagal nerve stimulation included a mixture of A#, A 6 and C fiber types. Responses elicited by stimulation of the vagal C fibers predominated in the medial vemis (Fig. 4A), with only an occasional response encountered in the lateral vermis (Fig. 4C) or intermediate regions.
In the lateral vermis, climbing fiber responses representing various body surfaces were frequently encountered in various sized patches representing the same general body area. In Fig. 4B, representation of the hindlimb, forelimb, and face were encountered in different patches as the electrode traversed from the surface to the deeper regions. Most patches contained a mixture of cells with and without vagal convergent input. The vagal input usually represented both A 6 and A# fiber types. For example, in Fig. 4B, only some of the responses representing the hindlimb included convergent vagal input whereas the responses representing either the forelimb or the face included convergent vagal input from both A 6 and A# afferent fibers. The intermediate region consisted mainly of cells with climbing fiber responses representing the forelimb (Fig. 5) and an occasional response representing the face, as well as scattering of unresponsive units. In this region, most of the vagal representation was convergent with somatic input, although an occasional response elicited only by vagal nerve stimulation was encountered. In some regions of particular animals, all the responses that were elicited by vagal nerve stimulation represented either A 6 or A# fibers. For example, in Fig. 5B and C, all the vagal input was mediated by A 6 at 4.0 mm from the midline and by Aa fibers at 5.0 mm. Thus, in this animal, there was a separation of the A 6 representation from the A# input. However, in other animals, the approximate same regions contained the opposite representation or a mixture of vagal nerve fiber types.
Vagal and somatic afferent convergence The proportion of cells with somatic and vagal convergence differed for various receptive fields (Fig. 6). All of the 20 climbing fiber responses with split receptive fields and 91% (42/46) of the responses representing the face had converging vagal input, but only half of the responses representing the ipsilateral extremities had converging input. Approximately 87% of the responses representing the face and the vagus nerve were encountered in the lateral vermis, whereas the responses with split receptive fields or representing the extremities were encountered throughout the lobule. The modality of cutaneous versus deep stimulation was different between responses having convergent vagal and face versus vagal and forelimb input (Fig. 7A). The responses with face convergent input were twice as likely to be elicited by cutaneous than deep stimulation, whereas the reverse was true for responses with forelimb convergent input, where there was a 1:3 cutaneous versus deep relationship. This is in contrast to the responses elicited only by somatic stimulation, where cutaneous and deep stimulation drove an equal number of responses
64 representing the face and forelimb (Fig. 7B). DISCUSSION The fine-grain mapping of the vagal climbing fiber representation in lobule V of this study revealed a larger representation of the vagus, a more complex spatial organization, and a more extensive convergence of somatic and vagal input than has been observed previously6'1s'24"25'33. This study demonstrated that approximately 40% of the climbing fiber responses isolated in lobule V were elicited by electrical stimulation of mainly the vagal A6 and A/~ afferent fibers. These fibers probably convey information from the cardiac and pulmonary organs via several brainstem nuclei to the vago-olivocerebellar projection. The vago-olivocerebellar input could substantially influence cerebellar activity through its distinct distribution and by the convergence of vagal and somatic representations.
Origin of vago-olivocerebellar fibers How information from the vagal afferents reaches the inferior olive, the origin of the climbing fibers, is not completely known. The range of the response latencies (18.5-68.8 ms) of the vagal climbing fiber responses suggest that several synaptic connections are possible. Anatomical studies demonstrate that vagal afferents project to the nucleus of the solitary tract (NTS), area postrema, and the dorsal motor nucleus of the vagus nerve in the cat 4'21'22. Only the NTS has a direct projection to the inferior olive 26, whereas the connections of other brainstem nuclei may provide vagal signals as an indirect pathway to the inferior olive. Many of the vagal afferents to the cerebellar cortex likely originate from the cardiac and pulmonary organs, since these organs provide the majority of myelinated axons of the vagal afferent system, whereas the C fibers mainly originate from the visceral organs 31. However, it has been shown previously that stimulation of abdominal vagal afferents at the level of the lower esophageal sphincter did not induce any cerebellar responses in the vermis 33, which was similar to the low proportion of responses elicited by C fiber stimulation in this study.
