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Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. I. Properties and synaptic localization of the excitatory component
342
Brain Research, 96 (1975) 342 34~ ~i~'/ Elsevier Scientific Publishing Company, Amstredam - Printed in The Netherlandr
Inputs from the pomrior lateral line nerves upon the goldfleh Mauthner cell. I. Properties and synapti¢ localization of the excitatory component
HENRI KORN* ANDDONALD S. FABER** University of Cincinnati Medical Center, Department o f Physiology, Cincinnati, Ohio 45219 (U.S.A.} and Laboratoire de Physiologie, C.H.U. Pitid-Salp~tri~re, Paris (France}
(Accepted June 27th, 1975)
The Mauthner cells (M-cells) of some cyclostomes, teleosts and amphibians receive a powerful excitatory input from the ipsilateral vestibular nerve afferents. The physiological properties of this input and its termination as club endings with mixed synapses on the distal portion of the lateral dendrite are clearly established 1,2,9,1°As,19. By a surprising contrast, relatively little has been known about the physiological and functional properties of the other afferents to the M-cells, specifically, about the second major part of the acoustico-lateral sensory system, the lateral line nerves which innervate the hair cells sensitive to water displacements 5,1a. Despite the prediction that the latter may be a sensory input to the M-cell zl, the fact that electrical stimulation of the lateral line nerves evokes a depolarization in this neuron was only noted in passing in studies with the sea lamprey Petrornyzon marinus 2o but was not further investigated in the past. We recently reported 17 that stimulation o f either the ipsi- or contralateral posterior lateral line nerves induce in the M-cell a mixed potential composed of an early excitatory postsynaptic potential (EPSP) partially masked by a chloride-dependent inhibitory postsynaptic potential (IPSP) which follows it at a brief interval; on the basis of its latency and conductance change, this response is likely to be mediated by way of chemical synapses (identical but smaller PSPs were also evoked by natural stimulation using jets of water applied to the body of the fish). Also, it was suggested that the involved afferents terminate mainly on the proximal part of the lateral dendrite. These basic observations were in agreement with those of Fukuda 8 who, under the same conditions, observed a monosynaptic EPSP and disynaptic and polysynaptic IPSPs, but who instead concluded that the input is restricted to the M-cell's soma. In the above mentioned reports, the composite nature of the lateral lineinduced PSP did not permit a study in isolation of the properties of its initial excitatory component. In this paper, we report the results of experiments designed to clarify * Maitre de Recherches/t I'I.N.S~E,R.M. ** Present address and address for reprint requests: Dr. Donald S. Faber, Research Institute on Alcoholism, 1021 Main Street, Buffalo, N.Y. 14203, U.S.A.
343 this situation as well as the question of the localization of the excitatory afferents upon the M-cell. For this purpose, the lateral line-evoked EPSP was separated from any further inhibitory components by blocking the latter pharmacologically with strychnine. This drug was used under the assumption that it could block these IPSPs as well as it suppresses the M-cell collateral inhibition4,11. The next paper 6 analyzes the network organization underlying the two antagonistic inputs from the lateral line and its possible functional significance. The experiments were conducted on common goldfish (Carassius auratus) 12-18 cm in length which were anesthetized with MS-222. Motor activity was paralyzed with either D-tubocurarine or Flaxedil (1 #g/g body wt.), and the fish were respired with a continuous flow of dechlorinated tap water through the mouth. The surgical and fixation techniques were similar to those employed previously10,16. Bipolar electrodes were placed on the posterior spinal cord for antidromic activation of the M-cell and on the exposed vestibular and posterior lateral line nerves (Fig. I A); the latter were stimulated approximately 1 cm caudal to the posterior margin of the opercula. Intracellular recordings were made from the M-cell soma and lateral dendrite with glass microelectrodes (2-6 Mr2 resistance) filled with 0.6 M K2SO4. When required, strychnine was injected intramuscularly (5 pg/g body wt.) in the form of its sulfate salt. Before any drug administration, the depolarizations induced in the M-cell by stimulation of either the ipsilateral or the contralateral posterior lateral line nerve (Fig. 1B-D, E4) are relatively small in amplitude (2-7 mV) and their time course is slow in comparison to that of the EPSP evoked by stimulation of the ipsilateral eighth nerve (Fig. 1Ea); peak time (measured as time from onset to peak) averaged 2.5 msec in 40 cells investigated