On neuronal homologies within the central nervous system of leeches

Camp. Biochem. Physiol., 1977, Vol. %A. pp. 285 to 297. Perganwn Press. Printed in Great Britain

ON NEURONAL HOMOLOGIES NERVOUS SYSTEM

WITHIN THE CENTRAL OF LEECHES

KENT T. KEYSERAND CHARLES M. LENT Department of Biology, State University of New York, Stony Brook, New York 11794, U.S.A. (Receioed 11 February 1977)

Abstract-l. We investigated the electrophysiological properties of 18 identifiable neurons in three leeches: Haemopis marmorata, Hirudo medicinalis and Macrobdella decora. 2. The paired serotonergic Retzius cells are indistinguishable among the species in terms of somata positions, branching patterns, electrophysiological parameters and muco-egector functions. 3. The T, P and N mechanosensory cells in these three species are virtually identical by virtue of their sizes, locations, axonal branching, electrophysiological properties, responses to dermal stimulation and receptive fields. 4. The L motor neurons of all the leeches are electrotonically coupled, have equivalent cell positions and axonal branches, and control the longitudinal muscles. 5. We have investigated several properties of 12 neurons in Hirudo and Haemopis and found no differences between their S interneuron, heart excitor motor neurons and their monoamine-containing neurons. 6. We found no differences between 30 homologous neurons in the C.N.S. of these leeches at any level of organization: membrane properties, central connectivity, morphology and axonal branching, peripheral connectivity or functional roles. The extremely conservative stereotypy of these cells is impressive.

INTRODUCIION Biological structures are homologous if they possess a common ancestor, and differences in these homologous structures are utilized in evaluating the evolu-

tionary relationships between and within taxonomic groups (taxa). The behavior of animals is held to be an important agent in shaping the course of evolution (Roe & Simpson, 1949); however, behavior is often difficult to systematically quantify. Kandel (1976) stipulates that “The discovery that particular cells mediate behavior has raised the possibility that the difficulties in establishing (behavioral) homologies might be overcome by using the neural architecture of behavior as the basic taxonomic characteristic”. Leeches (Annelida : Hirudinea) are particularly well-suited for investigations upon neuronal homologies as all have segmental nervous systems comprised of exactly 34 ganglia (Mann, 1953). Six ganglia are fused into an anterior brain (2 supra- and 4 sub-esophageal) and seven are fused into a caudal brain. The remaining 21 are distributed segmentally and communicate by means of longitudinal connnectives. Because leeches are limbless, all segmental ganglia are very similar except for the 5th and 6th which innervate their complex hermaphroditic genitalia (Zipser, 1976). Each of these segmental ganglia has six huge glial cell (Francois, 1885) which serve as packets enveloping its 35WtOO unipolar neurons (Coggeshall & Fawcett, 1964). Most of these neurons are in bilateral pairs in these packets producing anatomical relationships which are similar in several species of leech (Retzius. 1891 ; see Fig. 1). The largest pair of neurons in the leech is the serotonergic Retzius cells on the ventral surface of the ganglion. The histochemistry and geometry of these cells are very similar in 6 species of leech (5 genera) from two families: Hiru-

dinidae and Glossophoniidae (Lent, 1973a, 1977). Electrophysiological investigations of the Retzius cells in all these species find very similar resting and action potentials and that the pair of cells are electrotonitally coupled (see Lent, 1977). Such striking similarities in the morphology, physiology and chemistry of these the largest cells in all these species of leech lead to the conclusion that Retzius cells are neuronal homologues in the leech CNS. The European medicinal leech, Hi&o medicinalis, has been the subject of so many investigations upon its neuronal function and synaptic properties as to have become a “type specimen”. The relative constancy of neurosomata within the glia allows for a tentative visual identification of more than 30 identified neurons on its ventral ganglionic surface. These include three classes of primary mechanosensory neurons (Nicholls & Baylor, 1968). Three pairs of cells fire phasic impulse bursts when the skin is lightly touched (T cells). Two pairs respond to noxious derma1 stimulation with tonic impulses (N cells) and two pairs respond to pressure (P cells). Each class of mechano-sensory cell is morphologically and physiologically distinctive. Further each of the 14 cells has a discreet receptive field on the skin which is constant in size and shape, not only from segment to segment, but from animal to animal. The dorsal surface of each Hit-& ganglion has many pairs of motor neurons (Stuart, 1969). The L motor neurons are easily distinguished by their positions, small action potentials, and efficacious electrotonic interconnection. These cells each control the contralateral longitudinal musculature of their segment. This report is a comparison of the physiological properties of many neurons in the ganglia of the American bloodsucking leech, Macrobdella decora and in the carnivorous leech, Haemopis marmorata

285

Fig. I. Dark fieId photomicrographs of the ventral aspects of living segmental ganglia from Hirudo medicinalis (A) and Haemopis mrmorata (B). The identified neurons of Hirudo are labelled and the probable homologues in Haemopis are circled. T, P and N, mechanosensory cells; RZ. serotonergic mucoeffectors. Calibration mark = 250 pm.

