Camp. Biochem. Phgsiol.. 1977. Vol. 56A. pp. 509 fo 512. Prryamon Press. Printed in Great Britain

LACK OF ELECTROTONIC TRANSMISSION BETWEEN RETZIUS NERVE CELLS IN THE FIFTH AND SIXTH

FREE GANGLION OF HORSE LEECH HAEMOPIS SANGUISUGA

BOGDAN B. BELESLIN

Department of Pathological Physiology, Medical Faculty, Beograd, Yugoslavia

(Received 29 June 1976)

Abstract-l. The basic electrophysiological properties of the Retzius nerve cells in Vth and VIth free ganglion of horse leech (Haemopis sanguisuga) has been investigated.

2. Paired Retzius nerve cells in Vth and VIth free ganglion are not electrotonically coupled. Com- puted coupling resistance was too high to allow current flow from one cell to another.

3. The other membrane characteristics, such a resting, action potentials and the input resistances, are quite comparable to the values obtained with first four free ganglia.

INTRODUCTION

It is well known that the pair of Retzius nerve cells in the free (segmental) ganglia of medical leech (Hagiwara & Morita, 1962; Eckert, 1963) and horse leeches (Lent, 1972) influence each other by direct spread of electrical current from one cell to another. Histochemical investigations showed that 5-HT is present in the nerve cells of Retzius (Marsden & Ker- kut, 1969). Electromicroscopical studies demonstrated that accumulated cell organelles around the nucleus of leech neurons are not neurosecretory (Coggeshall & Fawcell, 1964). The action of acetylcholine, dopa- mine, 5-HT has been investigated on the spontaneous activity of Retzius nerve cells by Kerkut & Walker (1967). An increase in permeability to chloride ions has been shown to be involved in hyperpolarization to S-HT (Walker & Smith, 1973). A hypothetical model for the nature of 5-HT, M and D-receptors has been proposed (Smith & Walker, 1974, 1975). A voltage-sensitive slowly activated calcium current contributing to the inward current of the action potential, following application of TEA on either side of the membrane of Retzius nerve cells, has been demonstrated by Kleinhaus & Prichard (1975).

The greatly increased interest in the Retzius nerve cells itself stimulated investigation of basic electro- physiological properties of the giant cells in the first 10 free ganglia. Surprisingly it was found that Retzius nerve cells located in Vth and VIth free ganglion are not electrotoaically coupled.

A preliminary report describing some of these results has been presented (Beleslin, 1975a, b).

MATERIALS AND METHODS

Experiments were made on the Retzius nerve cells in the first 10 free ganglia of the horse leech Haemopis sanguisuga. Details of the dissection and experimental arrangement are described in the previous paper (Beleslin, 1971). Briefly. ganglia together with connectives and roots were removed from the anaesthetized leech (with 10% ethanol) and placed in leech Ringer solution. The composition of leech saline was (mM/1): NaCl 115, KC1 4. CaC1, 2, Na,HPO, 1.2.

NaH,PO, 0.3, pH = 7.2. Microelectrodes filled with 3M KC1 were used for intracellular recordings and for passing polarizing current intracellularly in these combinations: 2 or 4 microelectrodes were inserted into a pair of Retzius nerve cells or 3 micropippets in 3 Retzius nerve cells in 2 adjacent ganglia. Potentials were monitored through a high impedance preamplifier and displayed on a Tektronix storage 564 oscilloscope. The stimulus was delivered through a 150mQ series resistor to the stimulating elec- trode. Current was monitored across a 1 mR resistor. The input and coupling resistances were computed from the measured input resistances of directly polarised cells and transfer resistance (Bennett, 1966).

All experiments were done at 2&24”C.

