Brain Research, 96 (1975) 93-98 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
93
Two sites of axonal spike initiation in a bimodal interneuron
MICHAEL O'SHEA Department of Zoology, University of California, Berkeley, Calif. 94720 (U.S.A.) (Accepted May 28th, 1975)
Multiple sites of spike initiation have been demonstrated in moto- and interneuronal dendrites of both vertebrates and invertebrates. In most examples (alligator Purkinje cellslZ, 14, certain crab24, 25 and crayfish 28 motoneurons), single dendrite spikes do not initiate axon spikes and are, therefore, functionally equivalent to subthreshold EPSPs. There are examples, however, of neurons in which dendrite spikes have access to the axon. They include some alligator Purkinje cells13, ~4, oculomotor neurons in certain teleosts ~0-1~, cat neocortical neurons 2~ and the multisegmental tactile interneurons (MTIs) of crayfish4, 9. With the exception of the MTIs, axon spikes in these examples are initiated at a single point. Crayfish provide the most thoroughly studied case of central neurons (the MTIs) in which dendrite spikes initiate axon spikes at more than one site in a single dendritic arborization 4. The discovery of other examples of the same phenomenon will have interesting theoretical implications, some of which will be discussed here. This paper reports on an identified movement detector (MD) neuron in the brain of the locust (Schistocerca vaga) which generates spikes in response to both visual and auditory stimuli and which has two, modality-specific, sites of axon spike initiation, one located at each end of the axon. Action potentials initiated by stimuli in one modality travel along the major part of the neuron's axon in the opposite direction to those initiated by stimuli in the other. Bi- and multimodal neurons are widespread in both vertebrates and invertebrates. It is generally assumed that single action potentials in them are ambiguous, and their ubiquity is persuasive evidence, therefore, that at the level of the single neuron the nervous system is largely indeterministic s. The discovery of modality-specific initiation sites challenges the validity of the view that spikes in bimodal cells are unreliable because they cannot uniquely be interpreted. The lobular giant movement detector (LGMD) is an important integrative element in a small population of identified locust neurons which mediate escape jumping3,~,~7,18. It responds vigorously to novel and abrupt movement of small and contrasting objects anywhere in the visual field ~6. In addition, it has recently been found to generate one and sometimes two impulses in response to loud and transient sound. The auditory response is highly phasic and habituates rapidly on
94
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Fig. 1. A: a diagrammatic representation of the anatomy of the LGMD and DCMD as drawn from cobalt impregnation (see Refs. 17-19). The abrupt thickening of the L G M D axon is indicated by an arrow. The L G M D and D C M D make electrical synaptie contact in the brain and the D C M D extends its axon through the suboesophageal (SO) ganglion, the first (TI) and second (T2) thoracic ganglia to the third thoracic ganglion. Electrode placements, referred to throughout this paper, are X in the fan-like arborization of the LGMD, Y in the proximal part of the L G M D axon and Z on the thoracic nerve cord between TI and T2. B: upper trace is an intracellular recording from Y and the lower trace an extracellular recording of the D C M D at Z. Action potentials in the L G M D were initiated by injecting depolarizing current through the microelectrode. The oscilloscope was triggered by the L G M D spikes and about 30 sweeps are superimposed. Note that the D C M D follows 1:1 with practically no latency variation. Calibration: 0.5 msec and (trace Y only) 40 inV. C: an intracellular recording from site X in the fan-like arborization of the LGMD. The first impulse (v) was initiated by a visual stimulus and arises from a compound EPSP. The second (a), initiated by auditory stimulation, arises abruptly from baseline. At site X there is a constant background of EPSPs from presynaptic visual interneurons; any correspondence between auditory spikes and PSPs here is coincidental. Calibration: 40 msec and 10 inV. D: upper trace is an intracellular recording from the L G M D at site X and the lower an extracellular recording of the D C M D at Z. The oscilloscope was triggered from L G M D spikes and stimuli provided in both visual and auditory modalities. Two distinct classes of latency are evident. The latency in response to auditory stimulation (a) is shorter by 0.38 msec than to visual stimulation (v). In each modality 5 sweeps of the oscilloscope are superimposed. Calibration: 0.5 msec and (trace X only) 10 mV.