287

Neuronal homologies in the leech to those in the ganglia of Hi&o medicinalis. The T, P, and N mechanosensory cells in these three species are virtually identical in terms of cell size and location, axonal branching patterns, electrophysiological responses and receptive fields. The L motor neurons in all three species are electrotonically coupled, have similar axonal branches, and control the longitudinal muscles. The Retzius cells control the secretion of mucus from the skin of Hirudo (Lent, 1973b) and sub serve the same function in the other two genera. We also present data showing a remarkable constancy between twelve other identifiable neurons in Hirudo and Haemopis, the most distantly-related leeches. MATERIALS

Retzlus

H. medicinalis

cells

-

H. marmorata

w

AND METHODS

The

three species of leeches were obtained commercially and maintained at 20°C in 10% leech saline (Nicholls & Baylor, 1968) in distilled water. Specimens were chilled in 0°C saline for dissection and the CNS was exposed by a dorsal incision. Ganglia with and without a section of innervated body wall were secured to a layer of transparent Sylgard Resin (Dow Chemical) with stainless steel insect pins and trans-illuminated with a darkfield condenser. Microelectrodes were drawn from glass fiber-filled Kimax tubing (o.d. 0.8-l .Omm) on a Nastuk type puller (Industrial Associates, Inc.). These electrodes were backfilled with 4 M KAc and had tip resistances of 35-75 Ma. The electrodes were positioned with either MM-33 or “Aus Jena” sliding manipulators. Resting and action potentials were monitored with M-4A electrometers (W-P Instruments) whose outputs were fed into a Barrows Operational Amplifier manifold. Intracellular currents were generated with a Grass S48 stimulator and passed into the recording electrodes by means of balanced bridge circuits. Extracellular activities of the lateral roots were monitored with suction electrodes. These signals were amplified with Grass P-15 Amplifiers whose outputs were also fed into the Barrows amplifier. The outputs from the amplifier manifold were led simultaneously to a Tektonix 565 oscilloscope, a slave D-13 storage oscilloscope, an audio monitor, and a HewlettPackard 7404 Oscillographic Recorder. Permanent records were obtained either from the oscillos&ope face with a Grass Kymograph camera, from the storage scope with a Polaroid camera, or from the oscillographic pen writer. For morphological studies, electrodes were filled with a 56% solution of Procion Yellow dye (Stretton & Kravitz, 1968). The dye was iontophoretically injected into the cells with 500 msec lOOnA hyperpolarizing currents at 1 Hz. Intensely stained preparations were kept at 5°C for 24 hr and fixed in 10% formalin in leech saline for 12 hr. The ganglia were dehydrated in an ethanol series, and mounted whole in fluormount (Edward Gurr). The ganglia were viewed and photographed with a Zeiss Photomicroscope II. RESULTS

The two bloodsucking leeches, Hirudo and Macrob della, are in the same subfamily and are more closely related to one another than to the carnivorous Haemopis even though all three are in the family Hirudinidae (Sawyer, 1972, and personal communication, 1975). Therefore, the greatest taxonomic, and very probably evolutionary, distance occurs between Haemopis and either of the other two genera. Retzius cells (RZ) The largest pair of neurosomata is located within the anterior ventral packet of the segmental ganglia

M. decora

Fig. 2. Simultaneous intracellular and extracellular recordings from the Retzius cell bodies and the anterior, dorsal and posterior root branches (top to bottom). Recordings from all three species are taken in 20mM Mg’+ saline. The units in the roots occur at an invariant latency following a Retzius cell impulse. Calibrations: 50 mV. IOOpV, 10 msec. from these three genera of leeches as well as from Aulostoma and Placobdella (Tereshkov et al., 1969; Lent, 1973a). For each of these leeches, some evidence has been accrued which implies that these neurons contain serotonin as do the Retzius cells in Hiruah (Rude et al., 1969). Anatomical studies show that each of these three genera have RZ which send axons into all three major branches of the ipsilateral roots (Ehinger et al., 1968; Lent, 1973~). Simultaneous recordings from the Retzius neurosomata (microelectrode) and the anterior, dorsal and posterior branches of the lateral roots (suction electrodes) are shown for all three species by Fig. 2. An axonal impulse is invariably associated with a spontaneous intracellular action potential and is detected in all three roots. These axonal units occur at a fixed latency and persist in high Mg2+ saline. These root units are therefore axons from the RZ and not units driven by central chemical synapses. Stuart (1970) demonstrated that 20mM Mg2+ abolishes chemical synaptic transmission within the leech CNS. Thus. the Retzius cell location, axonal branching and efferent conduction are indistinguishable among these three leeches. Furthermore, we attempted to record at the same positions on the roots of each species and it can be seen in Fig. 2 that the axonal impulses of these three species have the same relative conduction velocities in the three roots. Table 1 presents the data on the electrophysiological parameters from thee three species. The mean resting potential of Haemopis is 34 mV and within 1 mV of Hit-ado. Macrobdella has an average resting potential of 28 mV; however, this value is well within the ranges of the other genera. There are no differences in the amplitudes of the action potentials (34-38 mV) seen in the RZ of these three leeches. The duration of the action potential was measured at the midpoint

