Electrophysiology and anatomy of the large neuron pairs in the subesophageal ganglion of the leech

Camp.Biochem.Physiol.,1973, Vol. 46A, pp. 301 to 309. Pergmwn Prcrs. Printed in Great Britain

ELECTROPHYSIOLOGY AND ANATOMY OF THE LARGE NEURON PAIRS IN THE SUBESOPHAGEAL GANGLION OF THE LEECH ANDREW

H. WILSON,

JR.* and CHARLES

M. LENT

Department of Zoology and Microbiology, Ohio University, Athens, Ohio 45701 U.S.A. (Received 13 December 1972)

Abstract-l. The functional and structural characteristics of the large neuron pairs located within each of the four subganglia which comprise the subesophageal ganglion of the horse leech, Haemopis marmorata (Say), were investigated by intracellular recording and iontophoretic injection of the fluorescent dye, Procion Yellow. 2. The large paired neurons of the three posterior subganglia are very similar to Retzius cell pairs in segmental ganglia: they are coupled by a bidirectional electrotonic junction and send their axons into their ipsilateral roots. 3. The pair of large neurons in the anterior subganglion have decussating axons which enter their contralateral neuropiles, but lack both electrotonic interconnections and peripheral axons. 4. The four large neuron pairs are probably serial homologues to Retzius cells and the anterior pair appears to have lost the peripheral function of Retzius cells-the control of mucus release by the skin.

INTRODUCTION THE ANTERIORbrain of leeches evolved by an embryonic fusion of six ganglia each of which is homologous to segmental ganglia of the ventral nerve cord. The supraesophageal ganglion is a fusion of two of these ganglia-referred to as subganglia I and II which innervate the first two segments in the head of the leech. The subesophageal ganglion (S.E.G.) is a fusion of four ganglia-subganglia III, IV, V and VI which innervate their respectively numbered segments (Mann, 1953). Thus the segmental ganglia and their component neurons are the evolutionary precursors, or prototypes, of the brain subganglia and their neurons. The neurons in brain subganglia should therefore be serially homologous to those in segmental ganglia and differences between brain neurons and their segmental prototypes might reflect modifications resulting from the formation of the larger neural mass. The largest neurons found within each of the twenty-one iterated segmental ganglia are the paired ‘Kolossal’ cells of Retzius (1891). The largest pair of neurons within each of the four subganglia of the S.E.G. have similar diameters and general

* Present address : Department of Biology, Clark University, Worcester, Massachusetts 01610. 301

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H. WILSON,JR.AND CHARLES M. LENT

location as the Retzius cells whose neurosomata range from 50 to 70 pm in diameter and are located on the most ventral aspect of the ganglion. Marsden & Kerkut (1969) utilized fluorescence histochemistry to show that these large neuron pairs in the S.E.G., which contain the same neurochemical photophore as Retzius cells, are not seen in the supraesophageai ganglion. This fluorescent neurochemical is S-hydroxytryptamine-5-HT or serotonin (Rude et al., 1969). Thus, the morphological and histochemical evidence suggests that these four pairs of large neurons in the S.E.G. are the serial homologues of Retzius cells and that their homologues in the supraesophageal ganglion cannot be identified. Studies of the electrophysiology and neuronal branching patterns of Retzius cells from the horse leech, Haemopis marmorata (Say), provide the physiological and anatomical data (Lent, 1972a, b) with which to compare the large paired neurons of the S,E.G. in the same species in order to ascertain, (1) if indeed they are homologous neurons, and (2) what structural or functional modifications may have occurred to Retzius cell homologues as a result of fusion into a brain. Our findings indicate that the axonal branching and electrophysiology of the three pairs of large neurons from the posterior subganglia IV, V and VI of the S.E.G. strongly resemble Retzius cells. The anatomy and physiology of the large neuron pair from the anterior subganglion III of the S.E.G. differ markedly from Retzius cells. As the four large neuron pairs are the probable serial homologues to Retzius cells, we conclude that those pairs toward the anterior end of the brain are modified from their prototypes. The modification appears as a loss of the peripheral role of Retzius cells-control of mucus release from the skin (Lent, 1973). MATERIALS

