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The Synaptic Link Between the Sensory and Motoneurones in the Eye Withdrawal Reflex of the Crab

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With 2 plates and 6 text-figures Printed in Great Britain

THE SYNAPTIC LINK

BETWEEN THE SENSORY AND MOTONEURONES IN THE

EYE-WITHDRAWAL REFLEX OF THE CRAB

BY D. C. SANDEMAN

Gatty Marine Laboratory and Department of Natural History University of St Andrews, Fife, Scotland

(Received 27 May 1968)

INTRODUCTION

The motor nerves causing the fast withdrawal of the crab's eye fire characteristic bursts of impulses following mechanical stimulation of the carapace. This reflex nerve discharge is not affected by the position of, or even presence of, the eye itself and can be recorded from the motor nerves of an isolated brain following electrical stimulation of the appropriate afferent inputs. The preparation provides an opportunity to study the link between the afferent and efferent pathways of a simple reflex system and, since the reflex is independent of peripheral feedback, the results of such a study should be relevant to the behaviour in the whole animal.

Previous study of this reflex system has shown that the afferent neurones causing the withdrawal of one eye he in each of the ipsilateral brain nerves (Sandeman, 1967). Only two motoneurones, one larger than the other, bring about the fast eye withdrawal (Burrows, 1968) and both lie in the optic tract. Both give a burst of spikes following afferent nerve stimulation, but as the extracellular response of the large motoneurone is about 10 times the amplitude of any other recordable activity and is easily recogni-zable it is the only one to have been studied so far.

A pacemaker is involved in the reflex system. This causes a burst of spikes in both motoneurones to appear usually about every 13 sec., although interburst intervals may vary from 3 to 50 sec. Electrical stimulation of the inside half of the ipsilateral oesophageal connective can inhibit the spontaneous firing of the motoneurones.

This paper describes part of the anatomy of the large motoneurone and attempts to answer the following questions about the reflex system. (1) Is there a simple summation of excitatory effect from all the afferent inputs? (2) Do the excitatory inputs converge on a common spike-initiating locus in the motoneurone? (3) Does the link between the sensory and motor neurones involve interneurones?

MATERIALS AND METHODS

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Anatomy

The axon of the large motoneurone, often clearly visible in fresh preparations, was impaled with a low-resistance glass micropipette filled with a mixture of 2-5 M-KC1 and 0-5 M potassium ferrocyanide (Kerkut & Walker, 1962) and the intracellular responses were compared with extracellular recordings to ensure penetration of the correct axon. Negative pulses of 1 sec. duration were applied to the electrode at about 40 pulses/min. and the intensity of the pulse was adjusted until the maximum current passing through the electrode was about 2 fiA., measured with a microammeter in series with the electrode. After 15-20 min. all electrodes and the perfusion cannula were removed from the preparation and the brain was allowed to dry out for 20-30 min. This enhances the spread of the potassium ferrocyanide in the axon. The brain was then de-sheathed and immersed in Carnoy's fixative (Humason, 1962) mixed 10:1 with a saturated solution of ferric chloride. The fixative penetrates the tissue very rapidly, allowing the ferric chloride to mix with the potassium ferrocyanide and precipitate the Prussian blue dye (potassium ferric ferrocyanide). Tissue lying over the stained axon was lifted away with a fine needle, and the brain was transferred directly to absolute alcohol, cleared in methyl benzoate and benzene and mounted in xylene Damar.

Physiology

Glass micropipettes, 20-40 M£i, filled with 3 M-KC1 were used to penetrate the motoneurone in de-sheathed brains and optic tracts. Intracellular spikes were recorded on an oscilloscope via a capacity-compensating unity-gain amplifier (Bak, 1958).

The sensory nerves and the oesophageal connectives were stimulated through bipolar suction electrodes with pulses from a Tektronix 161 pulse generator and radio-frequency isolating probes. Extracellular recordings from the motoneurone were made with a suction electrode and a Tektronix 122 low-level pre-amplifier.

RESULTS Anatomy

The crab's brain with its nerves is shown in Text-fig. 1. A cross-section of the optic tract shows an axon which is significantly larger than the rest (Plate 1), and intracellular recordings from this axon identified it as the large eye-withdrawal motoneurone. The axons of large motoneurones in Crustacea often have thick sheaths around them and are difficult to impale with micro-electrodes, but the axon of the eye-withdrawal motoneurone can be easily penetrated with fairly large micropipettes and the sheath surrounding it is unusually thin compared with that around the motor axons in the leg nerves of the same animal (Plate zb, c). The significance of the different sheath thicknesses around motor axons in the leg nerves and in the optic tract is not clear, for the motor axons from these two bundles give essentially similar responses to direct electrical stimulation. The thick sheath of the leg nerve axons may be simply of structural importance and prevents the motor axons from being too severely deformed during leg movements. The optic tract moves very little, is never bent back upon itself and needs much less structural reinforcement.

