Relation across the Receptor.afferent in a Tonic Electroreceptor of Marine Catfish

Transcription

1 No. 6] Proc. Japan Acad., 51 (1975) Input. output Synapses Relation across the Receptor.afferent in a Tonic Electroreceptor of Marine Catfish By Shun-ichi UMEKITA, Yoshiko SUGAWARA, and Shosaku OBARA Department of Physiology, Teikyo Univ., School of Med., Kaga , Itabashi-ku, Tokyo 173 (Comm. by Masahiro OKADA, M. J. A., June 3, 1975) In secondary sensory receptors it is generally accepted that after initial transduction the sensory information is transmitted across the afferent or sensory synapse from the receptor cell to the afferent nerve fiber where it is encoded into a respective pattern of afferent impulses.2~ Of necessity this sensory synapse must convey more or less gradedly variable presynaptic information, and has been often claimed as such, however, usually on the ground of single nerve recordings. This presumably graded transmission across the sensory synapse appears to stand in sharp contrast to that which occurs across the motor or efferent synapse5~ where the presynaptic inputs generally consist of well-coded signals in the form of nerve impulses. Previous reports have shown that the ampullae of Lorenzini of marine catfish, Plotosus anguillaris (Lacepede) are tonic electroreceptors in that their afferent nerve discharges are linearly increased or decreased upon small perturbations in extrinsic voltage fields impressed across the ampulla-the sensory epithelium7>>8) Intracellular recordings from an afferent nerve terminal reveal, in addition to nerve impulses, graded slow potential changes toward either polarity which have been termed the generator potential (GP). Qualitative correlation of the GP's with presynaptic, receptor potentials (RP) has led to proposition of a tonic mode of transmission involving a single transmitter substance for the Plotosus ampullae.7) The present report attempts to further define the input-output characteristics of this sensory synapse in a more quantitative form which could be directly compared with that of the efferent synapse. Materials and experimental conditions were similar to those of the IN SITU condition previously reported.8~ Dissection of the ampullary field was confined to an area only enough to expose ampulla, thus minimizing possible circulation disturbances within the field.7> The ampulla was stimulated by current pulses delivered through a glass microelectrode penetrating the duct close to the ampulla. The resulting potential changes in the ampulla were recorded by another

2 486 S. UMEKITA, Y. SUGAWARA, and S. OBARA [Vol. 51, Fig. 1. External GP's from afferent nerve terminal in Plotosus ampulla in situ, identified by simultaneous recording of intra-axonal GP's. Traces in each records are; I : combined stimulation given to the ampulla, N: external recording from afferent nerve bundle, N': simultaneous intra-axonal recording, and V : presynaptic response in the ampulla (also refer to the diagram in Fig. 2, C). A : concomitant occurrence of subthreshold depolarizing GP's (arrows). Dotted lines in traces N' and V indicate the afferent firing level and the ampullary reference level, respectively. B : evidence of complete disf acilitation during the conditioning pulse. C : highly synchronized afferent discharges upon suprathreshold stimulation (expanded sweep for test pulse). microelectrode (Fig. 2, C). Neural responses from the terminal afferent branch of the ampullary nerve were recorded externally with a pair of Pt wire electrodes. The nerve had been carefully separated from its duct, up to a point close to its supplying ampulla, and bathed in a pool of mineral oil. The terminal branch consisted of six to eight myelinated fibers, and usually exhibited a massive asynchronous spike discharge which was either facilitated or suppressed following ampullary potential variations. In addition, when recordings were taken close to the ampulla, small slow potential changes were often observed occurring concomitantly with changes in the spike discharge rate.8 The tonic mode of operation previously proposed for the Plotosus ampullae predicts a continually maintained release of excitatory transmitter substance even in the absence of stimuli.7~ To obviate any ambiguity, the spontaneous release of transmitter was initially suppressed by applying a long conditioning pulse to the ampulla. This induced a negative shift in the ampullary potential that was to hyperpolarize the presynaptic membrane-the basal or innervated surface of the receptor cells.1 8) This hyperpolarizing ampullary potential led, in turn, to a long hyperpolarizing GP in the nerve which suppressed the spontaneous afferent discharge (N' in Fig. 1). Over the conditioning pulse, shorter test pulses were superimposed to elicit graded positive shifts in the ampullary potential (presynaptic depolarization), and also clearly definable depolarizing GP's in the afferent terminal (Fig. 1, A).