AND METHODS

Haemopis of S-10 cm were maintained at 5 2 2°C in a S-10 7’; solution of leech saline in distilled water. For experiments in which chemical synaptic transmission was blocked, 20 mM MgCl, replaced a normally equivalent amount of NaCl in the leech physiological saline (Nicholls & Baylor, 1968 ; Stuart, 1970). An incision through the ventral body wall exposed the haemocoel which surrounds the ventral nerve cord. The supraesophageal ganglion, the S.E.G. and the first two segmental ganglia, together with approximately 1 mm of all their roots, were removed and placed into a chamber filled with saline at room temperature (21 -t 2°C). The preparation was secured with its ventral aspect upward. The neurons were viewed with lateral illumination and to facilitate their penetration by a microelectrode, the tough sheath of connective tissue enclosing the ganghon was removed. The large paired neurons are enumerated by their subganglion and position on the right or left side. Thus, the eight cells are called R3-L3, R4-L4, RS-R5 and R6-L6 (Fig. 1). Neuronal branching patterns were investigated by the iontophoretic injection of the fluorescent dye, Procion Yellow M4RS, into the large, paired cells (Stretton & Kravitz, 1968). Glass microelectrodes were prepared from tubing containing three to four glass fibers which act as a wick enabling these electrodes to be filled from the shank by a syringe containing a 4% solution of Procion in distilled water. Electrodes with tip resistances SO-120 MSZ passed dye readily. Penetration of the neural membrane by the electrode was monitored on an oscilloscope via a d.c. preamplifier. Hyperpolarizing pulses (300 msec, 1 Hz, 4-S x lo-‘A) carried the dye into the cell body and the current was measured as the 1R drop across a 1 MQ resistor.

LARGE NEURONS IN LEECH SUBESGPHAGEALGANGLION

303

Subganglion

Neurons

I

connectives

J R3,

111

R4,

i

FE*

ni

: Subesopbcgeof ganglion

1

E

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First!fe-gmentct ganglion

FIG. 1. A ventral view of the subesophageal ganglion (S&G.) of the horse leech, H. mtwwruta (Say), which shows the srrangement of the packets and the large neuron pairs with their axons in each of the four subganglia. The first segmental ganglion and its Retzius cells are illustrated for comparison.

The impaled cell would usually fill to a deep yellow-orange in 20-30 min, and four to five cells were tilled in each preparation. After dye injection, the preparation was maintained at 5 + 2’C for 24 hr in order to let the dye diffise into the processes of the neuron. The ganghon was fixed in 10% fo~~in-seine, dehydrated in a graded ethanol-water series, cleared in xylene and mounted in a depression slide with Lustrex (Dow Chemical). The whole mounts were viewed with a Leitz fluorescence microscope to prepare figures of the neuronal branching. Procion fluoresces orange which allows for easy differentiation of dye-filled processes from the green background fluorescence. Elec~ph~iolo~ of the large neurons was investigated with microelectrodes filled with 3 M KC1 and having tip resistances of 10-30 Ma. Resting and action potentials were monitored through d.c. preamplifiers whose outputs were displayed on a dual-beam oscilloscope. Hyper- and depolarizing currents were injected into the impaled neurons through one Ieg of a balanced bridge circuit which allows the use of a single microelectrode for simultaneous current injection and recording. Permanent records were prepared by photographing the oscilloscope trace with a kymograph camera. Physiological verification of the anatomical studies was obtained by simultaneously recording intracellular action potentials and extracellular units in the roots. The extracellular recordings were made with a suction electrode providing the differential input to a high gain a.c. preamplifier. To insure that the units were recorded from the axons of the large paired neurons and not from cells activated by chemical synapses, these experiments were conducted in a high Mgs+ saline. RESULTS

segmental ganglion is enclosed by 6 glial packets which envelope the neurosomata of ita approximately 175 pairs of bilaterally symmetrical neurons, Each