Ksr;r Table

1. The electrophysiological

Resting

Haemopis H irudo Macrobdella

Table

parameters of Retzlus cells in the three the number of cells sampled

potential X (range)

(mV)

Action

2. The electrophysiological

Haemopis Hirudo Macrobdella

potential X (range)

(mV)

Action

and the average from species to

species. Furthermore, the RZ in all three species are coupled by a bidirectional electrotonic junction. Direct current into either cell produces a d.c. shift in the other which persists in high Mg’+. Thus, this important pattern of central connectivity between Retzius cells is shared by all three species in this study. The Retzius cells control the secretion of mucus from the dermal glands of the medicinal leech by means of their transmitter serotonin (Lent, 1973b). The morphology of the RZ in these three species is so similar that it was predicted (Lent, 1973~) that the cells probably subserve similar functions. Intracellular stimulation of the Retzius cells in a given ganglion of both Haemopis and Macrohdella increases their T

Haemopis

marmorata

(mV)

parameters of touch cells in Ihe three number of cells sampled

- 40 (34 43) -41 (34-44) -39 (3(t42)

peak and undershoot is indistinguishable

potential X (range)

species

potential X (range)

(mV)

65 (5X 77) 64 (55~80) 62 (55 73)

of leeches.

N 15

Duration (msecl X (range)

Iv

7 (6 8) 6.X (6 XI 6.Y (6 XI

IS 17 IX

38 (25 4X) 36 (23-50) 34 (30~ 43)

- 34 (2@-40) -33 (17 42) -78 (21 37)

Resting

between the of 6%7.0msec

T. KEYSER ANU CHARLES M. Li.hr

species

of leeches.

N 1s the

Duration (msec) X (range)

!I

2.3 (1.9 7.4) 7.7 (I.‘) 2.3) 2.2 (1.X 7.4)

17 14 16

impulse frequency and results m the secretion of detectably large amounts of mucus from the skin of that particular segment. Thus. the RZ seen in the ganglia of these three genera of leech are morphologically similar, electrophysiologically indistinguishable and have a similar function. Thus. these cells are neuronal homologues in the ganglia of these three species of leech.

A. Touch crlls (T). Six neurons in each ganglion respond to gentle deformation of the ipsilateral skin within each of their receptive fields by firing phasic bursts of rapid impulses (Nicholls & Baylor, 1968). Segmental ganglia have three of these neurons in each of the paired anterior lateral packets along of Hirudo

P

II

Hirudo medicinalis

Fig. 3. Intracellular responses of T, P and N cells in the three species to depolarizing currents. With similar currents (0.5 nA) the T cells fire a phasic burst. the P cells fire a single, longer latency impulse and the N cell generates the longest latency impulse. The time constants and action potential we of each class of cell is indistinguishable across the species. Note also that the action potential amplitudes and durations as well as the size of the after potential increases in each class of cell (T. P. N). Calibrations are the same for each trace. IO msec. 40 mV and 4 nA.

289

Neuronal homologies in the leech Touch Medial

Latera

Mid

I

e

H. medicinalis

H. marmorata

cells

-z

M. decora &

WI

---++-+I

Fig. 4. Simultaneous intracellular and extracellular recordings from the three touch cells and the anterior, dorsal and posterior roots (top to bottom). For all three species, the recordings were made in 20 mM Mg2+ saline. The units in the roots of the three species occurred at invariant latencies following intracellular T cell impulses which were induced by current injection. The medial and mid T cells have axons in the anterior and posterior roots. The lateral T cell axon is found only in the dorsal branch. Calibrations: 50 mV. 200 pV, 10 msec.