RESULTS

Figure 1 illustrates the intracellular potential changes recorded simultaneously in a pair of Retzius nerve cells in the Vth free ganglion during spon- taneous spike discharges. The most striking finding was that spike potential aroused in one giant cell didn’t result in a postspike in adjacent one. Both cells fired completely independently. The same effect was obtained with Retzius nerve cells in VIth free gang- lion. The seemingly divers results obtained with giant cells in Vth and VIth free ganglia compared to others

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Fig. 1. Potential changes recorded simultaneously from a pair of Retzius nerve cells in Vth free ganglion.

509

510 BOGDAN B. BLLESLI~

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Fig. 2. Plot of voltage levels in the two cells at the termination of current against the current strength (left side) obtained from superimposed potential levels in directly polarizd and adjacent cell (right side). The cells were alternatively stimulated by hyperpolarizing current pulses (i, and i2) from left (CellI) and right side (CellII) of the junction. The straight lines were fitted to the point by eye. R,, and RzZ are input resistances of directly polarized cells. R, L and RL, arc transfer resistances.

R, and R, are input resistances. R, is coupling resistance. (e polarized cell. + nearby cell).

can be simply explained by supposing that these cells

are not electrotonically coupled. Figure 2 shows an example of one experiment test-

ing this prediction in a direct wdy. A pair of Retzius

CELL II

I

V22 J/W

v21 .n i2 e

Fig, 3. Potential changes in directly polarized Retrius nerve cells (V, , and V,,) and adjacent ones (V,z and V,,) in Vth free ganglion during outward current stimulation

(i, and iz).

nerve cells in Vth free ganglion were penetrated with four microelectrodes. Two of them were used for pass- ing constant current in a hyperpolarizing direction. and two for recording potential changes. Tracing of actual records are superimposed on the right side and potential levels in the two cells at the termination of current pulses are plotted against the amplitude of current on the left side of Fig. 2. Suprisingly, it was found that the electrotonic spread didn’t reach the adiacent cell. Commuted counling resistance was too high to allow current spread from one another.

cell to

Fig. 4. Simultaneous recording of spontaneous spike activity of Retzius nerve cells in Vth and Vlth free ganglion,

Uncoupling and leech neurones 511

Table 1. Major electrophysiological parameters of Retzius nerve cells in Vth and VIth free ganglion of Haemopis

identically ineffective in producing the electrotonic spread from one cell to another.

sanguisuga

Parameter

Resting potential Spike height Input resitance Coupling resistance

R (+ SE.) N

41.6 f 1.5 mV I 45.5 f 2.5 mV 16 13.8 + 1.6mQ 12

860.0 f 162.0 mc1 6

The data in Fig. 2 represents only a half of the complete experiment. In the second part, the cells were stimulated by depolarizing current. The repeti- tive spike activity could only be evoked in the directly polarized cell but not in the adjacent one (Fig. 3).

Table 1 summarises the main electrophysiological characteristic of Retzius nerve cells in Vth and VIth free ganglia. The values of resting potentials, spike heights and input resistances were comparable to the results obtained with the rest of first 10 ganglia. On the other hand computed coupling reisistance was sig- nificantly (P < 0.001) higher compared to the value (mean _t S.E.) of 16.0 k 0.9 mQ obtained with 37 Ret- zius nerve cells in the first 4 free ganglia.

The third possibility would be that Retzius nerve cells in Vth and VIth free ganglion are not electro- tonically coupled. This explanation is more probable since the computed coupling resistance is too high to allow current flow from one cell to another. At the moment it is very difficult to tell anything about significance of this finding. According to Lewis (1968) electrotonic conduction between a pair of spon- taneous firing neurons can produce a tendency toward spike synchrony, and under very special con- ditions; acceleration of spike production by means of circuit conduction. Considering more generally, junctional membrane with calcium ions play an im- portant role in regulation of cellular growth, differen- tiation and in response to injury (Loewenstein, 1974). The functional properties that distinguish electrotonic from chemical transmission are speed, intrinsic reci- procity, and ability to transmit graded potentials without impulse initiation (Bennett, 1966). Taking all these data into account it remain to be investigated if the Retzius nerve cells in the Vth and VIth free ganglion are involved in the release of mucus from globose gland which lie between the skin and muscle layer, as already has been proposed by Lent (1973).