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30 and 50 /i but tapers on entering the brain. In the brain it crosses diagonally to a point near the mid line where it bifurcates. A superficial branch extends laterally and dorsally over the tract of axons of the oesophageal connective, and a deeper branch passes ventro-laterally (Plate 2 a). The cell soma and axon terminals have not been seen and it is not yet known from the anatomy if the motoneurone can be included in the general arthropod pattern of having a cell body uninvolved in the transmission of signals. The study does, however, confirm the course taken by the axon through the brain previously plotted by electrophysiological means. In addition, no axon branch to the contralateral side of the brain is revealed, which agrees with the physiological finding that no excitatory effect follows normal electrical stimulation of the contra-lateral brain nerves.

Optic tract

Tegumentary nerve

Oesophageal connective

Text-fig. 1. Dorsal view of the brain and its nerves. The optic tracts and oculomotor nerves run out together to the eyecups. The tegumentary nerves extend laterally and dorsally to the carapace and the antennal nerves descend ventrally to innervate the muscles of the antennae. The antennal nerves also carry sensory neurones capable of producing the eye with-drawal. The nerves to the antennules come out of the brain ventrally and are not shown. The oesophageal connectives pass on either side of the oesophagus and extend posteriorly to the thoracic ganglion. The dotted lines mark the position of the globuli, discrete areas of neuropile.

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Physiology

The usual response of the large eye-withdrawal motoneurone following a single shock to the afferent nerves was a short high-frequency burst of impulses followed by an irregular train of impulses lasting for about ioo msec. The latency and number of spikes in the short burst depended upon the intensity of the stimulus and also upon the temporal relationship between the stimulus and the spontaneous bursts of the motoneurone. A stimulus which closely preceded a spontaneous burst sometimes produced a spike train of 80-100 impulses instead of the normal 1-10 impulses. The

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50 miec.

Text-fig. 2. Extracellular recordings from the optic tract, showing the response of the moto-neurone following stimulation of portions of the tegumentary nerve. The top record of each column is the response to stimulation of half the tegumentary nerve with a o-1 msec, shock at maximal intensity. Subsequent records in each column show the responses produced by thinner and thinner strands of the initial two halves. The stimulus intensity was adjusted in each case to produce a maximal response. The bottom records in each column show responses following simultaneous stimulation of all the strands in each half. Diminishing the number of stimu-lated afferent axons produced a proportional decrease in the number of spikes in the moto-neurone burst and an increase in the latency of the response. No spikes followed stimulation of the thinnest bundles. Gathering all the strands of each half together and stimulating them simultaneously produced a response jimilar to the initial one and indicates that the changes in the response were not caused by damaging the fibres when splitting the nerve.

tegumentary nerve often produced a longer initial burst of spikes than the oculomotor nerve but latencies were similar, ranging from 4-5 to 7 msec.

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|he number of long bursts but these rapidly adapted, the number of motor axon spikes dropping to one or two. Single spikes consistently followed with constant latency a stimulus frequency of between 5 and 10 impulses/sec. (Text-fig. 3/) but quickly dropped out at frequencies in excess of 50 or 60 impulses/sec. Failure of one afferent pathway due to repetitive stimulation did not result in failure of the others and spon-taneous bursts still occurred in preparations in which both afferent pathways had failed.

(e) (0

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4 msec. •40 mV.

Tort-fig. 3. Intracellular and extracellular (lower trace in a and b) recordings from the peripheral end of the motor axon, showing the summation of excitatory effect produced by the two afferent nerves. A subthreshold stimulus to the oculomotor nerve (a) produced a short burst in the motoneurone if it was preceded by a suprathreshold stimulus to the tegumentary nerve (6). Simultaneous stimulation of both afferents with suprathreshold shocks produced a short high-frequency burst in the motoneurone (c), which lengthened as the two stimuli were moved further apart {d and e). Repetitive stimulation of the tegumentary nerve at 7/sec. produced a single spike with fairly constant latency as shown by a number of superimposed records (/).

(1) Summation of the excitatory inputs

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evoke motoneurone spikes but simultaneous stimulation of two such bundles would do so. Intracellular stimulation of single axons in the tegumentary nerve never produced a response in the motoneurone but there is no evidence that the axons tested in this way were connected to the motoneurone. Also behavioural studies have shown that the eye withdrawal is not produced by large hair-receptor organs on the carapace but by sensilla responsive to mechanical deformation of the carapace although both types of re-ceptor have their axons in the tegumentary nerve (A. P. Scott, personal communication).

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20 msec.

«mV.