while the average half decay time was 2.75 msec in the same cells. Their latencies (which were not modified by strychnine injections) ranged from 0.8 to 2.92 msec after stimulation of the ipsilateral posterior lateral line nerve (m = 1.42 msec, n ----- 34) and from 0.8 to 3.1 msec after stimulation of the contralateral one (m -- 1.89 msec, n ~ 30). The 0.47 msec difference in average latency is most likely due to a longer conduction distance for the contralateral input, since at least the initial phase of both responses is evoked monosynaptically s,lv. Presumably, the latency variations reflect differences in fish size and location of the stimulating electrodes. As described earlier, this postsynaptic response has both excitatory and inhibitory components; its falling phase is enhanced by intracellular CI- injections, and there is a significant conductance increase during that phase, which was best demonstrated as a reduction in the antidromic spike height following a conditioning stimulation of either lateral line nerve (Fig. IB). Simultaneously, the activation of the M-cell by stimulation of the ipsilateral eighth nerve is blocked (Fig. 1C), presumably due to a shunting of the vestibular-induced EPSP. Finally, no conductance increase can be detected in the axon, even though a small electrotonically conducted depolarization evoked by the lateral line stimulus is still recorded at that site (Fig. 1D). In contrast, the apparently brief initial excitatory phase of the lateral line-evoked depolarization never succeeded to bring the cell to firing level, neither when combined with a stimula-
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Fig. l. Postsynaptic responses evoked in the M-cell by lateral line nerve stimulation and their modifications by strychnine. A: schematic illustration of the experimental arrar~ement and of the basic neuronal networks involvin8 the M-cell. A microelectrode (ME) was inserted in the cell's soma or in the lateral dendrite (Lat. Dendr.). Intracellular potentials were monitored following stimulation (Stim.) of the axon (M. axon), of the posterior lateral line nerves (Lat. L.N.) and of the vestibular nerve (VIIIth N.). E (white circle) and I (black circle) are, respectively, excitatory and inhibitory interneurons of the recurrent collateral inhibitory network. B: effect of the lateral line-evoked PSPs on antidromic spike height, as recorded in the M-cell soma. Stimulations of the ipsilateral posterior lateral line nerve which induced a depolarization were followed at different time intervals by antidromic stimuli; the PSP is associated with a conductance change, as indicated by the reduction in the antidromic spike height. C: inhibitory effect of the lateral line-evoked PSPs. Several superimposed sweeps as in B. Stimuli applied to the ipsilateral posterior lateral line nerve were followed, at different intervals, by suprathreshold stimulations of the ipsilateral vestibular nerve. The depolarizing potential evoked by the former blocks spike initiation by the eighth nerve input, and redt~ced underlying EPSPs (arrows) are thereby unmasked. Recordings were obtained from the M-cell soma. D: same experiment and procedure as in B; the recordings are from the axon of the same Mcell. The lateral line stimulation produced a small residual depolarization but no measurableconductance change in this part of the neuron. E1-ET: effect of strychnine upon lateral line nerveevoked PSPs. All traces are from the soma of the same M-cell; the intensities of the different stimuli were kept constant throughout the experiment. El-E4: control records. El: paired stimuli were applied to the spinal cord. The amplitude of the second antidromic spike is reduced, due to the conductance change during collateral inhibition. Ea: orthodromic spike evoked by ipsitateral eighth nerve stimulation. E3: the same stimulus as in E2 was presented after a spinal cord stimulation. Initiation of the synaptic spike is blocked by the collateral inhibition, and a subthreshold EPSP is revealed. E4: a depolarizing PSP evoked by stimulation of the contralateral posterior lateral line nerve blocks orthodromic spike initiation (superimposed sweeps with and without eighth nerve stimulation). Es-ET: records obtained 30 min after strychnine. Es: same procedure as in E.~. The vestibular stimulus now evokes a spike, indicating that the collateral inhibition has been removed by strychnine. E6: the depolarization evoked by lateral line nerve stimulation is increased in size, presumably due to a blockage of the inhibitory component which was also induced by this stimulus before strychnine (compare with corresponding traces in E4). ET" in contrast with E4, the spike evoked by a stimulation of the vestibular nerve is not blocked by a preceding lateral line nerve stimulation. Upper and lower traces in B-D. El, E4, E6; high gain AC. and low gain D C recordings, respect ively.