The Retzius nerve cells in Vth and VIth free gang- lion are not electrotonically coupled. Figure 4 shows the intracellular potential changes in three neurons recorded simultaneosly during spontaneous discharge. Two microelectrodes were inserted into a pair of Ret- zius nerve cells in Vth, and one in the giant cell in VIth free ganglion. Complete asynchronous activity was obtained. Actually each cell had its own rhythm.

Acknowledgement-This work was supported in part by the Research Council of Serbia.

REFERENCES

BELE~LIN B. B. (1971) Effects of different external media on the leech ganglion cells interaction. Period. hiol. 73. 6367.

DISCUSSION

The present results showed that Retzius nerve cells located in Vth and VIth free ganglia are in many respects electrophysiologically comparable to that found in other ganglia. The values of resting, action potentials and input resistances are very similar to the values found in medical and horse leeches (Hagiw- ara & Morita, 1962; Eckert, 1963; Kuffler & Potter, 1964; Lent, 1972). On the other hand the coupling resistance was extremely high. There are several explanantions for such unexpected data.

BELE~LIN B. B. (1975a) Lack of electrotonic transmission between Retzius nerve cells in Vth and VIth free gang- lion of leech. Proc. of Vth Inter. Biophys. Cong. (Copen- hagen), P-5 13.

One possibility is that the identification of Retzius nerve cells was not correct. The central nervous sys- tem of medical .and horse leeches consists of the head, tail and free ganglia. The morphology of the ganglia has been studied by light microscopy (Retzius, 1891) and by electromicroscopy (Coggeshall & Fawcet, 1964). The free ganglia are very similar. The cells are segregated into 6 discrete groups. The position of the cells are constant so that with experience one can identify by inspection very easily 2 giant Retzius nerve cells. The size, shape and position as has been pointed out by Nicholls & Baylor (1968) provide valuable clues, and unmistakable evidence for its identification.

BELE~LIN B. B. (1975b) Basic electrophysiological properties of Retzius nerve cells in fifth and sixth free ganglion of horse leech Haemopis sanguisuga. Period. hiol. 77. Il.

BENNETT M. V. L. (1966) Physiology of electrotonic junc- tions. Ann. N.Y. Acad. Sci. 137. 509-539.

C~GGE~HALL R. E. & FAWCE?~ D. W. (1964) The fine struc- ture of the central nervous system of the leech, Hirudo medicinalis. J. Neurophysiol. 27. 229-289.

ECKERT R. (1963) Electrical interaction of paired ganglion cells in the leech. 1. gen. Physiol. 46. 573-587.

HAGIWARA S. & MORITA H. (1962) Electrotonic transmis- sion between two nerve cells in leech ganglion. J. Neuro- physiol. 25, 721-731.

KLEINHAUS A. L. & PRICHARD J. W. (1975) Calcium depen- dent action potentials produced in leech Retzius cells by tetraethylammonium chloride. J. Physiol., Land. 246. 351-361.

KERK~JT G. A. & WALKER R. J. (1967) The action of acetyl- choline, dopamine and 5-hydroxytryptamine on the spontaneous activity of the cells of Retzius of the leech, Hirudo medicinalis. Br. J. Pharmac. Chemother. 30, 644 654.

The second possibility would be that junctional membrane has the properties of an electrical rectifier. Such explanation cannot be accepted since transmis- sion didn’t occur no matter from which side or in what direction the cells were stimulated. Actually, depolarizing and hyperpolarizing current pulses were

KUFFLER S. W. & POTTER D. D. (1964) Glia in the leech central nervous system: physiological properties and neuron-glia relationship. J. Neuropkysiol. 27. 29G320.

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LENT C. M. (1973) Retzius cells: neuroeffectors controlling mucus release by the leech. Science, Wash. 179, 693696.

512 BOGDAN B. BELSLIN

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