3 No. 6] Synaptic Transfer in Sensory Synapse 487 Fig. 2. Input-output relation across the sensory synapse of the Plotosus ampulla in the presence of TTX. A: the ordinate indicates the relative amplitude of the external GP's, and abscissa the presynaptic ampullary potential change measured with respect to the reference level. B : sample records. The arrows in traces N and V indicate the depolarizing external GP and ampullary potential change, respectively. C : diagram of experimental set-up for stimulation and for recording in in situ ampul] ae. Abbreviations in B and C are the same as in Fig. 1. The conditioning pulse was of sufficient strength to completely turn off any on-going transmitter release. Evidence of the complete disfacilitation7~ could be shown by superimposing an additional hyperpolarizing test pulse to the ampulla. This test pulse evoked no further hyperpolarizing GP in the nerve despite the corresponding ampullary potential change (Fig. 1, B). Simultaneous intra-axonal recordings with an additional microelectrode indicated that the externally recorded slow potentials (N) closely agreed, in time and also in amplitude, with the intracellular GP's (N'), in the range subthreshold for spike initiation (Fig. 1, A). With suprathreshold stimuli, the triggered afferent spikes obscured the time course of the underlying GP's. Agreement between the intra- and extracellular slow potential, however, appeared to hold at least for their initial peaks in that the first few afferent spikes were highly synchronized in both records (Fig. 1, C). Thus, the externally recorded slow potentials will hereafter be referred to as the external GP's. The AC amplifier used for external recordings had a long time constant (1.5 sec), thus no serious distortion was introduced in the GP's under discussion. The control or reference level previously defined as the ampullary potential measured prior to stimulation8 could be assessed by

4 488 S. UMEKITA, Y. SUGAWARA, and S. OBARA [Vol. 51, observing the steady rate of `resting' afferent discharges, which was ultimately restored following each test stimuli. In preliminary experiments, similar to those in Fig. 1, simultaneous recordings from a single nerve fiber and from the whole terminal branch were compared in terms of the time course of recovery following various test pulses. The hyperpolarizing external GP's in response to finite conditioning pulses were found to recover with a time course similar to that of the rate of discharges in the single nerve fiber. Thus, the conditioning hyperpolarizing GP's should serve as a good indicator in assessing restoration of the reference level in the ampullary potential following test stimuli. Depolarizing external GP's in response to various test pulses superimposed over a finite conditioning pulse, were recorded in the presence of tetrodotoxin (TTX) at 10-7 g/ml. This concentration of TTX suppressed afferent spike initiation completely, but it had no effect on the ampullary responses.1 The external GP's attained a maximum with increasing stimuli, that might amount to about 40 mv depolarization by intracellular recording (unpublished data). Upon supramaximal stimulation, the ampulla began giving oscillatory responses with corresponding peaks in the GP's.8~ The relative amplitudes of the initial peak of the external GP's were plotted against the ampullary potential, the latter being measured with respect to the reference level (Fig. 2). The input-output relation for this receptorafferent synapse was found to follow an approximately sigmoidal curve, similar to that reported for the efferent synapse.5~,6~ Significant differences from the efferent transfer curve, however, may be pointed out. First, the curve of the input-output relation is clearly shifted along the presynaptic voltage axis toward the negative region. With no stimulus the ampullary reference level corresponds to about 20% maximum in the output, indicating again the maintained effect of depolarizing transmitter in the absence of stimuli.7~ The apparent `threshold' ampullary potential, at which GP's are just detectable, varies among preparations. However, this potential is always negative to the reference level, at less than 200,uV. The corresponding presynaptic `threshold' depolarization in the squid giant synapse is reported to range between 25 and 40 mv above the resting membrane potential.5> Secondly, the input-output relation around the reference level is quite linear and very steep. An increment of 1-2 mv in the ampullary potential results in a tenfold change in the external GP. This transfer slope may well be even steeper, since the ampullary potential represents voltage change across the sensory epithelium7> and the