r e p e a t e d s t i m u l a t i o n . B o t h visual a n d a u d i t o r y spikes are t r a n s m i t t e d 1:1 f r o m the L G M D to t h e d e s c e n d i n g c o n t r a l a t e r a l m o v e m e n t d e t e c t o r ( D C M D ) , w h i c h e x t e n d s its a x o n f r o m the b r a i n to the t h i r d t h o r a c i c g a n g l i o n w h e r e it m a k e s o u t p u t c o n n e c t i o n s w i t h m o t o n e u r o n s i n v o l v e d w i t h j u m p i n g ~. R e c o r d i n g s w e r e m a d e i n t r a c e l l u l a r l y f r o m the L G M D a n d e x t r a c e l l u l a r l y f r o m the D C M D . G l a s s m i c r o e l e c t r o d e s filled w i t h 3 M p o t a s s i u m a c e t a t e a n d h a v i n g resistances f r o m 50 to 80 m~) were e m p l o y e d for r e c o r d i n g i n t r a c e l l u l a r l y f r o m the L G M D in the n e u r o p i l e o f t h e o p t i c l o b e a n d brain. Silver h o o k e l e c t r o d e s p l a c e d o n the t h o r a c i c n e r v e c o r d served to m o n i t o r the a c t i v i t y o f the D C M D , w h i c h is t h e
95 most conspicuous (3-4 mV) and easily identified unit when recorded in this way. Visual excitation to the MD neurons was provided by hand movements in the LGMD's receptive field. Auditory responses to pure tones produced by a wave form generator were weak and unreliable; a far more effective stimulus was found to be a loud brief 'hiss' by the author. The anatomy of the DCMD and LGMD, as drawn from intracellular cobalt impregnation, and electrode positions are represented diagrammatically in Fig. 1A. The relative positions of sites of spike initiation in the L G M D were determined by comparing conduction times for action potentials generated by visual and auditory stimulation between a recording site in the L G M D and another on the DCMD. This is possible because the two cells are connected by an electrical synapse which transmits spikes 1 : 1 with no measurable variation in latency (Fig. 1B). Furthermore, assuming there are no local and anomalous variations in conduction velocity on the L G M D axon, it is possible to calculate with some precision the spatial separation of initiation sites from measurements of conduction times. The validity of this assumption is founded on anatomical grounds. The spike supporting part of the L G M D axon has throughout its length a rather constant diameter (15 ~m). Estimates of propagation velocity, determined by two-point recording on the L G M D axon, lie between 2.9 and 3.1 m/sec at room temperature (20 °C). The close correspondence with the conduction velocity of the D C M D axon 8 (3.1 m/sec) and the similarity of L G M D and D C M D axonal diameters, suggests that large, local variations in conduction velocity would have clear anatomical correlates. The conduction time for a visually induced spike from recording site X in the L G M D to site Z on the D C M D axon is invariable (Fig. 1D). It is not measurably altered by recording at different locations in the fan-like arborization of the L G M D but is reduced by recording proximally to an abrupt thickening of the axon from 5 #m to 15 ~m, which occurs at the junction of optic lobe and brain (Fig. 1A). The dendrites of the fan arborization are not spike supporting. Action potentials in their proximal regions reach 25 mV and are considerably smaller in more distal regions which suggests that they are conducted passively and distally (or centrifugally) from their site of initiation into the fan. Since the abrupt axonal thickening is the most distal part of the L G M D where overshooting action potentials can be recorded, it is proposed to be the site of visual spike initiation. Auditory action potentials have the same shape and amplitude when recorded at site X as those induced by visual stimulation but differ from them in two important respects. First, they do not rise from EPSPs (Fig. 1C), suggesting that the auditory synapse onto the L G M D is remote from recording site X. Second, they arrive at site Z earlier (Fig. 1D), showing that they are initiated closer to Z than visual spikes and are propagated along the L G M D axon to the site of visual spike initiation. If X is indeed distal to the site of visual spike initiation, the difference in arrival times of auditory and visual spikes at Z is twice the temporal separation of the two initiation sites. In Fig. 1D, which is data taken from an adult female, this difference is 0.38 msec and the temporal separation of initiation sites is, therefore, 0.19 msec. Using 3 m/sec as the axonal conduction velocity of the LGMD, the spatial separation is 576 #m.