345 tion of the lateral line nerve of the opposite side, nor when paired with subthreshold eighth nerve EPSPs or with strong depolarizing current pulses. As shown in the following paper 6, this is partly due to the fact that the early phase of depolarization is mixed with electrical inhibition. Thus, in all cases the inhibitory components of the lateral line inputs were dominant over the excitatory one. The mixed response of the M-cell to the posterior lateral line nerve stimulation was generally not associated with a membrane hyperpolarization, as is also often the case with the collateral IPSP evoked by the cell's antidromic activation12; even when there was a hyperpolarizing collateral IPSP, there was no similar observable hyperpolarization evoked by the lateral line stimulus, which suggests that there is not a strict separation in time between the two components. That is, the EPSP duration approximates that of the IPSP. However, no definite information could be obtained about the former: the excitatory and inhibitory components could not be separated by varying stimulus strength, and they both appeared to have the same thresholds and qualitatively similar stimulus-response characteristics. Thus, further experiments were designed to isolate the EPSP pharmacologically. As expected, the lateral line-evoked IPSP was blocked by strychnine and the associated EPSP was thereby revealed. This is shown in the records of Fig. 1E1-Ev which were obtained from the same M-cell before (El-E4) and 30 rain after (E5-[!7) injection of this drug. In the control records the collateral IPSP produces a conductance change (El) which blocks the orthodromic activation of the cell by eighth nerve stimulation (E2-E3). The lateral line-evoked PSP is only 2 mV in amplitude, and the response to the vestibular stimulus is inhibited during its falling phase (E4). Strychnine suppressed both these inhibitory mechanisms and their associated membrane permeability changes (E5 and ET). At the same time the EPSP from the lateral line was approximately doubled in amplitude (E6). (It should also be pointed out that, due to M-cell refractoriness, a slight reduction of the vestibular-evoked spike height could still be observed in the records of Es.) Comparable results were observed in 14 investigated M-cells: the average EPSP amplitude increased from 2.82 to 5.68 mV and, in all cases, strong stimuli then induced PSPs which were large enough to bring the cell to its firing level (Fig. 2A1-A4). Furthermore, strychnine also increased both the PSP's average time to summit (2.97 vs. 5.07 msec, n -- 9) and its average half decay time (3.71 vs. 6.23 msec, n = 8). We assume that the prolonged EPSP observed in the strychninized preparation is comparable to the underlying one masked by the IPSP in the control situation and does not primarily reflect either enhanced excitatory input to the M-cell or alterations in its membrane properties 1~. This statement is based on the observations that: (1) the first 0.8-1.2 msec of the rising phase of the EPSP is not altered by strychnine (this corresponds to the latency differential for the excitatory and inhibitory components and the transition point seen after CI- injections); (2) strychnine has no effects on the M-cell's membrane resistance or spike height; (3) the conductance change associated with the IPSP disappears as well. The EPSPs isolated in this manner had a clear facilitatory effect on other excitatory synaptic potentials (Fig. 2B1-B2 and C). As illustrated by Fig. 2B1-B2, if subthreshold EPSPs from ipsi-and contralateral posterior lateral nerves were paired,
346
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Fig. 2. Distribution and properties of the excitatory input from the lateral line to the M-cell. All the responses were obtained 60 min after injection of strychnine. A1-A4: comparison of the EPSPs recorded in the cell's soma and laferal dendrite. Upper and middle traces: high gain AC and low gain D C intracellular recordings, respe.etively. Lower traces: corresponding extracellular fields. Potentials were monitored dose to the M-cell soma for A1-A~, and 100 u m away, in the lateral dendrite, for A3-A4. A1, An: stimuli were applied to the spinal cord for antidromic activation of the cell. As, A 4: EPSPs evoked by stimulation of the contralateral posterior lateral line nerve. Note that although antidromic spike height decreases as the recording site is shifted more laterally, the EPSP amplitude remains fairly constant and is s u ~ i e n t to initiate a spike, B1-B2, C: facititatory properties of the lateral line input. B1-B~: summation of lateral line-evoked EPSPs. Bz: subthreshold EPSP evoked in an M-cell by a stimulation of the ipsilateral posterior lateral line nerve. Bz: the same stimulus as in BI was paired with a conditioning stimulation of the contralateral posterior lateral line nerve which also generated a below threshold EPSP; when combined together, the two potentials summate and induce a spike (superimposed traces with and without stimulation of the ipsilateral lateral line nerve; upper and lower traces: high gain A C and low gain D C recordings, respectively). C: facilitation of vestibular-evoked EPSP by a preceding lateral line nerve stimulation. Upper trace: a stimulus applied to the ipsilateral eighth nerve evokes an EPSP and a relatively long latency spike. Lower trace:when the vestibular response occurs at the peak of a PSP evoked by stimulation of the contralateral posterior lateral line nerve, the latency of the first M-cell spike is reduced, and the neuron discharges repetitively (superimposed traces with and without eighth nerve stimulation). B~-B~ and C are from different experiments. D, upper: diagram of the M-cell indicating that in the non-strychninized preparation, the distribution of the maximum amplitude lateral line-evoked depolarization is restricted to the lateral dendrite (hatched area), proximal to the region of the eighth nerve input. A chemical synapse from the lateral line nerve (Lat. L.N.) and an electrotonie synapse from the vestibular nerve (VIIIth N.) are also illustrated. The arrow indicates the initial segment where spikes are initiated. Lower: same diagram with hatched area showing that when mapped under strychnine, the maximum size EPSPs are recorded from the soma and lateral dendrite as welt.