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A subthreshold stimulus to one of the afferent nerve bundles would produce spikes in the motoneurone if it was preceded by a suprathreshold shock to the other (Text-fig. 3). The interval between the two stimuli could be as long as 100 msec. Gradually decreasing the interval between two suprathreshold sequential stimuli initially shortened the latency and increased the number of spikes following the second stimulus. Even-tually, when the two stimuli were close together, the two separate bursts blended into one even train. Pulses applied simultaneously to two different afferent inputs often gave a shorter but higher-frequency burst than two stimuli separated by 4 or 5 msec. (Text-fig. 3).

(2) Common spike-initiation point

Stimulation of the afferent nerves during a spontaneous burst produced a similar smooth summation of excitatory effect. The frequency of the spike train was raised but not accompanied by any break in the even spike pattern (Text-fig. 4). The absence of a break in the spike train is indicative of a common spike-initiation point on the motoneurone shared by the three inputs, but two other possibilities are (1) separate spike-initiation points one behind the other, and (2) separate spike-initiation points on different branches of the main axon trunk. The spike occlusion which would be ex-pected to occur if either these two situations prevails was demonstrated by intra-cellularly stimulating the motoneurone near the periphery in the following way:

(1) Two sequential spike-initiating sites on the motoneurone could be reproducibly demonstrated if the axon was driven repetitive with a depolarizing pulse of 50 msec, duration. Extracellular records from the periphery show the spontaneously generated burst to be interrupted by a high-frequency burst followed by a break in the spike train (Text-fig. 4 c).

(2) To simulate different spike sites on different branches of the axon and the con-sequent mixing of two separately generated spike trains, motor axon spikes were initiated with single short depolarizing pulses through the intracellular electrode. The spontaneously generated train was interrupted by the additional spikes and an irregular pattern appeared at the periphery (Text-fig. 4/). Neither of the above two irregularities follows afferent nerve stimulation combined with the spontaneous discharge.

(3) The sensory/motor link

The results of recording from the peripheral end of the axon suggest: (1) a long-lasting post-synaptic excitatory state within the motoneurone following a single afferent volley, (2) the excitatory effects of the afferent inputs and the spontaneous burst generator are summed before affecting a common spike-initiating locus on the moto-neurone.

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94

humps but did not alter their amplitude or duration (Text-fig. 5). These small unitary fluctuations in the membrane potential occurred irregularly for up to 100 msec, after a single pre-synaptic stimulus and, in some cases when the excitatory depolarization was large enough, erupted into conducted spikes. The separate excitatory depolari-zations in the motoneurone caused by repetitive stimulation of one afferent nerve, or by stimulation of both afferents together, combined to initiate a motoneurone discharge.

b c

b c

110 mV.

10 msec

Text-fig. 5. Intracellular records (upper traces) from the motoneurone within the brain {A in Plate 2 a) showing the excitatory depolarizations which follow stimulation of the tegumentary nerve. The depolarization increases with the stimulus intensity (1-8) until it produces a spike which is conducted to the periphery. Extracellular records (lower traces) show the peripheral response of the motoneurone and confirm that the microelectrode was in the correct axon. A number of smaller humps (a—f) are superimposed on the over-all excitatory depolarization and change their latencies with different stimulus intensities but do not alter in duration or amplitude. Voltage scales apply to the upper traces only.

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all-or-none activity since the amplitude of the potentials decreased at high frequencies, a characteristic of action potentials. The small potentials were, however, never con-ducted as spikes to the periphery.

50 msec. 10 msec.

Text-fig. 6. Two amplitudes of action potential recorded from the game axon. Upper traces show intracellular responses from the axon within the brain (B in Plate 2 a) and lower traces the extracellular peripheral response of the same axon. (a) The beginning of a spontaneous burst, showing a decrease in the size of the small potentials coinciding with a high frequency of discharge. The small potentials are not conducted to the periphery, (b) The same phenomenon on a faster time scale, showing the position of the small spikes on the falling phase of the preceding large spikes. Small spikes are always separated from one another by at least one large spike. Voltage scales apply to the upper traces only.

DISCUSSION

The response of the motoneurone following a single pre-synaptic stimulus is characterized by an initial short spike burst followed by an irregular tail of spikes. This could indicate two parallel pathways joining the sensory nerves with the moto-neurone; a direct, possibly monosynaptic link, and an indirect link involving an interneurone chain.

A direct monosynaptic link between all the sensory fibres and an extensive moto-neurone arborization would explain the temporal spread of initially synchronous pre-synaptic action potentials and the production of a smooth 10-20 msec, long post-synaptic depolarization in the motoneurone. Spike latencies of 5-7 msec, may seem too long for a single synaptic delay but intracellular recordings show the excitatory potential in the motoneurone to begin about 2 msec, after a subthreshold pre-synaptic stimulus. If the electrode was relatively distant from the fine synaptic terminals, true synaptic delays are shorter than 2 msec, which would not exclude a monosynaptic link. The slow rise time of the excitatory post-synaptic depolarization, and thus com-paratively long spike latencies, argue for a diffuse synaptic field some distance from the spike-initiation point. The system would act as a high-frequency filter and prevent regular one-to-one following of the post-synaptic neurone to a high rate of pre-synaptic firing, in spite of the ability of the post-synaptic axon to fire at 400 impulses/sec, to a maintained depolarization.

