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Olfactory nerve projections to the olfactory bulb in rabbit: demonstration by means of a simplified ammoniacal silver degeneration method
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Olfactory nerve projections to the olfactory bulb in rabbit: demonstration by means of a simplified ammoniacal silver degeneration method Physiological 1 and anatomical2, 3 studies have suggested some degree of topographical organization in the projection of nerves from olfactory receptors in the nose to the glomeruli of the olfactory bulb in mammals. Thus far there has been no attempt to study this pathway by making restricted lesions in the olfactory mucosa and examining, light microscopically, the resultant degenerated olfactory nerve fibers and their terminals in the bulb. The present investigation in rabbits has been undertaken, firstly, to determine the stainability of these degenerating axons and terminals in the olfactory bulb with selective silver methods, and, secondly, to describe more precisely the localization of the olfactory nerve projection, as a complement to physiological analyses of bulbar responses to olfactory stimulation, Since the olfactory nerve is composed of unmyelinated axons only 4,9, this system has provided an especially good opportunity to test the feasibility of using selective silver methods to study degenerating central nervous system pathways composed primarily or wholly of unmyelinated fibers. Unilateral olfactory nerve lesions were made, under aseptic conditions, in white male rabbits. In several animals a small opening was made in the nasal bone near the midline and anterior to the bulb. Through this exposure small bundles of olfactory nerves lying in the submucosa of the 'dorsal recess'", between the nasoturbinate and the septum, could be clearly visualized. In each case a few of these bundles were transected with the sharp point of a scalpel blade. In other animals an opening was made over the olfactory bulb and extended anteriorly by careful rongeuring. This procedure exposed a relatively large and constant dorsal bundle of olfactory nerve fibers in the ethmoid bone (@ Shepherd 1'~) in which lesions were made by transection or electrocautery. Dissections have shown us that this dorsal bundle contains fibers from the dorsal recess and from the neighboring nasoturbinate and dorsal area of the septum. Following postoperative recovery for 5-9 days, the animals were perfused with either 10~,q phosphate-buffered formalin or 10~,£ formal-saline, preceded in each case by 0.9 ~ saline. Bulbs were removed and stored for at least several days in 10°,~ buffered formalin with sucrose (30°/(i) after which they were cut into 30/~na frozen sections in the transverse plane. Sections were stained either by the following simplified ammoniacal silver method 5,6 or by the Fink-Heimer method II (ref. 8) as a control: (I) Distilled water rinse; (2) 2.5g~; uranyl nitrate (5 rain)" (3) distilled water rinse; (4) ammoniacal silver (3-15 min) (40 ml of 1.5~o silver nitrate, 24 ml of 95°/0 ethyl alcohol, 4 ml of concentrated ammonium hydroxide, 3.6 ml of 2.5 o/,;sodium hydroxide); (5) reducer (2-5 min) (810 ml of distilled water, 90 ml of 100%o ethyl alcohol, O/ 27 ml of I o/citric acid, 27 ml of 10~o formalin); (6) distilled water rinse: (7) 0.5/o //0 sodium thiosulfate (2 rain); (8) distilled water. Following silver reactions, sections were bleached by short (10-15 sec) treatment in l ~ potassium ferricyanide, mounted and counterstained with cresyl violet. Both silver methods produced selective impregnation of the degenerating Brain Research, 23 (1970) 250-254
Fig. 1. Degeneration restricted to ipsilateral dorsal glomeruli (boundaries marked by arrows) following lesion of nerves in dorsal recess of left nasal cavity. Eager method (see text), 5 days degeneration. Section bleached with I }o~ potassium ferricyanide and counterstained with cresyl violet. Abbreviations: ON, olfactory nerve layer; GL, glomerular layer; EP, external plexiform layer: MB, mitral cell body layer; GR, granule cell layer..< 100. Fig. 2. Degenerated olfactory nerve fibers entering (large arrow) and terminating in two dorsal glomeruli following lesion of nerves in dorsal recess of left nasal cavity. Normal glomeruli (NG) lie on either side, and portions of the affected glomeruli (small arrows) are without degeneration. Fink Heimer method II, 5 days degeneration. Bleached and counterstained as in Fig. I. 350.