347 they summated and could generate a spike. Also, a powerful facilitation of the response to eighth nerve stimulation could be obtained. For example, in the upper record of Fig. 2C a moderate vestibular stimulus evokes a spike in the M-cell with a latency of approximately 3.0 msec; when, as in the lower record of Fig. 2C, the same response is paired with a subthreshold EPSP from the ipsilateral posterior lateral line nerve, the cell generates a high frequency burst of impulses and the latency of the first spike is reduced to about 1 msec. These different results strongly suggest that the startle reflex which is initiated in the M-cell by acoustical or visual inputs3, 22 can be facilitated or triggered, under certain conditions, by some of the lateral line afferents as well. In our initial mapping experiments iv, the amplitude of the mixed lateral lineevoked depolarization was maximal in the proximal regions of the M-cell's lateral dendrite (Fig. 2D, upper diagram). We have found that the EPSP amplitude after strychnine injection is relatively constant from the level of the soma to about 200 /~m out on the dendrite: comparable size EPSPs, whether subthreshold or maximal (Fig. 2A2-A4), were recorded in these different parts of the cell, provided only that the lateral line stimulus was kept at a fixed intensity. In the same experiments, antidromic spike height was maximal in the soma (Fig. 2A1 vs. A3), in accordance with previous investigators TM who demonstrated that active spike generation fails at the level of the axon hillock. We, therefore, conclude that the excitatory synapses from the posterior lateral line nerves are distributed over the cell soma and proximal regions of the lateral dendrite (Fig. 2D, lower diagram), and our earlier results can be explained by postulating that, as in other neurons, the inhibitory synapses are concentrated on the somatic membrane. This statement is not necessarily in conflict with that of Fukuda s who localized the lateral line input to the M-cell's soma primarily on the basis of the extracellular field potential distribution in the axon cap surrounding the axon hillock, since: (a) these prominent fields appear to be generated by the presynaptic fibers mediating also the somatic inhibition, and (b) the fact that they are not recorded in more lateral regions in part reflects the unique electrical properties of the axon cap TM. Thus, comparable extracellular potentials need not be associated with a diffuse excitatory input onto the dendrite. Only two types of excitatory synapses cover both the M-cell's perikaryon and the proximal part of the lateral dendritO s, the numerous large vesicle boutons which are postulated to be in part terminals of eighth nerve neurons, and the sparsely distributed small myelinated club endings of unknown origin. It is not yet possible to conclude which endings mediate the excitatory input from the lateral line nerves, but because of their greater number the large vesicle boutons appear to be the most likely ones. Finally, the excitatory and inhibitory components originate from one population of afferent fibersS; this raises the question of whether the two antagonistic synaptic potentials are evoked by different afferents, for example, by fibers innervating hair cells with opposed directional sensitivitiesV, 14. In this context, certain physiological inputs from the lateral line would inhibit the startle reflex while under different conditions other lateral line detectors could be expected to exert a predominant facilitatory influence upon the M-cell and to be capable of triggering this reflex.
348 T h i s w o r k was s u p p o r t e d in p a r t by N I N D S
G r a n t N o s . N S 11313-01 a n d N S
12132-01.
1 BARTLEMEZ,G. W., Mauthner's cell and nucleus motorius tegmenti, J. comp. NeuroL, 25 (1915) 87-128. 2 BODIAN,D., The structure of the vertebrate synapse. A study of the axon endings on Mauthner's cell and neighboring centers in the goldfish, J. comp. Neurol., 58 (1937) 11%159. 3 DIAMOND,J., The Mauthner cell. In W, S. HOAR AND D. J. RANDALL (Eds.), Fish Physiology, Vol. V, Academic Press, New York, 1971, pp. 265-346. 4 DIAMOND,J., ROPER, S., AND YASARGIL,G. M., The membrane effects, and sensitivity to strychnine, of neural inhibition of the Mauthner cell, and its inhibition by glycine and GABA, J. Physiol. (Lond.), 232 (1973) 87-111. 5 DUKORAAF, S., The function and significance of the lateral line organs. Biol. Rev., 38 (1963) 51-105. 6 FArtER, D. S., ANO KORN, H., Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. II. Evidence that the inhibitory components are mediated by interneurons of the recurrent collateral network, Brain Research, 96 (1975) 349-356. 7 FLOCK, A., The lateral line organ mechanoreceptors. In W. S. HOAR AND D. J. RANDALL(Eds.), Fish Physiology, VoL V, Academic Press, New York, 1971, pp. 241-263. 8 FUKUDA,J., Termination and synaptic activities of posterior lateral-line nerve fibers upon Mauthner cells of goldfish, J. Neurophysiol., (1975) in press. 