96
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Fig. 2. A: upper trace is an intracellular recording from site Y (see Fig. IA), the middle trace an extracellular recording from Z and the lower trace a recording of a loud sound by the author. The single auditory action potential (a) in the LGMD is preceded by a compound EPSP and is followed in the middle trace by a spike in the DCMD (arrowed). Action potentials in the L G M D at site Y are large (80-90 mV) and overshooting and are clipped in this recording. Compare with Fig. 1C and Fig. 2B. Calibration: 10 msec and (trace Y only) 10 inV. B: upper trace is an intracellular recording from Y and the middle trace an extracellular recording from Z. Three spikes induced by visual stimulation (v) are shown and they are followed 1:1 by DCMD spikes (arrowed). Note that the L G M D spikes arise abruptly from baseline without preceding PSP. Compare with Fig. IC and Fig. 2A. Calibration: 10 msec and (trace Y only) 10 mV. C: subthreshold auditory EPSPs in the LGMD. The upper trace is an intracellular recording from site Y, the middle trace an extracellular recording of a brief 'hiss'. Spikes from auditory neurons ascending from the third thoracic ganglion, where sensory afferents from the tympanic organs enter the central nervous system, are seen in trace Z. None appear to be directly presynaptic to the LGM D. Calibration: 20 msec and (trace Y only) 5 mV. D: upper trace is an intracellular recording from the L G M D at Y and the lower trace an extracellular recording of the D C M D at Z. The oscilloscope was triggered by L G M D spikes and stimuli provided in both visual and auditory modalities. Two visual (v) and one auditory (a) response are superimposed. The auditory D C M D spike precedes fractionally the visual spikes, showing that site Y is very close to the initiation site for auditory spikes. This difference in latency to auditory and visual spikes should be compared to that in Fig. 1D. Absolute latency measurements and the apparent duration of the extracellularly recorded D C M D spike depend upon the location of the bipolar electrodes on the nerve cord between T1 and T2 (see Fig. 1A). Calibration: 0.5 msec and (trace Y only) 25 mV.
D i r e c t m e a s u r e m e n t s t a k e n f r o m c o b a l t i m p r e g n a t i o n o f the L G M D in a d u l t f e m a l e s s h o w the d i s t a n c e f r o m the p r o p o s e d site o f visual spike i n i t i a t i o n to t h e p r o x i m a l e n d o f the a x o n in t h e b r a i n to be a b o u t 580 # m . T h e r e is a r e m a r k a b l e c o r r e s p o n d e n c e b e t w e e n t h e c a l c u l a t e d s e p a r a t i o n o f i n i t i a t i o n sites a n d the a x o n a l length, s h o w i n g t h a t the a s s u m p t i o n o f c o n s t a n t a x o n a l v e l o c i t y a n d t h e p r o p o s a l t h a t visual spikes are initiated at t h e a b r u p t a x o n a l t h i c k e n i n g are v e r y p r o b a b l y c o r r e c t . C o n f i r m a t i o n t h a t a u d i t o r y spikes are i n i t i a t e d at t h e p r o x i m a l e n d o f t h e a x o n was p r o v i d e d b y r e c o r d i n g i n t r a c e l l u l a r l y f r o m the L G M D the DCMD
close to its s y n a p s e w i t h
(site Y in Fig. 1A). H e r e o n e r e c o r d s E P S P s in r e s p o n s e to a u d i t o r y sti-
97 mulation (Fig. 2A and C) which can give rise to spikes. Visual spikes at this site arise abruptly without preceding PSPs (Fig. 2B). The conduction times for auditory and visual spikes from Y to Z are practically identical, which indicates that electrodes placed at the proximal end of the L G M D are very close to the site of axonal spike initiation for auditory stimulation (Fig. 2D). Two consequences of separating modality-specific sites of initiation will be considered. First, as suggested by Calabrese and Kennedy 4, postsynaptic inhibition can easily be directed selectively at specific modalities of excitation within a single cell. The L G M D provides an example of this phenomenon; spike initiation in response to rapid whole-field movement but not sound is prevented by postsynaptic inhibition directed at a small branch of the L G M D (subfield C of O'Shea and Williams la) which arises between the fan-like arborization and the site of visual spike initiation 16. These inhibitory potentials are not seen at the site of auditory spike initiation. Second, an action potential in a bimodal interneuron does not necessarily transmit ambiguous information. It