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unmyelinated axons in the olfactory glomeruli. In cases with lesions in the dorsal recess of the nasal cavity, degeneration was found in glomeruli in the dorsal aspect of the ipsilateral bulb (Figs. I 4). Some background staining was discernible even in those glomeruli judged to be without experimentally p r o d u c e d degeneration, but the level was no greater than that o f glomeruli in the contralateral normal bulb and only slightly greater than that seen in bulbs from a normal, unoperated animal used as a further control. It is possible that the staining in ' n o r m a l ' glomeruli is a consequence o f degeneration resulting from chronic rhinitis, to which rabbits are notoriously subject. Olfactory nerve degeneration stained by the procedure outlined above:,, ~; was indistinguishable from that obtained with the widely-used, and considerably more complex, Fink--Heimer method II (ref. 8), employed as a control procedure in this study (of. Figs. 2 and 3). In addition, the degeneration was very similar to that seen in other parts o f the central nervous system to which this procedure has been applied ~,~. There is g o o d reason to believe that the smallest impregnated structures observed within the glomeruli include degenerated axon terminals since they correspond well in size with similarly degenerated terminal boutons observed in recent fine structural investigations o f this pathwa3, in rats ~:'. These observations give added support to recent data '5,~,11 which have sho~ n that selective staining o f degenerating axons and their terminals with the amtnoniacal silver and reducer solutions above is in no way dependent upon 'suppression' of normal fiber impregnation by the pretreatments suggested in recent modifications o f the N a u t a technique s,~e. In the present cases pretreatment with uranyl nitrate was necessary to maximize the impregnation o f degenerated axons, but it could be eliminated without jeopardizing the selectivity o f the method (Fig. 4). One must conclude, then, that in this degenerating system, as in others'5,6, ~l, the selectivity o f the ammoniacal silver reaction is dependent s o m e h o w upon the differential availability of reactive sites in degenerating axons as opposed to normal ones. One striking feature o f the present results was the observed wide variation in intensity of degeneration from glomerulus to glomerulus in affected dorsal regions. It was often the case that a glomerulus containing a dense accumulation o f particles was flanked by others with no more than a b a c k g r o u n d level o f staining (Figs. 2 and 3). Instances o f this were found in the middle o f the dorsal region as well as at
Fig. 3. Degenerated olfactory nerve fibers (large arrow) entering two ipsilateral dorsal glomeruli following lesion of nerves in dorsal recess of left nasal cavity. The 'finger-like' distribution of degeneration is especially apparent in the right glomerulus (see text for discussion). Part of the middle glomerulus is without degeneration (small arrow), and a normal glomerulus (NG) lies to the left in the field. The selectivity and intensity of staining are indistinguishable from that obtained with the Fink-Heimer method II (C~ Fig. 2). Eager method (see text), 5 days degeneration. Bleached and counterstained as in Fig. 1. ~ 450. Fig. 4. Degeneration in an ipsilateral dorsal glomerulus (arrow) following lesion of nerves in dorsal recess of left nasal cavity. Stained by Eager method (see text) but without pretreatment with uranyl nitrate, There is some diminution in amount of stainable degeneration but no decrease in selectivity. Normal glomeruli (NG) lie on either side. Five days degeneration. Bleached and counterstained as in Fig. I. ~: 450. Brain Research, 23 (1970) 250-254
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Brain Research, 23 (1970)250-254
SHOR[ COMMUNI(TATIONS