9 FURSrtPAN, E. J., 'Electrical transmission' at an excitatory synapse in a vertebrate brain, Science, 144 (1964) 878-880. 10 FURSHPAN, E. J., AND FURUKAWA, T., Intracellular and extraceilular responses of the several regions of the Mauthner cell of the goldfish, J. Neurophysiol., 25 (1962) 732-771. 11 FLIRUKAWA,T., FUKAMI, Y., AND ASADA, Y., Effects of strychnine and procaine on collateral inhibition of the Mauthner cell of goldfish, Jap. J. Physiol., 14 (1964) 386-399. 12 FURUKAWA,T., AND FURSHPAN,E. J., Two inhibitory mechanisms in the Mauthner neurons of goldfish, J. NeurophysioL, 26 (1963) 140-176. 13 HARRIS,G. G., AND VAN BEgGEIJK,W. A., Evidence that the lateral line organs respond to nearfield displacements of sound sources in water, J. acoust. Soc. Amer., 34 (1962) 1831-1841. 14 HARRIS,G. G., FRISHKOPF,L., AND FLOCK. A.. Receptor potentials from hair cells of the lateral line, Science, 167 (1970) 76-79. 15 KEEL,M. R., FAn,R, D. S.. AND HEISS, W. D., Strychnine- and pentylenetetrazol-induced changes of excitability in Aplysia neurons, Science, 179 (1973) 1133-1136. 16 KORN, H., AND FAnEa, D. S., An electrically mediated inhibition in the goldfish medulla, J. Neurophysiol., 38 (1975) 452-471. 17 KORN, H., FAaER, D. S., ET MARIANI,J., Existence de projections des nerfs post6rieurs de la ligne lat~rale sur la cellule de Mauthner; leur effet antagoniste sur ractivation de ce neurone par les aff6rences vestibulaires, C.R. Acad, Sci. (Paris), 279 (1974) 413-416. 18 NAKAJIMA,Y., Fine structure of the synaptic endings on the Mauthner cell of the goldfish, J. comp. NeuroL, 156 (1974) 375-402. 19 ROnERTSON,J. D., BODENmme~R, T. S., AND STAGE, D. E., The ultrastructure of Mauthner celt synapses and nodes in goldfish brains, J. Cell BioL, 19 (1963) 159-199. 20 ROVAINEN,C. M., Physiological and anatomical studies on large neurones of the central nervous system of the sea lamprey (Petromyzon marinus). I. Mliller and Mauthner's cells. J. Neurophysiok, 30 (1967) 1000-1023. 21 STEFANELLI,A., The Mauthnerian apparatus in the ichtyopsida; its nature and function and correlated problems of the neurohistogenesis, Quart. Rev. BIOL. 26 (1951) 17-34. 22 WILSON, D. M., Function of giant Mauthner's neurons in the lungfish, Science. 129 (1959) 841 842.
Brain Research, 96 (1975) 342 34~ ~i~'/ Elsevier Scientific Publishing Company, Amstredam - Printed in The Netherlandr
Inputs from the pomrior lateral line nerves upon the goldfleh Mauthner cell. I. Properties and synapti¢ localization of the excitatory component
HENRI KORN* ANDDONALD S. FABER** University of Cincinnati Medical Center, Department o f Physiology, Cincinnati, Ohio 45219 (U.S.A.} and Laboratoire de Physiologie, C.H.U. Pitid-Salp~tri~re, Paris (France}
(Accepted June 27th, 1975)
The Mauthner cells (M-cells) of some cyclostomes, teleosts and amphibians receive a powerful excitatory input from the ipsilateral vestibular nerve afferents. The physiological properties of this input and its termination as club endings with mixed synapses on the distal portion of the lateral dendrite are clearly established 1,2,9,1°As,19. By a surprising contrast, relatively little has been known about the physiological and functional properties of the other afferents to the M-cells, specifically, about the second major part of the acoustico-lateral sensory system, the lateral line nerves which innervate the hair cells sensitive to water displacements 5,1a. Despite the prediction that the latter may be a sensory input to the M-cell zl, the fact that electrical stimulation of the lateral line nerves evokes a depolarization in this neuron was only noted in passing in studies with the sea lamprey Petrornyzon marinus 2o but was not further investigated in the past. We recently reported 17 that stimulation o f either the ipsi- or contralateral posterior lateral line nerves induce in the M-cell a mixed potential composed of an early excitatory postsynaptic potential (EPSP) partially masked by a chloride-dependent inhibitory postsynaptic potential (IPSP) which follows it at a brief interval; on the basis of its latency and conductance change, this response is likely to be mediated by way of chemical synapses (identical but smaller PSPs were also evoked by natural stimulation using jets of water applied to the body of the fish). Also, it was suggested that the involved afferents terminate mainly on the proximal part of the lateral dendrite. These basic observations were in agreement with those of Fukuda 8 who, under the same conditions, observed a monosynaptic EPSP and disynaptic and polysynaptic IPSPs, but who instead concluded that the input is restricted to the M-cell's soma. In the above mentioned reports, the composite nature of the lateral lineinduced PSP did not permit a study in isolation of the properties of its initial excitatory component. In this paper, we report the results of experiments designed to clarify * Maitre de Recherches/t I'I.N.S~E,R.M. ** Present address and address for reprint requests: Dr. Donald S. Faber, Research Institute on Alcoholism, 1021 Main Street, Buffalo, N.Y. 14203, U.S.A.