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following a single pre-synaptic volley. The activity of one of these interneurones appears as small fluctuations on the falling phase of the post-synaptic excitatory poten-tial. The constant amplitude and duration of these fluctuations suggests that each one is a post-synaptic potential generated by a single pre-synaptic spike in the interneurone which has a relatively localized contact with the motoneurone. The eruption of these potentials into spikes making up the irregular tail of the motoneurone burst confirms their excitatory function and it is unlikely that they represent the activity of the primary afferents, because repetitive activity of the afferents would not normally follow a single o*i msec, shock.

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(a)

100//

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Journal of Experimental Biology, Vol. 50, No.

Plate 2

(a)

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SUMMARY

1. The axon of the larger of the two motoneurones in the optic tract of Carcinus which bring about the rapid eye withdrawal is 30—50 fi in diameter and has an unusually thin sheath compared with crab leg neurones of similar diameter.

2. Within the brain the axon of the large eye-withdrawal motoneurone tapers fairly rapidly to 5-10/1. It extends diagonally across one side of the brain and branches in two near the mid line but does not pass the contralateral side of the brain. The axon terminals and the cell body have not been seen.

3. A single stimulus to the afferent inputs of the motoneurone produces a charac-teristic burst of impulses followed by an irregular train of impulses.

4. The excitatory effects of all tested afferent inputs to the motoneurone are sum-med before affecting a common spike-initiating locus on the motoneurone.

5. Intracellular recordings from the motoneurone within the brain show two types of subthreshold activity to follow a single pre-synaptic volley: a smooth graded depo-larization and small superimposed depodepo-larizations of constant amplitude and duration. 6. The synaptic link between afferents and the eye-withdrawal motor neurone is thought to be via a direct, possibly monosynaptic pathway and also by way of inter-neurone collaterals.

REFERENCES

BAK, A. E. (1958). A unity gain cathode follower. Electroenceph. din. Neurophysiol. 10, 745-8. BURROWS, M. (1967). Reflex withdrawal of the eyecup in the crab Carcimu. Nature, Land. 215, 56-7. CHTJ, L. W. (1954). A cytological study of anterior horn cells isolated from human spinal cord. J. comp.

Neurol. 100, 381—414.

FURSHPAN, E. J. & FURUKAWA, T. (1962). Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish. J. Neurophysiol. 35, 732-71.

HORRIDGE, G. A. & CHAPMAN, R. A. (1964). Sheaths of the motor axons of the crab Carcinus. Q. J.

microsc. Sci. 105, 175-81.

HUMASON, G. L. (1962). Animal Tissue Techniques. San Francisco and London: Freeman.

KERKUT, G. A. & WALKER, R. J. (1962). Marking individual nerve cells through electrophoresis of ferrocyanide from a microelectrode. Stain Technol. 37, 216—19.

MATTHEWS, P. B. C , PHILLIPS, C. G. & RUSHWORTH, G. (1958). Afferent systems converging upon cerebellar Purkinje cells in the frog. Q. J. exp. Physiol. 43, 38-60.

MELLON, DE F. & KENNEDY, D. (1964). Impulse initiation and propagation in a bipolar sensory neuron.

J. gen. Physiol. 47,

487-99-PABST, H. and KENNEDY, D. (1967). Cutaneous mechanoreceptors influencing motor output in the cray-fish abdomen. Z. vergl. Physiol. 57, 190-208.

RAMON Y CAJAL, S. (1933). Histology, transl. M. Fernan-Nunez. Baltimore: William Wood and Co. SANDEMAN, D. C. (1967).Excitation and inhibition of the reflex eye withdrawal of the crab Carcinus. J.

exp. Biol. 46, 475-85.

TAKEDA, K. & KENNEDY, D. (1965). The mechanism of discharge pattern formation in crayfish inter-neurons. J. gen. Physiol. 48, 435-53.

EXPLANATION OF PLATES

PLATE I

(a) Thin transverse section of the optic tract stained with toluidine blue, showing the spectrum of dif-ferent diameter axons, one of which is larger than the others (mn). None of the axons is heavily sheathed. (6) Whole mount of the optic tract with the motoneurone (mn) stained after injection with potassium ferrocyanide and treatment with ferric chloride. The Prussian blue dye was always precipitated on the inside of the axon membrane and not throughout the cytoplasm.

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PLATE 2

References

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