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its borders. Within a glomerulus, the d e g e n e r a t i o n , especially when it was heavy, often formed, patches or 'fingers' in restricted p a r t s o f the g l o m e r u l u s (Figs. 2 and 3), the r e m a i n i n g parts being relatively free o f d e g e n e r a t i o n . In f a v o r a b l e sections, these areas could be seen to be related to a single b u n d l e o f d e g e n e r a t i n g o l f a c t o r y nerve axons entering from the nerve layer (Figs. 2 and 3). These areas a p p e a r to c o r r e s p o n d to the d a r k l y stained p r e s y n a p t i c regions d e s c r i b e d by others 7,~4. The d e m o n s t r a t i o n o f a t o p o g r a p h i c a l r e l a t i o n between the d o r s a l recess o f the nasal cavity and the d o r s a l aspect o f the b u l b is consistent with p r e v i o u s reports'~, 3 and c o r r e s p o n d s well to o u r o b s e r v a t i o n s on n a s a l dissections. This o r g a n i z a t i o n m a y have i m p o r t a n t i m p l i c a t i o n s for an u n d e r s t a n d i n g o f the o l f a c t o r y input to the bulb. The present results also raise the possibility t h a t a small p a r t o f the d o r s a l nasal m u c o s a projects to selective g r o u p s o f glomeruli in the d o r s a l bulb, and even to selective g r o u p s o f p o s t s y n a p t i c elements within a glomerulus. This g r o u p i n g could p r o v i d e the a n a t o m i c a l basis for potentials r e c o r d e d across i n d i v i d u a l g l o m e r u l i which show selective responses to nasal s t i m u l a t i o n with different o d o r s a0. F i n a l l y , it will be o f interest to relate these findings to the responses o f b u l b a r units to electrical s t i m u l a t i o n o f o l f a c t o r y nerve bundles. This w o r k was s u p p o r t e d , in part, by G r a n t s NS-04760 and NS-07609 from the N a t i o n a l Institute o f N e u r o l o g i c a l Diseases a n d Stroke, U.S. Public H e a l t h Service. The technical assistance o f Miss N a n c y M a r g i o t t a is gratefully a c k n o w l e d g e d . Departments of Biology, Anatomy, attd Physiology, Yale University, New Haven, Conn. (U.S.A.)
L.J. LAND R.P. EAGER G. M. SHEPHERD
1 ADRIAN,E. D., Sensory discrimination, Brit. med. Bull., 6 (1950) 330-333. 2 CLARK,W. E. LEG., The projection of the olfactory epithelium on the olfactory bulb in the rabbit, J. Neurol. Neurosurg. Psychiat., 14 (1951) 1-10. 3 CLARK, W. E. LEG., Inquiries into the anatomical basis of olfactory discrimination, Proc, roy. Soc. B, 146 (1957) 299-319. 4 DE LORENZO, A. J., Electron microscopic observations of the olfactory mucosa and olfactory nerve, J. biophys, bioehem. Cytol., 3 (1957) 839-850. 5 EAGER,R. P., The stainability of mossy and climbing fiber degeneration in the cerebellum of the cat: an evaluation of current silver degeneration methods, Anat. Ree., 166 (1970) 405. 6 EAGER, R. P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 7 ESTABLE-Pum,J. F., Light and electron microscopy of the olfactory bulb of normal and experimentally treated rats, Ph.D. Dissertation, Stanford University, 1968. 8 FINK,R. P., AND HE1MER,L., Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 9 GASSER,H. S., Olfactory nerve fibers, J. gen. Physiol., 39 (1956) 473-496. 10 LEVETEAU,J., .aND MAcLEOD, P., Olfactory discrimination in the rabbit olfactory glomerulus, Science, 153 (1966) 175-176. 11 LOEWY, A. D., Ammoniacal silver staining of degenerating axons, Acta neuropath. (Berl.), 14 (1969) 226 236. 12 NAUTA,W. J. H., Silver impregnation of degenerating axons. In W. F. WINDLE (Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, pp. 17-26. 13 PINCHING, A. J., Persistence of post-synaptic membrane thickenings after degeneration of olfactory nerves, Brain Research, 16 (1969) 277-281. 14 REESE,T. S., ANDBRIGHTMAN,M. W., Olfactory surface and central olfactory connections in some vertebrates. In Ciba Symposium on Taste and Olfaction, in press. 15 SHEPHERD,G. M., Responses of mitral cells to olfactory nerve volleys in the rabbit. J. Physiol. (Lond.), 168 (1963) 89-100. (Accepted July 31st, 1970)