Distribution of the vagal representation Previous studies indicated that the vagal climbing fiber responses in the vermis are distributed to the medial parts 6'33 or throughout the caudal part of Iobule V and the rostral part of lobule V133. However, the mapping techniques of this study showed that the vagal climbing fiber representation was not only distributed throughout the whole vermis but was significantly increased in two longitudinal sectors (Fig. 2). These longitudinal sectors
may coincide with some of the spino-olivocerebetlar longitudinal zones in the vermis, which have been previously defined by the latencies of field potentials and the body areas represented 1'1<3s'39. The sectors at 1.0 and 1.5 mm lateral from the midline, in this study, may correspond to the medial part of the b zone, since this region contained long-latency responses to stimulation of ipsilateral forelimb receptive fields and contained mixed representation of the forelimb and hindlimb, which was similar to previously reported representations 1;39. If the 1.0 and 1.5 mm sectors correspond to the medial b zone, then the 0.5 mm sector, which contained a significantly increased proportion of vagal climbing fiber input, would correspond to zone x. Although the 0.5 sector is close to the midline, there appears to be considerable variability in the reported width of the most medial zone (e.g. compare the width of the zones of Figs. 1F and 213 of ref. 39). The most medial sector presumably corresponds to zone a. The medial sector mainly contained a distinct population of responses elicited by just the vagus nerve. The low incidence of convergent vagal and somatic input in this region is partly due to the limited representation of the body surface that is characteristic of this zone 15"34. However, vagal convergence may occur for proprioceptive 7, vestibular 37, or visual 9 input that also project to this region. The concentration of climbing fiber input from vagal afferents and from the various receptors related to postures suggests the possibility that the vagal afferents may participate in regulating the cardiovascular activity during the orthostatic reflex or may be associated with visceral activity during motion sickness. Vagal climbing fiber responses were also encountered throughout the intermediate zone of lobule V, although a significantly increased proportion of responses occurred in the sector at 4.0 mm lateral from the midline. The occurrence of a vagal representation throughout the intermediate cortex rather than a single longitudinal zone may be due to the chloralose anesthesia used in this study and by Perrin and Crousillat 33 instead of the sodium pentobarbital used by Hennemann and Rubia 18. The barbiturate anesthesia may have interfered with vagal afferent input ~9. The increased vagal representation in the sector at 4.0 mm of this study probably corresponds to the 500-800-/,m-wide longitudinal zone of vagal representation identified by Hennemann and Rubia TM in the intermediate cortex. It was not possible to identify any spatial correspondence of the vagal representation with various spino-olivocerebellar longitudinal zones in the intermediate cortex 16'3s, since no significant differences in the response latencies to vagal or somatic stimulation were identified and the representation of body surface did not match the zonal somatic represen-
65 tations of E k e r o t and L a r s o n 16. H o w e v e r , the absence of clearly defined longitudinal zones in the intermediate cortex for the somatic r e p r e s e n t a t i o n , in this study, is similar to previous studies that have used natural stimulation to elicit the responses 15'27'34'36.
Convergence o f vagal and somatic representations T h e large p r o p o r t i o n of climbing fiber responses with convergent vagal and somatic input from the extremities suggests an interaction b e t w e e n autonomic and somatom o t o r activity. Various postural adjustments or increases in m o t o r activity, such as running, require changes in the c a r d i o p u l m o n a r y and o t h e r visceral systems that may be partly r e g u l a t e d by the vagal afferents to the cerebellum 29'32. H o w e v e r , it is not clear what the significance is for the strong association between the vagal input and the somatic r e p r e s e n t a t i o n s of the face. Possibly, the cutaneous stimulation of the cat's face m a y effectively trigger orienting responses that are a c c o m p a n i e d by a u t o n o m i c changes. T h e consequences of vagal nerve and somatic interactions on the discharge p r o p e r t i e s of the Purkinje cell have yet to be elucidated. Climbing fiber responses elicited by vagal afferents m a y have a short-term influence on REFERENCES 1 Andersson, G. and Eriksson, L., Spinal, trigeminal, and cortical climbing fiber paths to the lateral vermis of the cerebellar anterior lobe in the cat, Exp. Brain Res., 41 (1981) 71-81. 2 Armstrong, D.M., Functional significance of connections of the inferior olive. Physiol. Rev., 54 (1974) 358-417. 3 Bradley, D.J., Ghelarducci, B., Paton, J.F.R. and Spyer, K.M., The cardiovascular responses elicited from the posterior cerebellar cortex in the anaesthetized and decerebrate rabbit, J. Physiol. (Lond.), 383 (1987) 537-550. 4 Cottle, M.K.W., Degeneration studies of primary afferents of the IXth and Xth cranial nerves in the cat, J. Comp. Neurol., 122 (1964) 329-345. 5 Del Bo, A., Sved, A.F. and Reis, D.J., Fastigial stimulation releases vasopressin in amounts that elevate arterial pressure, Am. J. Physiol., 224 (1983) H687-H694. 6 Dell, D. and Olson, R., Projections thalamiques, corticales et cerebeleuses des afferences viscerales vagales, C.R. Soc. Biol. (Paris), 45 (1951) 1084-1088. 7 Denoth, E, Magherini, P.C., Pompeiano, O. and Stanojevic, M., Responses of Purkinje cells of cerebellar vermis to sinosoidai rotation of the neck, J. Neurophysiol., 43 (1980) 46-59. 8 Dietrichs, E. and Haines, D.E., Interconnections between hypothalamus and cerebellum, Anat. Embryol., 179 (1989) 207-220. 9 Donaldson, I.M.L. and Hawthorne, M.E., Coding of visual information by units in the cat cerebellar vermis, Exp. Brain Res., 34 (1979) 27-48. 10 Dormer, K.J., Andrezik, J.A., Person, R.J., Braggio, J.T. and Foreman, R.D., Fastigial nucleus cardiovascular response and brainstem lesions in the beagle, Am. J. Physiol., 250 (1986) 231-239. 11 Dow, R.S., Kramer, R.E. and Robertson, L.T., Disorders of the cerebellum. In R.J. Joynt (Ed.), Clinical Neurology, Lippincott, Philadelphia, in press. 12 Dowdy, S. and Wearden, S., Statistics for Research, Wiley, New
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