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and the Retzius cell bodies are always located within the anterior ventral packet (Gray & Guillery, 1963). The neurosomata of the pair of large neurons in the posterior subganglion of the S.E.G., R6 and L6, are found consistently within a similar anterior, ventral packet. However, in the anterior subganglion of the S.E.G., the neurosomata of the large neuron pair, R3 and L3, are consistently found in separate packets. The packets of the anterior subganglion have a lateral orientation rather than an axial one and the boundary between them occupies the most ventral position (Fig. 1). There is variability between individual animals in the packet locations of the large neuron pairs in the middle subganglia IV and V. Most often, the neurosomata of both pairs are located within separate right and left packets as is illustrated in Fig. 1. Most rarely, the neurosomata of both pairs are each found within single packets ; however, if only one of the pairs has its somata in a single packet, it is invariably the more posterior pair, R5 and L5. When the neurosomata of the large neuron pairs in subganglia IV and V are within a single packet, the boundary between the packets is oblique-intermediate between the lateral and axial boundaries of the packets of the posterior and anterior subganglia. Furthermore there is a general tendency for the neurosomata of the large neuron pairs to become smaller and more laterally displaced from the posterior to the anterior of the S.E.G. Figure 1 is a composite of all the anatomical data showing the branching of the four large neuron pairs in the S.E.G. as well as that of the Retzius cell pair in the first segmental ganglion. The patterns of the posterior three pairs of large neurons from subganglia IV, V and VI are nearly identical to Retzius cells from both Haemopis (Lent, 1972b) and Hirudo (Marsden & Kerkut, 1969). Their neurites enter the dorsal neuropile and there bifurcate into two major branches which leave their subganglia by paired ipsilateral roots. The paired lateral roots of subganglion IV have fused into a single structure (Hanke, 1948) and the two major branches of data are confirmed both R4 and L4 enter their ipsilateral one. These morphological by recording an axonal unit in the lateral root which corresponds with action potentials in the large neurosomata in each of these three subganglia. The 1 : 1 correspondence between these units and neural action potentials is reminiscent of that reported for Retzius cells and their axonal units in Haemopis (Lent, 1972a). The branching of the anterior pair of large neurons contrasts strongly with Retzius cells and with the three posterior pairs. The axons of R3 and L3 decussate and enter their dorsal contralateral neuropiles (Fig. 1). No observations were made of these axons branching or entering any roots. Thus, these Procion studies substantiate the results of Methylene blue histology (Retzius, 1891), showing that R3 and L3 have decussating axons, as well as those results of fluorescence histochemistry (Marsden & Kerkut, 1969), showing that their axons can be traced into the central neuropile. Suction electrode recordings from all roots in the anterior brain failed to exhibit any units corresponding with the action potentials of R3 and L3. Not every axon in a nerve can be detected by suction electrode; however, since units are detected from Retzius cell axons (Lent, 1972a) and from the axons of the other three large paired neurons, their absence from R3 and L3 indicates that they lack peripheral axons.

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The electrophysiology of the three posterior large neuron pairs from subganglia IV, V and VI is very similar to that of Retzius cells in Huemqpis(Lent, 1972a) and Hido (Hagiwara & Morita, 1962; Eckert, 1963 ; Gerasimov, 1967). Their resting potentials ranged from 30 to 40 mV and their action potentials of 20-25 mV do not actively invade the neurosomata, Both action potentials and d,c. potentials are transferred bidirectionally with attenuation through an electrotonic junction which

5OmVL 500 msec

Currrnt tntc left “UJurO” ~~

-40

FIG. 2. Electrophysiological characteristics of the three large neuron pairs from subganglia IV, V and VI of the S.E.G. a. Typical recordings from R5 and L5 which show their simultaneous action potentials and the electrotonic transfer of depolarizing and hyperpolarizing currents. In the top pair of traces subthreshoid depolarizations are transferred to LS from the action potentials arising in RS. b. Voltage-voltage relationships for R4-L4, RS-L5 and Rd-L6 to direct currents. The non-rectifying responses of the electrical junctions between each of the three pairs is represented by squares for R4-L4, open circles for RS-L5 and filled circles for R6-L6.

couples the cell pair (Fig. 2a). Spiking by the cell pair within a subganglion is usually synchronous: an action potential in one cell produces a suprathreshold depolarization in the other giving rise to an action potential. Occasionally, the depolarization in the follower cell is insufficient to produce an action potential and these observations (Fig. 2a) are utilized to calculate the action potential attenuation factor-action potential (mV)/sub~reshold electrotonic depola~zation (mV).