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Olfactory nerve projections to the olfactory bulb in rabbit: demonstration by means of a simplified ammoniacal silver degeneration method Physiological 1 and anatomical2, 3 studies have suggested some degree of topographical organization in the projection of nerves from olfactory receptors in the nose to the glomeruli of the olfactory bulb in mammals. Thus far there has been no attempt to study this pathway by making restricted lesions in the olfactory mucosa and examining, light microscopically, the resultant degenerated olfactory nerve fibers and their terminals in the bulb. The present investigation in rabbits has been undertaken, firstly, to determine the stainability of these degenerating axons and terminals in the olfactory bulb with selective silver methods, and, secondly, to describe more precisely the localization of the olfactory nerve projection, as a complement to physiological analyses of bulbar responses to olfactory stimulation, Since the olfactory nerve is composed of unmyelinated axons only 4,9, this system has provided an especially good opportunity to test the feasibility of using selective silver methods to study degenerating central nervous system pathways composed primarily or wholly of unmyelinated fibers. Unilateral olfactory nerve lesions were made, under aseptic conditions, in white male rabbits. In several animals a small opening was made in the nasal bone near the midline and anterior to the bulb. Through this exposure small bundles of olfactory nerves lying in the submucosa of the 'dorsal recess'", between the nasoturbinate and the septum, could be clearly visualized. In each case a few of these bundles were transected with the sharp point of a scalpel blade. In other animals an opening was made over the olfactory bulb and extended anteriorly by careful rongeuring. This procedure exposed a relatively large and constant dorsal bundle of olfactory nerve fibers in the ethmoid bone (@ Shepherd 1'~) in which lesions were made by transection or electrocautery. Dissections have shown us that this dorsal bundle contains fibers from the dorsal recess and from the neighboring nasoturbinate and dorsal area of the septum. Following postoperative recovery for 5-9 days, the animals were perfused with either 10~,q phosphate-buffered formalin or 10~,£ formal-saline, preceded in each case by 0.9 ~ saline. Bulbs were removed and stored for at least several days in 10°,~ buffered formalin with sucrose (30°/(i) after which they were cut into 30/~na frozen sections in the transverse plane. Sections were stained either by the following simplified ammoniacal silver method 5,6 or by the Fink-Heimer method II (ref. 8) as a control: (I) Distilled water rinse; (2) 2.5g~; uranyl nitrate (5 rain)" (3) distilled water rinse; (4) ammoniacal silver (3-15 min) (40 ml of 1.5~o silver nitrate, 24 ml of 95°/0 ethyl alcohol, 4 ml of concentrated ammonium hydroxide, 3.6 ml of 2.5 o/,;sodium hydroxide); (5) reducer (2-5 min) (810 ml of distilled water, 90 ml of 100%o ethyl alcohol, O/ 27 ml of I o/citric acid, 27 ml of 10~o formalin); (6) distilled water rinse: (7) 0.5/o //0 sodium thiosulfate (2 rain); (8) distilled water. Following silver reactions, sections were bleached by short (10-15 sec) treatment in l ~ potassium ferricyanide, mounted and counterstained with cresyl violet. Both silver methods produced selective impregnation of the degenerating Brain Research, 23 (1970) 250-254
Fig. 1. Degeneration restricted to ipsilateral dorsal glomeruli (boundaries marked by arrows) following lesion of nerves in dorsal recess of left nasal cavity. Eager method (see text), 5 days degeneration. Section bleached with I }o~ potassium ferricyanide and counterstained with cresyl violet. Abbreviations: ON, olfactory nerve layer; GL, glomerular layer; EP, external plexiform layer: MB, mitral cell body layer; GR, granule cell layer..< 100. Fig. 2. Degenerated olfactory nerve fibers entering (large arrow) and terminating in two dorsal glomeruli following lesion of nerves in dorsal recess of left nasal cavity. Normal glomeruli (NG) lie on either side, and portions of the affected glomeruli (small arrows) are without degeneration. Fink Heimer method II, 5 days degeneration. Bleached and counterstained as in Fig. I. 350.