343 this situation as well as the question of the localization of the excitatory afferents upon the M-cell. For this purpose, the lateral line-evoked EPSP was separated from any further inhibitory components by blocking the latter pharmacologically with strychnine. This drug was used under the assumption that it could block these IPSPs as well as it suppresses the M-cell collateral inhibition4,11. The next paper 6 analyzes the network organization underlying the two antagonistic inputs from the lateral line and its possible functional significance. The experiments were conducted on common goldfish (Carassius auratus) 12-18 cm in length which were anesthetized with MS-222. Motor activity was paralyzed with either D-tubocurarine or Flaxedil (1 #g/g body wt.), and the fish were respired with a continuous flow of dechlorinated tap water through the mouth. The surgical and fixation techniques were similar to those employed previously10,16. Bipolar electrodes were placed on the posterior spinal cord for antidromic activation of the M-cell and on the exposed vestibular and posterior lateral line nerves (Fig. I A); the latter were stimulated approximately 1 cm caudal to the posterior margin of the opercula. Intracellular recordings were made from the M-cell soma and lateral dendrite with glass microelectrodes (2-6 Mr2 resistance) filled with 0.6 M K2SO4. When required, strychnine was injected intramuscularly (5 pg/g body wt.) in the form of its sulfate salt. Before any drug administration, the depolarizations induced in the M-cell by stimulation of either the ipsilateral or the contralateral posterior lateral line nerve (Fig. 1B-D, E4) are relatively small in amplitude (2-7 mV) and their time course is slow in comparison to that of the EPSP evoked by stimulation of the ipsilateral eighth nerve (Fig. 1Ea); peak time (measured as time from onset to peak) averaged 2.5 msec in 40 cells investigated while the average half decay time was 2.75 msec in the same cells. Their latencies (which were not modified by strychnine injections) ranged from 0.8 to 2.92 msec after stimulation of the ipsilateral posterior lateral line nerve (m = 1.42 msec, n ----- 34) and from 0.8 to 3.1 msec after stimulation of the contralateral one (m -- 1.89 msec, n ~ 30). The 0.47 msec difference in average latency is most likely due to a longer conduction distance for the contralateral input, since at least the initial phase of both responses is evoked monosynaptically s,lv. Presumably, the latency variations reflect differences in fish size and location of the stimulating electrodes. As described earlier, this postsynaptic response has both excitatory and inhibitory components; its falling phase is enhanced by intracellular CI- injections, and there is a significant conductance increase during that phase, which was best demonstrated as a reduction in the antidromic spike height following a conditioning stimulation of either lateral line nerve (Fig. IB). Simultaneously, the activation of the M-cell by stimulation of the ipsilateral eighth nerve is blocked (Fig. 1C), presumably due to a shunting of the vestibular-induced EPSP. Finally, no conductance increase can be detected in the axon, even though a small electrotonically conducted depolarization evoked by the lateral line stimulus is still recorded at that site (Fig. 1D). In contrast, the apparently brief initial excitatory phase of the lateral line-evoked depolarization never succeeded to bring the cell to firing level, neither when combined with a stimula-
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Fig. l. Postsynaptic responses evoked in the M-cell by lateral line nerve stimulation and their modifications by strychnine. A: schematic illustration of the experimental arrar~ement and of the basic neuronal networks involvin8 the M-cell. A microelectrode (ME) was inserted in the cell's soma or in the lateral dendrite (Lat. Dendr.). Intracellular potentials were monitored following stimulation (Stim.) of the axon (M. axon), of the posterior lateral line nerves (Lat. L.N.) and of the vestibular nerve (VIIIth N.). E (white circle) and I (black circle) are, respectively, excitatory and inhibitory interneurons of the recurrent collateral inhibitory network. B: effect of the lateral line-evoked PSPs on antidromic spike height, as recorded in the M-cell soma. Stimulations of the ipsilateral posterior lateral line nerve which induced a depolarization were followed at different time intervals by antidromic stimuli; the PSP is associated with a conductance change, as indicated by the reduction in the antidromic spike height. C: inhibitory effect of the lateral line-evoked PSPs. Several superimposed sweeps as in B. Stimuli applied to the ipsilateral posterior lateral line nerve were followed, at different intervals, by suprathreshold stimulations of the ipsilateral vestibular nerve. The depolarizing potential evoked by the former blocks spike initiation by the eighth nerve input, and redt~ced underlying EPSPs (arrows) are thereby unmasked. Recordings were obtained from the M-cell soma. D: same experiment and procedure as in B; the recordings are from the axon of the same Mcell. The lateral line stimulation produced a small residual depolarization but no measurableconductance change in this part of the neuron. E1-ET: effect of strychnine upon lateral line nerveevoked PSPs. All traces are from the soma of the same M-cell; the intensities of the different stimuli were kept constant throughout the experiment. El-E4: control records. El: paired stimuli were applied to the spinal cord. The amplitude of the second antidromic spike is reduced, due to the conductance change during collateral inhibition. Ea: orthodromic spike evoked by ipsitateral eighth nerve stimulation. E3: the same stimulus as in E2 was presented after a spinal cord stimulation. Initiation of the synaptic spike is blocked by the collateral inhibition, and a subthreshold EPSP is revealed. E4: a depolarizing PSP evoked by stimulation of the contralateral posterior lateral line nerve blocks orthodromic spike initiation (superimposed sweeps with and without eighth nerve stimulation). Es-ET: records obtained 30 min after strychnine. Es: same procedure as in E.~. The vestibular stimulus now evokes a spike, indicating that the collateral inhibition has been removed by strychnine. E6: the depolarization evoked by lateral line nerve stimulation is increased in size, presumably due to a blockage of the inhibitory component which was also induced by this stimulus before strychnine (compare with corresponding traces in E4). ET" in contrast with E4, the spike evoked by a stimulation of the vestibular nerve is not blocked by a preceding lateral line nerve stimulation. Upper and lower traces in B-D. El, E4, E6; high gain AC. and low gain D C recordings, respect ively.