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H. WILSON, JR.AND CHARLESM. LENT

These three cell pairs had action potential attenuation factors of 3-4 which is similar for the 2-3 for Retzius cells in this species (Lent, 1972a), but lower than the 4-7 reported for Hz&do (Hagiwara & Morita, 1962). Direct current injected into one neuron of the pair produces a hyper- or depolarization, Yi, and an attenuated polarization, B,, in the other member. D.c. attenuation factors, YJYa, were linear over a range of 60 mV and ranged from 6 to 8 for these three large neuron pairs (Fig. 2b). Retzius cells from Haemopis have similar d.c. attenuation factors of 6-7 which is higher than those of 2-3.5 for Hirudo (Hagiwara & Morita, 1962). The d.c. attenuations for Hamopis Retzius cells were measured by an identical procedure to that used in this study, while those for Himdo Ret&us cells were measured by utilizing a third current-injecting microelectrode. The attenuations for these three large neuron pairs were not influenced by whether their neurosomata were located within a single packet or sequestered between two packets. The large neurons lack synaptic interactions with those in adjacent subganglia. Likewise, Retzius cells in adjacent ganglia show no electrophysiolo~cal interactions in either ~ae~opis (Lent, 1972a) or Himdo (Gerasimov, 1967). The electrophysiology of the anterior cell pair RJ and L3 also contrasts strongly with Retzius cells and the three posterior pairs. The range of their action potentials is lo-15 mV implying that the site of electrogenesis is further in space constants from their neurosomata than it is in Ret&us cells. Their spontaneous action potentials are not synchronous, but they tend to occur together (Fig. 3a). A cross-correlation histogram is useful in measuring weak neural interactions and one is shown for 93 spike pairs from R3 and L3 in Fig. 3(c). As can be seen in the figure, the histogram has a bimodal distribution with a low tendency for perfectly synchronous action potentials. The majority of the spike pairs (62 per cent) were initiated in R3 and the delay to the action potential in L3 had a mode of about 70 msec. The remaining 38 per cent were initiated by L3 and the model delay was about 30 msec. This pattern suggests that R3 and L3 are mutually excitatory and not driven by a common neural input (Kristan, 1973). Furthermore, the failure of either of these neurons to discharge rapidly when the other is depolarized suggests that their mutually excitatory synapses are weak ones (Fig. 3b). The synapse between R3 and L3 does not appear to be electrotonic as direct current producing a Yl of up to 50 mV failed to produce a V, of 1 mV. Any electrotonic interaction that may exist between R3 and L3 has an attenuation greater than 50. Thus, R3 and L3 differ from Retzius cells and twenty-five other pairs of bilaterally symmetrical neurons in the segmental ganglia of Himdo each of which possesses electrotonic junctions between them (Fomina 8t Tereshkov, 1972). DISCUSSION

Several morphological and physiological characteristics of the anterior brain of the leech are modified along its posterior to anterior axis. In the S.E.G., the ventral packets of the subganglia are laterally displaced, the paired roots fuse

LARGE NEURONS

IN LJD%H SURRSOPHAGRAL

and the large neuron pairs become smaller and sequestered packets.

(b) R3Tt I.3

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GANGLION

laterally into separate

+----F 504_

500msec

Iniervel from I_3 spikes to R3spikes, msec FIG. 3. Electrophysiological characteristics of R3 and L3. a. Spontaneous action potentials of R3 and L3 which tend to occur at similar periods. b. The impulse activities of R3 and L3 to depolarizations showing a lack of electrotonic transfer and the weak activation of the unstimulated neuron. c. A cross correlation histogram of ninety-three pairs in R3 and L3. Spike pairs with positive time values are those initiated in L3 and those with negative values are initiated in R3. Perfectly synchronous spike pairs would occur at time zero (dashed line).

The large neuron pairs within subganglia IV, V and VI are each very similar to Ret&us cells in size, location, neurochemistry, axonal branching, resting and action potentials and electrotonic interactions. These six neurons are in all probability the serial homologues to Retzius cells. They have been modified minimally, if at all, from their prototypes by being incorporated into the brain. No identifiable neurons corresponding to Retzius cells are found in subganglia I and II: no neurons in the supraesophageal ganglia contain 5-HT (Marsden 8t Kerkut, 1969) and no large neurosomata were observed in this study. The Retzius cell homologues in these subganglia are either smaller and synthesize a different neurochemical or they no longer exist. In either case, they are highly modified from their prototypes. The large neuron pair R3 and L3 of subganglia III are similar to Retzius cells in size, location and neurochemistry, but they differ in axonal branching, action