5 No. 61 Synaptic Transfer in Sensory Synapse 489 effective presynaptic depolarization may be still smaller. In comparison, synaptic transfer in the squid giant synapse is markedly non-linear, and requires a much larger presynaptic depolarization. A presynaptic increment of mv for a tenfold postsynaptic change is reported, but only for the initial logarithmically rising part of the curve,5>,6) which may amount to less than 40% maximum. In the present study, the analysis of synaptic transfer has been based upon correlation of the ampullary potential to the external GP. Namely, both pre- and postsynaptic potentials represent mass responses recorded externally, while both quantities have been obtained by intracellular recording in the squid giant synapse. Despite these differences, the general features of synaptic transfer appear to have been characterized for sensory and efferent synapses. As has been often presumed,9~ a sigmoidal transfer exists in sensory as well as efferent synapses, suggesting common characteristics for the chemical transmission in general. Transfer efficacy of the Plotosus sensory synapse is significantly higher than that of the efferent synapse. The higher incremental transfer ratio appears to be common among sensory synapses. For example, in goldfish macula, intracellular EPSP's recorded from the eighth nerve terminal increase gradedly in response to sound stimuli.4~ From their data (Fig. 3, B, ref. 4), an increment of about 200 pv in microphonics (extracellular recording) may be estimated to be similarly effective for the first peak of the EPSP's. Conceivably, the lower transfer efficacy in the efferent synapse is complemented by larger presynaptic inputs of nerve impulse. The shift of the transfer curve found in the Plotosus ampullae is of functional significance, and may also apply, though probably with varying degree, to sensory receptors of various modalities in that an extremely small `absolute threshold' has been reported. A mechanism of presynaptic biasing9~ has been described as the basis of this shift in the Plotosus ampullae.81 The higher incremental transfer ratio, however, may also be ascribed to higher postsynaptic sensitivity. The excitatory transmitter substance previously proposed for the Plotosus ampullae, has not been identified. However, the afferent impulses can be significantly facilitated by L-glutamate at concentration as low as M/1, even when the natural transmitter has been completely turned off. The dose-response curve shows a steep increase in the firing rate of afferent discharges, prior to saturation at over 300 impulses/sec, within a narrow concentration range of 10.7 to 10-6 M/1.3) Postsynaptic sensitivity to L-glutamate is reminiscent of the steep slope found in the synaptic transfer.

6 490 S. UMEKITA, Y. SUGAWARA, and S. OBARA [Vol. 51, Presumably both can be explained by the extensive convergence proposed to this sensory synapse.8~ Acknowledgments. The authors wish to thank Mito Aquarium, Numazu-City and Shima Marine Land, Mie-Pref. for the generous supply of fish. Thanks are also due to Dr. Dominic W. Hughes, Department of Otorhinolaryngology, Teikyo University, for his discussion and for correcting manuscript. The work has been supported in part by grants from the Ministry of Education of Japan, and References 1) 2) 3) 4) 5) 6) 7) 8) 9) Akutsu, Y., and Obara, S. (1974) : Proc. Japan Acad., 50, 247. Davis, H. (1965) : Cold Spr. Harb. Symp. Quant. Biol., 30, 181. Higuchi, T., Umekita, S., and Obara, S. (1974) : J. Physiol. Soc. Japan (English Abst.), 36, 309. Ishii, Y., Matsuura, S., and Furukawa, T. (1971) : Jap. J. Physiol., 21, 91. Katz, B., and Miledi, R. (1967) : J. Physiol., 192, 213. Kusano, K. (1970) : J. Neurobiol., 1, 435. Obara, S., and Oomura, Y. (1973) : Proc. Japan Acad., 49, 213. Obara, S. (1974) : Proc. Japan Acad., 51, 386. Steinbach, A. B. (1974) : Synaptic Transmission and Neuronal Interaction (ed, by Bennett, M. V. L.), pp Raven Press, New York.