is possible for an interneuron to encode unambiguously the sensory origin of each spike. This depends upon the ability of postsynaptic units to detect the temporal differences corresponding to the two sites; these could be detected by a single postsynaptic neuron and several models of suitable mechanisms are available (e.g., see Rail22). The complexities of regional differentiation of vertebrate neurons 27 provide the anatomical requirements necessary for multiplexing or distinguishing between individual action potentials in a single cell. Axoaxonal synapses are widespreadt, 5,6, 19,20,26, and although presynaptic inhibition is the function most often attributed to them, it is not implausible that some initiate impulses. Axodendritic synapses at the nodes of Ranvier 2 and dendrodendritic synapses 7,2a could form the anatomical substrate of a readout system. While it is true that interpretation of individual action potentials is not possible when collisions occur or when, between readout points, the axon carries more than one impulse in the same direction e..t the same time, their probability, which depends on axon length and conduction velocity, may be low and have a small effect on reliability. In the absence, therefore, of detailed information about spike initiation and anatomy of individual neurons, there is no compelling reason to assume that spikes in bimodal cells are necessarily ambiguous and unreliable. The author wishes to thank Dr. Hugh Rowell, Corey Goodman, Dr. A. Leiman, Dr. D. Kennedy and Dr. R. Calabrese for stimulating discussion and their criticisms of the manuscript. This work was supported by a grant from the U.S. Public Health Service.
1 BENNETT,M. V. L., NAKAJIMA, V., AND PAPPA8, G. D., Physiology and ultrastructure of electrotonic junctions. I. Supramedullary neurons, J. Neurophysiol., 30 (1967) 161-179. 2 BODIAN, D.~ AND TAYLOR, N., Synapse arising at a central node of Ranvier, and a note on fixation in the central nervous system, Science, 139 (1963) 330-332. 3 BURROWS,M., AND ROWELL, C. H. F., Connections between descending visual interneurones and metathoracic motoneurons in the locust, J. comp. Physiol., 85 0973) 221-234.
98 4 CALABRESE, R. L., AND KENNEDY, D., Multiple sites of spike initiation in a single dendritic system, Brain Research, 82 (1974) 316-321. 5 COLLONNIER, M., AND GUILLERY, R. W., Synaptic organisation of the lateral geniculate nucleus of the monkey, Z. Zellforsch., 62 (1964) 333-335. 6 GRAY, E. G., A morphological basis for presynaptic inhibition, Nature (Lond.), 193 (1962) 82-83. 7 HIRATA, Y., Some observations on the fine structure of the olfactory bulb of the mouse, with particular reference to the atypical synaptic configuration, Arch. Histol. Japan, 24 (1964) 293. 8 JOHN, E. R., Switchboard versus statistical theories of learning and memory, Science, 177 (1972) 850-864. 9 KENNEDY, D., AND MELLON, DE F., Synaptic activation and receptive fields in crayfish interneurons, Comp. Biochem. Physiol., 13 (1964) 275-300. 10 KORN, H., AND ~ENNETT, M. V. L., Dendritic and somatic impulse initiation in fish oculomotor neurons during vestibular nystagmus, Brain Research, 27 (1971) 169-175. I I KORN, H., AND BENNETT, M. V. L., Vestibular nystagmus and teleost oculomotor neurons: functions of electrotonic coupling and dendritic impulse initiation, J. Neurophysiol., 38 0975) 403451. 12 KRIEBEL, M. E., BENNET, M. V. L., WAXMAN, S. G., AND PAPPAS, G. O., Oculomotor neurons in fish: electrotonic coupling and multiple sites of impulse initiation, Science, 166 (1969) 520-524. 13 LLINA.S, R., AND NICHOLSON, C., Electrophysiological properties of dendrites and somata in alligator Purkinje cells, J. Neurophysiol., 34 (1971) 532-551. 14 LLIN.~S, R., NICHOLSON, C., FREEMAN, J. A., AND HILLMAN, D.E., Dendritic spikes and their inhibition in alligator Purkinje cells, Science, 160 (1968) 1132-1135. 15 O'SHEA, M., AND ROWELL, C. H. F., A spike transmitting electrical synapse between visual interneurons in the locust movement detector system, J. comp. Physiol., 97 (1975) 143-158. 16 O'SHEA, M., AND ROWELL, C. H. F., Protection from habituation by lateral inhibition, Nature (Lond.), 254 (1975) 53-55. 17 O'SHEA, M., ROWELL, C. H. F., AND WILLIAMS, J. L. D., The anatomy of a locust visual interneurone; the descending contralateral movement detector, J. exp. Biol., 60 (1974) 1-12. 18 O'SHEA, M., AND WILLIAMS, J. L. D., The anatomy and output connection of a locust visual interneurone; the lobular giant movement detector (LGMD) neurone, J. comp. Physiol., 91 (1974) 257-266. 