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unmyelinated axons in the olfactory glomeruli. In cases with lesions in the dorsal recess of the nasal cavity, degeneration was found in glomeruli in the dorsal aspect of the ipsilateral bulb (Figs. I 4). Some background staining was discernible even in those glomeruli judged to be without experimentally p r o d u c e d degeneration, but the level was no greater than that o f glomeruli in the contralateral normal bulb and only slightly greater than that seen in bulbs from a normal, unoperated animal used as a further control. It is possible that the staining in ' n o r m a l ' glomeruli is a consequence o f degeneration resulting from chronic rhinitis, to which rabbits are notoriously subject. Olfactory nerve degeneration stained by the procedure outlined above:,, ~; was indistinguishable from that obtained with the widely-used, and considerably more complex, Fink--Heimer method II (ref. 8), employed as a control procedure in this study (of. Figs. 2 and 3). In addition, the degeneration was very similar to that seen in other parts o f the central nervous system to which this procedure has been applied ~,~. There is g o o d reason to believe that the smallest impregnated structures observed within the glomeruli include degenerated axon terminals since they correspond well in size with similarly degenerated terminal boutons observed in recent fine structural investigations o f this pathwa3, in rats ~:'. These observations give added support to recent data '5,~,11 which have sho~ n that selective staining o f degenerating axons and their terminals with the amtnoniacal silver and reducer solutions above is in no way dependent upon 'suppression' of normal fiber impregnation by the pretreatments suggested in recent modifications o f the N a u t a technique s,~e. In the present cases pretreatment with uranyl nitrate was necessary to maximize the impregnation o f degenerated axons, but it could be eliminated without jeopardizing the selectivity o f the method (Fig. 4). One must conclude, then, that in this degenerating system, as in others'5,6, ~l, the selectivity o f the ammoniacal silver reaction is dependent s o m e h o w upon the differential availability of reactive sites in degenerating axons as opposed to normal ones. One striking feature o f the present results was the observed wide variation in intensity of degeneration from glomerulus to glomerulus in affected dorsal regions. It was often the case that a glomerulus containing a dense accumulation o f particles was flanked by others with no more than a b a c k g r o u n d level o f staining (Figs. 2 and 3). Instances o f this were found in the middle o f the dorsal region as well as at
Fig. 3. Degenerated olfactory nerve fibers (large arrow) entering two ipsilateral dorsal glomeruli following lesion of nerves in dorsal recess of left nasal cavity. The 'finger-like' distribution of degeneration is especially apparent in the right glomerulus (see text for discussion). Part of the middle glomerulus is without degeneration (small arrow), and a normal glomerulus (NG) lies to the left in the field. The selectivity and intensity of staining are indistinguishable from that obtained with the Fink-Heimer method II (C~ Fig. 2). Eager method (see text), 5 days degeneration. Bleached and counterstained as in Fig. 1. ~ 450. Fig. 4. Degeneration in an ipsilateral dorsal glomerulus (arrow) following lesion of nerves in dorsal recess of left nasal cavity. Stained by Eager method (see text) but without pretreatment with uranyl nitrate, There is some diminution in amount of stainable degeneration but no decrease in selectivity. Normal glomeruli (NG) lie on either side. Five days degeneration. Bleached and counterstained as in Fig. I. ~: 450. Brain Research, 23 (1970) 250-254
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Brain Research, 23 (1970)250-254
SHOR[ COMMUNI(TATIONS