345 tion of the lateral line nerve of the opposite side, nor when paired with subthreshold eighth nerve EPSPs or with strong depolarizing current pulses. As shown in the following paper 6, this is partly due to the fact that the early phase of depolarization is mixed with electrical inhibition. Thus, in all cases the inhibitory components of the lateral line inputs were dominant over the excitatory one. The mixed response of the M-cell to the posterior lateral line nerve stimulation was generally not associated with a membrane hyperpolarization, as is also often the case with the collateral IPSP evoked by the cell's antidromic activation12; even when there was a hyperpolarizing collateral IPSP, there was no similar observable hyperpolarization evoked by the lateral line stimulus, which suggests that there is not a strict separation in time between the two components. That is, the EPSP duration approximates that of the IPSP. However, no definite information could be obtained about the former: the excitatory and inhibitory components could not be separated by varying stimulus strength, and they both appeared to have the same thresholds and qualitatively similar stimulus-response characteristics. Thus, further experiments were designed to isolate the EPSP pharmacologically. As expected, the lateral line-evoked IPSP was blocked by strychnine and the associated EPSP was thereby revealed. This is shown in the records of Fig. 1E1-Ev which were obtained from the same M-cell before (El-E4) and 30 rain after (E5-[!7) injection of this drug. In the control records the collateral IPSP produces a conductance change (El) which blocks the orthodromic activation of the cell by eighth nerve stimulation (E2-E3). The lateral line-evoked PSP is only 2 mV in amplitude, and the response to the vestibular stimulus is inhibited during its falling phase (E4). Strychnine suppressed both these inhibitory mechanisms and their associated membrane permeability changes (E5 and ET). At the same time the EPSP from the lateral line was approximately doubled in amplitude (E6). (It should also be pointed out that, due to M-cell refractoriness, a slight reduction of the vestibular-evoked spike height could still be observed in the records of Es.) Comparable results were observed in 14 investigated M-cells: the average EPSP amplitude increased from 2.82 to 5.68 mV and, in all cases, strong stimuli then induced PSPs which were large enough to bring the cell to its firing level (Fig. 2A1-A4). Furthermore, strychnine also increased both the PSP's average time to summit (2.97 vs. 5.07 msec, n -- 9) and its average half decay time (3.71 vs. 6.23 msec, n = 8). We assume that the prolonged EPSP observed in the strychninized preparation is comparable to the underlying one masked by the IPSP in the control situation and does not primarily reflect either enhanced excitatory input to the M-cell or alterations in its membrane properties 1~. This statement is based on the observations that: (1) the first 0.8-1.2 msec of the rising phase of the EPSP is not altered by strychnine (this corresponds to the latency differential for the excitatory and inhibitory components and the transition point seen after CI- injections); (2) strychnine has no effects on the M-cell's membrane resistance or spike height; (3) the conductance change associated with the IPSP disappears as well. The EPSPs isolated in this manner had a clear facilitatory effect on other excitatory synaptic potentials (Fig. 2B1-B2 and C). As illustrated by Fig. 2B1-B2, if subthreshold EPSPs from ipsi-and contralateral posterior lateral nerves were paired,
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Fig. 2. Distribution and properties of the excitatory input from the lateral line to the M-cell. All the responses were obtained 60 min after injection of strychnine. A1-A4: comparison of the EPSPs recorded in the cell's soma and laferal dendrite. Upper and middle traces: high gain AC and low gain D C intracellular recordings, respe.etively. Lower traces: corresponding extracellular fields. Potentials were monitored dose to the M-cell soma for A1-A~, and 100 u m away, in the lateral dendrite, for A3-A4. A1, An: stimuli were applied to the spinal cord for antidromic activation of the cell. As, A 4: EPSPs evoked by stimulation of the contralateral posterior lateral line nerve. Note that although antidromic spike height decreases as the recording site is shifted more laterally, the EPSP amplitude remains fairly constant and is s u ~ i e n t to initiate a spike, B1-B2, C: facititatory properties of the lateral line input. B1-B~: summation of lateral line-evoked EPSPs. Bz: subthreshold EPSP evoked in an M-cell by a stimulation of the ipsilateral posterior lateral line nerve. Bz: the same stimulus as in BI was paired with a conditioning stimulation of the contralateral posterior lateral line nerve which also generated a below threshold EPSP; when combined together, the two potentials summate and induce a spike (superimposed traces with and without stimulation of the ipsilateral lateral