19 PAPPAS, G. D., COHEN, E. B., AND PURPURA, D. P., Fine structure of synaptic and non-synaptic relations in the thalamus of the cat. In D. P. PURPURA AND M. YAHR (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 47-57. 20 PETERS, A., AND PALAY S. L., The morphology of laminae A and A1 of the dorsal nucleus of the lateral geniculate body of the cat, J. Anat. (Lond.), 100 (1966) 451486. 21 PURPURA, D . P . , Comparative physiology of dendrites. In G. C. QUARTON, T. MELNECHUCK AND F. O. SCHMITT (Eds.), The Neurosciences: a Study Program, Rockefeller University Press, New York, 1967, pp. 372-393. 22 RALL, W., Theoretical significance of dendritic trees for neuronal input-output relations. In R. F. REISS (Ed.), Neural Theory and Modeling, Stanford University Press, Stanford, Calif., 1964, pp. 73-97. 23 REES, T. S., AND SHEPHERD, G. M., Dendro-dendritic synapses in the central nervous system. In G. D. PAPPAS AND D. P. PURPURA (Eds.), Structure and Function of the Synapse, Raven Press, New York, 1972, pp. 121-136. 24 SANDEMAN,D. C., The site of synaptic activity and impulse initiation in an identified motoneurone in the crab brain, J. exp. BioL, 50 (1969) 771-784. 25 SANOEMAN,D. C., Integrative properties of a reflex motoneurone in the brain of the crab Carcinus maenus, Z. vergl. Physiol., 64 (1969) 450--464. 26 WALBER6, F., Axoaxonic contacts in the cuneate nucleus, probable basis for presynaptic depolarization, Exp. Neurol., 13 (1965)218-231. 27 WAXMAN, S.G., Regional differentiation of the axon: a review with special reference to the concept of the multiplex neuron, Brain Research, 47 (1972) 269-288. 28 ZOCKER, R. S., Crayfish escape behavior and central synapses. III. Electrical junctions and dendritic spikes in fast flexor motoneurons, J. NeurophysioL, 35 (1972) 638-651.
93
Two sites of axonal spike initiation in a bimodal interneuron
MICHAEL O'SHEA Department of Zoology, University of California, Berkeley, Calif. 94720 (U.S.A.) (Accepted May 28th, 1975)
Multiple sites of spike initiation have been demonstrated in moto- and interneuronal dendrites of both vertebrates and invertebrates. In most examples (alligator Purkinje cellslZ, 14, certain crab24, 25 and crayfish 28 motoneurons), single dendrite spikes do not initiate axon spikes and are, therefore, functionally equivalent to subthreshold EPSPs. There are examples, however, of neurons in which dendrite spikes have access to the axon. They include some alligator Purkinje cells13, ~4, oculomotor neurons in certain teleosts ~0-1~, cat neocortical neurons 2~ and the multisegmental tactile interneurons (MTIs) of crayfish4, 9. With the exception of the MTIs, axon spikes in these examples are initiated at a single point. Crayfish provide the most thoroughly studied case of central neurons (the MTIs) in which dendrite spikes initiate axon spikes at more than one site in a single dendritic arborization 4. The discovery of other examples of the same phenomenon will have interesting theoretical implications, some of which will be discussed here. This paper reports on an identified movement detector (MD) neuron in the brain of the locust (Schistocerca vaga) which generates spikes in response to both visual and auditory stimuli and which has two, modality-specific, sites of axon spike initiation, one located at each end of the axon. Action potentials initiated by stimuli in one modality travel along the major part of the neuron's axon in the opposite direction to those initiated by stimuli in the other. Bi- and multimodal neurons are widespread in both vertebrates and invertebrates. It is generally assumed that single action potentials in them are ambiguous, and their ubiquity is persuasive evidence, therefore, that at the level of the single neuron the nervous system is largely indeterministic s. The discovery of modality-specific initiation sites challenges the validity of the view that spikes in bimodal cells are unreliable because they cannot uniquely be interpreted. The lobular giant movement detector (LGMD) is an important integrative element in a small population of identified locust neurons which mediate escape jumping3,~,~7,18. It responds vigorously to novel and abrupt movement of small and contrasting objects anywhere in the visual field ~6. In addition, it has recently been found to generate one and sometimes two impulses in response to loud and transient sound. The auditory response is highly phasic and habituates rapidly on
94
B
v C