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ANDREW

Since R3 and L3 are the most probable potential size and electronic interactions. homologues to Retzius cells in subganglion III, we conclude that they are in an intermediate condition between the unmodified homologues from subganglia IV, V and VI and the highly modified homologues from I and II. The 5-HT from Retzius cells mediates the release of mucus from the skin of the leech, and a single pair of these neurons innervates eight to nine annuli in the middle body segments of Hirudo (Lent, 1973). Each body segment consists of five annuli; therefore, there is a functional overlap at the periphery of three to four annuli from each Retzius cell pair. The modification of the more anterior Retzius cell homologues has not reduced the amount of mucus released from the head of the leech perhaps because when its segments are compared to those of the body, each has a reduced number of annuli with a correspondingly smaller surface area. The six segments comprising the head have a total of ten annuli (Mann, 1953) each with an average surface area Thus, the three pairs of unmodified Retzius about half that of a body annulus. cell homologues from subganglia IV, V and VI could control mucus release from the entire head and each homologue would innervate a surface area only one-sixth that of a Retzius cell in a segmental ganglion. Retzius cells have minor axonal branches ending in the neuropile (Lent, 1972b) implying an interneuronal function in addition to the peripheral role. R3 and L3 appear to have lost their peripheral role, but retained, and perhaps enhanced, their interneuronal function. Acknowledgements-We thank Doctors C. H. Page, G. S. Stent and W. B. Kristan for advice and assistance with the manuscript. Part of this work is from an M.S. thesis (A. H. W.) submitted to Ohio University.

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Electrical

interaction

of paired

ganglion

cells in the leech.

r. gen. Physiol.

46, 573-587. FOMINA M. S. & TERESHKOV 0. D. (1972) Electrical transmission between symmetrical neurons in leech ganglia. Neurosci. Behav. Physiol. 5, 91-96. GERA~IMOV V. D. (1967) Electrical properties and connections of CNS giant nerve cells of Hirudo medicinalis. In Neurobiology of Invertebrates (Edited by SALANKI J.), pp. 285-292. Plenum Press, New York. GRAY E. G. & GUILLERY R. W. (1963) An electron microscopical study of the ventral nerve cord of the leech. 2. Zellforsch. Mikrosk Anat. 60, 826-849. HACIWARA S. & MORITA H. (1962) Electrotonic transmission between two nerve cells in leech ganglion. J. Neuvophysiol. 25, 721-731. HANKE R. (1948) The nervous system and the segmentation of the head in the Hirudinea.

Microentomology

13, 57-64.

LENT C. M. (1972a) Electrophysiology of Retzius cells of segmental ganglia in the horse leech, Haemopis marmorata (Say). Comp. Biochem. Physiol. 42A, 857-862. LENT C. M. (197213) Retzius cells from segmental ganglia of four species of leeches: comparative neuronal geometry. Comp. Biochem. Physiol. 44A. 35-40. LENT C. M. (1973) Retzius cells: neuroeffectors controlling mucus release by the leech.

Science, Wash. 179,693-696.

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KRISTANW. B. (1973) Characterization of connectivity among invertebrate motor neurons by cross correlation of spike trains. In The Neurosciences Third Study Program (Edited by SCHMITT F. 0. et. al.) M.I.T. Press Cambridge, Mass. MANN K. H. (1953) The segmentation of leeches. Biol. Rew. 28, l-15. MAR~DENC. A. & KIXRKUT G. A. (1969) Fluorescence microscopy of the S-HT and catecholamine containing cells in the central nervous system of the leech Hirudo medicinalis. Cmp. Biochem. Physiol. 31, 851-862. NICHOLLSJ. G. & BAYLORD. A. (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. J. Neurophysiol. 16, 740-756. R~TZIUSG. (1891) Biologische untersuchungen, New FoZge II. Sampson and Wallin, Stockholm RUDE S., COGGESHALLR. E. & VAN ORDEN L. S. (1969) Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neuron. J. Cell Biol. 41, 832-854. STRETTONA. 0. W. & KRAVITZE. A. (1968) Neuronal geometry: determination with a technique of intracellular dye injection. Science, Wash. 162, 132-134. STUARTA. E. (1970) Physiological and morphological properties of motorneurons in the central nervous system of the leech. J. Physiol., Land. 209, 627-646. Key Word Index-Retzius cells; leech CNS; Procion Yellow; electrotonic coupling; Haemopis marmorata; Hirudo; giant neurons; subesophageal ganglion; brain.