line nerve; upper and lower traces: high gain A C and low gain D C recordings, respectively). C: facilitation of vestibular-evoked EPSP by a preceding lateral line nerve stimulation. Upper trace: a stimulus applied to the ipsilateral eighth nerve evokes an EPSP and a relatively long latency spike. Lower trace:when the vestibular response occurs at the peak of a PSP evoked by stimulation of the contralateral posterior lateral line nerve, the latency of the first M-cell spike is reduced, and the neuron discharges repetitively (superimposed traces with and without eighth nerve stimulation). B~-B~ and C are from different experiments. D, upper: diagram of the M-cell indicating that in the non-strychninized preparation, the distribution of the maximum amplitude lateral line-evoked depolarization is restricted to the lateral dendrite (hatched area), proximal to the region of the eighth nerve input. A chemical synapse from the lateral line nerve (Lat. L.N.) and an electrotonie synapse from the vestibular nerve (VIIIth N.) are also illustrated. The arrow indicates the initial segment where spikes are initiated. Lower: same diagram with hatched area showing that when mapped under strychnine, the maximum size EPSPs are recorded from the soma and lateral dendrite as welt.
347 they summated and could generate a spike. Also, a powerful facilitation of the response to eighth nerve stimulation could be obtained. For example, in the upper record of Fig. 2C a moderate vestibular stimulus evokes a spike in the M-cell with a latency of approximately 3.0 msec; when, as in the lower record of Fig. 2C, the same response is paired with a subthreshold EPSP from the ipsilateral posterior lateral line nerve, the cell generates a high frequency burst of impulses and the latency of the first spike is reduced to about 1 msec. These different results strongly suggest that the startle reflex which is initiated in the M-cell by acoustical or visual inputs3, 22 can be facilitated or triggered, under certain conditions, by some of the lateral line afferents as well. In our initial mapping experiments iv, the amplitude of the mixed lateral lineevoked depolarization was maximal in the proximal regions of the M-cell's lateral dendrite (Fig. 2D, upper diagram). We have found that the EPSP amplitude after strychnine injection is relatively constant from the level of the soma to about 200 /~m out on the dendrite: comparable size EPSPs, whether subthreshold or maximal (Fig. 2A2-A4), were recorded in these different parts of the cell, provided only that the lateral line stimulus was kept at a fixed intensity. In the same experiments, antidromic spike height was maximal in the soma (Fig. 2A1 vs. A3), in accordance with previous investigators TM who demonstrated that active spike generation fails at the level of the axon hillock. We, therefore, conclude that the excitatory synapses from the posterior lateral line nerves are distributed over the cell soma and proximal regions of the lateral dendrite (Fig. 2D, lower diagram), and our earlier results can be explained by postulating that, as in other neurons, the inhibitory synapses are concentrated on the somatic membrane. This statement is not necessarily in conflict with that of Fukuda s who localized the lateral line input to the M-cell's soma primarily on the basis of the extracellular field potential distribution in the axon cap surrounding the axon hillock, since: (a) these prominent fields appear to be generated by the presynaptic fibers mediating also the somatic inhibition, and (b) the fact that they are not recorded in more lateral regions in part reflects the unique electrical properties of the axon cap TM. Thus, comparable extracellular potentials need not be associated with a diffuse excitatory input onto the dendrite. Only two types of excitatory synapses cover both the M-cell's perikaryon and the proximal part of the lateral dendritO s, the numerous large vesicle boutons which are postulated to be in part terminals of eighth nerve neurons, and the sparsely distributed small myelinated club endings of unknown origin. It is not yet possible to conclude which endings mediate the excitatory input from the lateral line nerves, but because of their greater number the large vesicle boutons appear to be the most likely ones. Finally, the excitatory and inhibitory components originate from one population of afferent fibersS; this raises the question of whether the two antagonistic synaptic potentials are evoked by different afferents, for example, by fibers innervating hair cells with opposed directional sensitivitiesV, 14. In this context, certain physiological inputs from the lateral line would inhibit the startle reflex while under different conditions other lateral line detectors could be expected to exert a predominant facilitatory influence upon the M-cell and to be capable of triggering this reflex.
348 T h i s w o r k was s u p p o r t e d in p a r t by N I N D S
G r a n t N o s . N S 11313-01 a n d N S
12132-01.
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