L
ja
i'
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[D
Fig. 1. A: a diagrammatic representation of the anatomy of the LGMD and DCMD as drawn from cobalt impregnation (see Refs. 17-19). The abrupt thickening of the L G M D axon is indicated by an arrow. The L G M D and D C M D make electrical synaptie contact in the brain and the D C M D extends its axon through the suboesophageal (SO) ganglion, the first (TI) and second (T2) thoracic ganglia to the third thoracic ganglion. Electrode placements, referred to throughout this paper, are X in the fan-like arborization of the LGMD, Y in the proximal part of the L G M D axon and Z on the thoracic nerve cord between TI and T2. B: upper trace is an intracellular recording from Y and the lower trace an extracellular recording of the D C M D at Z. Action potentials in the L G M D were initiated by injecting depolarizing current through the microelectrode. The oscilloscope was triggered by the L G M D spikes and about 30 sweeps are superimposed. Note that the D C M D follows 1:1 with practically no latency variation. Calibration: 0.5 msec and (trace Y only) 40 inV. C: an intracellular recording from site X in the fan-like arborization of the LGMD. The first impulse (v) was initiated by a visual stimulus and arises from a compound EPSP. The second (a), initiated by auditory stimulation, arises abruptly from baseline. At site X there is a constant background of EPSPs from presynaptic visual interneurons; any correspondence between auditory spikes and PSPs here is coincidental. Calibration: 40 msec and 10 inV. D: upper trace is an intracellular recording from the L G M D at site X and the lower an extracellular recording of the D C M D at Z. The oscilloscope was triggered from L G M D spikes and stimuli provided in both visual and auditory modalities. Two distinct classes of latency are evident. The latency in response to auditory stimulation (a) is shorter by 0.38 msec than to visual stimulation (v). In each modality 5 sweeps of the oscilloscope are superimposed. Calibration: 0.5 msec and (trace X only) 10 mV.
r e p e a t e d s t i m u l a t i o n . B o t h visual a n d a u d i t o r y spikes are t r a n s m i t t e d 1:1 f r o m the L G M D to t h e d e s c e n d i n g c o n t r a l a t e r a l m o v e m e n t d e t e c t o r ( D C M D ) , w h i c h e x t e n d s its a x o n f r o m the b r a i n to the t h i r d t h o r a c i c g a n g l i o n w h e r e it m a k e s o u t p u t c o n n e c t i o n s w i t h m o t o n e u r o n s i n v o l v e d w i t h j u m p i n g ~. R e c o r d i n g s w e r e m a d e i n t r a c e l l u l a r l y f r o m the L G M D a n d e x t r a c e l l u l a r l y f r o m the D C M D . G l a s s m i c r o e l e c t r o d e s filled w i t h 3 M p o t a s s i u m a c e t a t e a n d h a v i n g resistances f r o m 50 to 80 m~) were e m p l o y e d for r e c o r d i n g i n t r a c e l l u l a r l y f r o m the L G M D in the n e u r o p i l e o f t h e o p t i c l o b e a n d brain. Silver h o o k e l e c t r o d e s p l a c e d o n the t h o r a c i c n e r v e c o r d served to m o n i t o r the a c t i v i t y o f the D C M D , w h i c h is t h e
95 most conspicuous (3-4 mV) and easily identified unit when recorded in this way. Visual excitation to the MD neurons was provided by hand movements in the LGMD's receptive field. Auditory responses to pure tones produced by a wave form generator were weak and unreliable; a far more effective stimulus was found to be a loud brief 'hiss' by the author. The anatomy of the DCMD and LGMD, as drawn from intracellular cobalt impregnation, and electrode positions are represented diagrammatically in Fig. 1A. The relative positions of sites of spike initiation in the L G M D were determined by comparing conduction times for action potentials generated by visual and auditory stimulation between a recording site in the L G M D and another on the DCMD. This is possible because the two cells are connected by an electrical synapse which transmits spikes 1 : 1 with no measurable variation in latency (Fig. 1B). Furthermore, assuming there are no local and anomalous variations in conduction velocity on the L G M D axon, it is possible to calculate with some precision the spatial separation of initiation sites from measurements of conduction times. The validity of this assumption is founded on anatomical grounds. The spike supporting part of the L G M D axon has throughout its length a rather constant diameter (15 ~m). Estimates of propagation