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its borders. Within a glomerulus, the d e g e n e r a t i o n , especially when it was heavy, often formed, patches or 'fingers' in restricted p a r t s o f the g l o m e r u l u s (Figs. 2 and 3), the r e m a i n i n g parts being relatively free o f d e g e n e r a t i o n . In f a v o r a b l e sections, these areas could be seen to be related to a single b u n d l e o f d e g e n e r a t i n g o l f a c t o r y nerve axons entering from the nerve layer (Figs. 2 and 3). These areas a p p e a r to c o r r e s p o n d to the d a r k l y stained p r e s y n a p t i c regions d e s c r i b e d by others 7,~4. The d e m o n s t r a t i o n o f a t o p o g r a p h i c a l r e l a t i o n between the d o r s a l recess o f the nasal cavity and the d o r s a l aspect o f the b u l b is consistent with p r e v i o u s reports'~, 3 and c o r r e s p o n d s well to o u r o b s e r v a t i o n s on n a s a l dissections. This o r g a n i z a t i o n m a y have i m p o r t a n t i m p l i c a t i o n s for an u n d e r s t a n d i n g o f the o l f a c t o r y input to the bulb. The present results also raise the possibility t h a t a small p a r t o f the d o r s a l nasal m u c o s a projects to selective g r o u p s o f glomeruli in the d o r s a l bulb, and even to selective g r o u p s o f p o s t s y n a p t i c elements within a glomerulus. This g r o u p i n g could p r o v i d e the a n a t o m i c a l basis for potentials r e c o r d e d across i n d i v i d u a l g l o m e r u l i which show selective responses to nasal s t i m u l a t i o n with different o d o r s a0. F i n a l l y , it will be o f interest to relate these findings to the responses o f b u l b a r units to electrical s t i m u l a t i o n o f o l f a c t o r y nerve bundles. This w o r k was s u p p o r t e d , in part, by G r a n t s NS-04760 and NS-07609 from the N a t i o n a l Institute o f N e u r o l o g i c a l Diseases a n d Stroke, U.S. Public H e a l t h Service. The technical assistance o f Miss N a n c y M a r g i o t t a is gratefully a c k n o w l e d g e d . Departments of Biology, Anatomy, attd Physiology, Yale University, New Haven, Conn. (U.S.A.)
L.J. LAND R.P. EAGER G. M. SHEPHERD
1 ADRIAN,E. D., Sensory discrimination, Brit. med. Bull., 6 (1950) 330-333. 2 CLARK,W. E. LEG., The projection of the olfactory epithelium on the olfactory bulb in the rabbit, J. Neurol. Neurosurg. Psychiat., 14 (1951) 1-10. 3 CLARK, W. E. LEG., Inquiries into the anatomical basis of olfactory discrimination, Proc, roy. Soc. B, 146 (1957) 299-319. 4 DE LORENZO, A. J., Electron microscopic observations of the olfactory mucosa and olfactory nerve, J. biophys, bioehem. Cytol., 3 (1957) 839-850. 5 EAGER,R. P., The stainability of mossy and climbing fiber degeneration in the cerebellum of the cat: an evaluation of current silver degeneration methods, Anat. Ree., 166 (1970) 405. 6 EAGER, R. P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 7 ESTABLE-Pum,J. F., Light and electron microscopy of the olfactory bulb of normal and experimentally treated rats, Ph.D. Dissertation, Stanford University, 1968. 8 FINK,R. P., AND HE1MER,L., Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 9 GASSER,H. S., Olfactory nerve fibers, J. gen. Physiol., 39 (1956) 473-496. 10 LEVETEAU,J., .aND MAcLEOD, P., Olfactory discrimination in the rabbit olfactory glomerulus, Science, 153 (1966) 175-176. 11 LOEWY, A. D., Ammoniacal silver staining of degenerating axons, Acta neuropath. (Berl.), 14 (1969) 226 236. 12 NAUTA,W. J. H., Silver impregnation of degenerating axons. In W. F. WINDLE (Ed.), New Research Techniques of Neuroanatomy, Thomas, Springfield, Ill., 1957, pp. 17-26. 13 PINCHING, A. J., Persistence of post-synaptic membrane thickenings after degeneration of olfactory nerves, Brain Research, 16 (1969) 277-281. 14 REESE,T. S., ANDBRIGHTMAN,M. W., Olfactory surface and central olfactory connections in some vertebrates. In Ciba Symposium on Taste and Olfaction, in press. 15 SHEPHERD,G. M., Responses of mitral cells to olfactory nerve volleys in the rabbit. J. Physiol. (Lond.), 168 (1963) 89-100. (Accepted July 31st, 1970)