velocity, determined by two-point recording on the L G M D axon, lie between 2.9 and 3.1 m/sec at room temperature (20 °C). The close correspondence with the conduction velocity of the D C M D axon 8 (3.1 m/sec) and the similarity of L G M D and D C M D axonal diameters, suggests that large, local variations in conduction velocity would have clear anatomical correlates. The conduction time for a visually induced spike from recording site X in the L G M D to site Z on the D C M D axon is invariable (Fig. 1D). It is not measurably altered by recording at different locations in the fan-like arborization of the L G M D but is reduced by recording proximally to an abrupt thickening of the axon from 5 #m to 15 ~m, which occurs at the junction of optic lobe and brain (Fig. 1A). The dendrites of the fan arborization are not spike supporting. Action potentials in their proximal regions reach 25 mV and are considerably smaller in more distal regions which suggests that they are conducted passively and distally (or centrifugally) from their site of initiation into the fan. Since the abrupt axonal thickening is the most distal part of the L G M D where overshooting action potentials can be recorded, it is proposed to be the site of visual spike initiation. Auditory action potentials have the same shape and amplitude when recorded at site X as those induced by visual stimulation but differ from them in two important respects. First, they do not rise from EPSPs (Fig. 1C), suggesting that the auditory synapse onto the L G M D is remote from recording site X. Second, they arrive at site Z earlier (Fig. 1D), showing that they are initiated closer to Z than visual spikes and are propagated along the L G M D axon to the site of visual spike initiation. If X is indeed distal to the site of visual spike initiation, the difference in arrival times of auditory and visual spikes at Z is twice the temporal separation of the two initiation sites. In Fig. 1D, which is data taken from an adult female, this difference is 0.38 msec and the temporal separation of initiation sites is, therefore, 0.19 msec. Using 3 m/sec as the axonal conduction velocity of the LGMD, the spatial separation is 576 #m.
96
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r~T.,r. . . . . . .
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......
,,~~.V Z
Fig. 2. A: upper trace is an intracellular recording from site Y (see Fig. IA), the middle trace an extracellular recording from Z and the lower trace a recording of a loud sound by the author. The single auditory action potential (a) in the LGMD is preceded by a compound EPSP and is followed in the middle trace by a spike in the DCMD (arrowed). Action potentials in the L G M D at site Y are large (80-90 mV) and overshooting and are clipped in this recording. Compare with Fig. 1C and Fig. 2B. Calibration: 10 msec and (trace Y only) 10 inV. B: upper trace is an intracellular recording from Y and the middle trace an extracellular recording from Z. Three spikes induced by visual stimulation (v) are shown and they are followed 1:1 by DCMD spikes (arrowed). Note that the L G M D spikes arise abruptly from baseline without preceding PSP. Compare with Fig. IC and Fig. 2A. Calibration: 10 msec and (trace Y only) 10 mV. C: subthreshold auditory EPSPs in the LGMD. The upper trace is an intracellular recording from site Y, the middle trace an extracellular recording of a brief 'hiss'. Spikes from auditory neurons ascending from the third thoracic ganglion, where sensory afferents from the tympanic organs enter the central nervous system, are seen in trace Z. None appear to be directly presynaptic to the LGM D. Calibration: 20 msec and (trace Y only) 5 mV. D: upper trace is an intracellular recording from the L G M D at Y and the lower trace an extracellular recording of the D C M D at Z. The oscilloscope was triggered by L G M D spikes and stimuli provided in both visual and auditory modalities. Two visual (v) and one auditory (a) response are superimposed. The auditory D C M D spike precedes fractionally the visual spikes, showing that site Y is very close to the initiation site for auditory spikes. This difference in latency to auditory and visual spikes should be compared to that in Fig. 1D. Absolute latency measurements and the apparent duration of the extracellularly recorded D C M D spike depend upon the location of the bipolar electrodes on the nerve cord between T1 and T2 (see Fig. 1A). Calibration: 0.5 msec and (trace Y only) 25 mV.
D i r e c t m e a s u r e m e n t s t a k e n f r o m c o b a l t i m p r e g n a t i o n o f the L G M D in a d u l t f e m a l e s s h o w the d i s t a n c e f r o m the p r o p o s e d site o f visual spike i n i t i a t i o n to t h e p r o x i m a l e n d o f the a x o n in t h e b r a i n to be a b o u t 580 # m . T h e r e is a r e m a r k a b l e c o r r e s p o n d e n c e b e t w e e n t h e c a l c u l a t e d s e p a r a t i o n o f i n i t i a t i o n sites a n d the a x o n a l length, s h o w i n g t h a t the a s s u m p t i o n o f c o n s t a n t a x o n a l v e l o c i t y a n d t h e p r o p o s a l t h a t visual spikes are initiated at t h e a b r u p t a x o n a l t h i c k e n i n g are v e r y p r o b a b l y c o r r e c t . C o n f i r m a t i o n t h a t a u d i t o r y spikes are i n i t i a t e d at t h e p r o x i m a l e n d o f t h e a x o n was p r o v i d e d b y r e c o r d i n g i n t r a c e l l u l a r l y f r o m the L G M D the DCMD
close to its s y n a p s e w i t h
(site Y in Fig. 1A). H e r e o n e r e c o r d s E P S P s in r e s p o n s e to a u d i t o r y sti-
97 mulation (Fig. 2A and C) which can give rise to spikes. Visual spikes at this site arise abruptly without preceding PSPs (Fig. 2B). The conduction times for auditory and visual spikes from Y to Z are practically identical, which indicates that electrodes placed at the proximal end of the L G M D are very close to the site of axonal spike initiation for auditory stimulation (Fig. 2D). Two consequences of separating modality-specific sites of initiation will be considered. First, as suggested by Calabrese and Kennedy 4, postsynaptic inhibition can easily be directed selectively at specific modalities of excitation within a single cell. The L G M D provides an example of this phenomenon; spike initiation in response to rapid whole-field movement but not sound is prevented by postsynaptic inhibition directed at a small branch of the L G M D (subfield C of O'Shea and Williams la) which arises between the fan-like arborization and the site of visual spike initiation 16. These inhibitory potentials are not seen at the site of auditory spike initiation. Second, an action potential in a bimodal interneuron does not necessarily transmit ambiguous information. It is possible for an interneuron to encode unambiguously the sensory origin of each spike. This depends upon the ability of postsynaptic units to detect the temporal differences corresponding to the two sites; these could be detected by a single postsynaptic neuron and several models of suitable mechanisms are available (e.g., see Rail22). The complexities of regional differentiation of vertebrate neurons 27 provide the anatomical requirements necessary for multiplexing or distinguishing between individual action potentials in a single cell. Axoaxonal synapses are widespreadt, 5,6, 19,20,26, and although presynaptic inhibition is the function most often attributed to them, it is not implausible that some initiate impulses. Axodendritic synapses at the nodes of Ranvier 2 and dendrodendritic synapses 7,2a could form the anatomical substrate of a readout system. While it is true that interpretation of individual action potentials is not possible when collisions occur or when, between readout points, the axon carries more than one impulse in the same direction e..t the same time, their probability, which depends on axon length and conduction velocity, may be low and have a small effect on reliability. In the absence, therefore, of detailed information about spike initiation and anatomy of individual neurons, there is no compelling reason to assume that spikes in bimodal cells are necessarily ambiguous and unreliable. The author wishes to thank Dr. Hugh Rowell, Corey Goodman, Dr. A. Leiman, Dr. D. Kennedy and Dr. R. Calabrese for stimulating discussion and their criticisms of the manuscript. This work was supported by a grant from the U.S. Public Health Service.
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