their ventro-medial margins (Fig. 1). We have impaled and recorded from six neurosomata in the same positions in Haemopis and Macrobdella ganglia and find that their electrophysiological properties are indistinguishable from those of Hirudo (Table 2). The average resting potential ranges between 39 and 41 mV, the action potentials from 62 to 65 mV, and their duration from 2.2 to 2.3 msec. That the electrophysiological parameters of these cells are virtually identical among the three species can be clearly seen in Fig. 3. These neurons in Haemopis and Macrobdella ganglia respond to gentle touch of the ipsilateral skin by firing impulses in phasic bursts. Most often the bursts occur at the application (on response) and cessation (off response) of the touch stimulus. Movements of the bathing saline are often sufficient to excite the cells. Therefore, these cells are touch responsive mechanosensory cells as they are in Hirudo. We investigated the axonal branching of these T cells with simultaneous intra- and extracellular recordings. The data for all three T cells in the three leeches is shown by Fig. 4. Their axonal units have fixed latency, Mg2 + resistant impulses. The most lateral T cell has an axon in the dorsal root of all three species. The most medial and intermediate (mid) T cells each have axons in the anterior and posterior lateral roots. As was true for the RZ, the branching and relative conduction velocities of these axons are identical across the species. Figure 5 is a photograph of a Procion-filled mid T cell from Haemopis which has axons projecting into both homolateral roots. Thus, in these three leeches, the six T cells are homologous as adjudged by the criteria of cell size and position, axonal branching, resting and action potential properties, phasic firing patterns, and responsiveness to “touch” stimulation. The receptive fields of these cells are also very similar between these leeches and will be discussed in detail below. B. Pressure cells (P). Four neurons in each segmental Hirudo ganglion respond to a maintained mechanical pressure on the ipsilateral skin by firing

slowly-adapting impulse bursts. These P cells sometimes continue to fire after the stimulus ceases (Nicholls & Baylor, 1968). Their somata are the two largest within the ventral aspect of each posterior lateral packet. Similar large somata are found in these positions in Haemopis and Macrobdella (Fig. 1). We have recorded from these somata and they have average resting potentials of 4&42mV, action potential of 75-19mV and durations of amplitudes 3.94.0msec (Table 3). These action potentials have more undershoot than those in T cells, and are indistinguishable from species to species (Fig. 3). The cells in Haemopis and Macrobdella ganglia are stimulated to fire impulses by the application of pressure on the homolateral skin. Therefore, these neurons are P cells in all three species. Figure 6 shows simultaneous recordings from P cell bodies and the lateral roots in all three species. The medial P cell has an invariant axonal unit in the dorsal branch of the posterior root. The lateral P cell has axons in both the anterior and posterior roots. The morphology of the P cell can be discerned from Fig. 7 which is a fluorescence micrograph of a lateral P cell from Haemopis which has been filled with Procion Yellow. This neuron has axons in the anterior and posterior roots which are responsible for the units detected with the suction electrodes. Thus, all three of these leeches have two pairs of pressuresensitive cells which are homologous. These neurons have very similar cell sizes and locations, axonal branches, resting and action potentials, as well as slowly-adapting responsiveness to dermal pressure. Furthermore, the receptive fields of the P cells are indistinguishable between the species and will be discussed in detail below. C. Nociceptive cells (N). Four neurons in each Hirado segmental ganglion respond to noxious stimulation of the skin by firing tonic impulses. These large impulses usually continue following the cessation of noxious stimulation (Nicholls & Baylor, 1968). Two N cells are located near the T cells within each of

KENT T. KEYSERAND CHARLES M. LENT

290

Fig. 5. A fluorescence photomicrograph of a whole-mounted segmental ganghon trom Haemopis murA Procion-filled mid T cell is the fluorescing cell in this preparation. This T cell sends one process into the anterior connectives and an axon into the anterior and posterior lateral roots. The morphology of this Haemopis cell is very similar to that of homologous cells in Hirdo. Calibration:

moruta.

the anterior lateral packets. We find similarly-sized cell bodies in these packets in both Macrobdella and Haemopis (Fig. I), and when impaled these cells demonstrate resting and action potentials which are virtually identical to those of Hirudo (Fig. 3). The electrophysiological parameters for these cells in all three species are given by Table 4. They have resting potentials of 4@41 mV, and their 83-85 mV action potentials have very large undershoots and last 464.1 msec. In Macrobdella and Haemopis, these neurons respond to noxious stimulation of the skin (e.g. cutting or pinching) by firing tonic impulses. Therefore these cells are N cells in all three leeches. Simultaneous recordings from the N cell bodies and lateral roots are shown by Fig. 8. The axonal Table 3. The clectrophysiological

parameters of pressure cells in the three species of leeches. N IS the number of cells sampled

Resting potential (mV) X (range) Haemopis Hirudo Mucrobdullu

branching is invariant in all three species: The medial N cells each have an axon in the dorsal branch of the posterior root and each lateral N has axons in all three roots. Despite contrary reports on Hirudo. we detect an invariant. Mg’+ resistant unit in the dorsal branch from the lateral N: however, this axon is not responsive to stimulation of the dorsal skin. Figure 9 is a fluorescence micrograph of a lateral N cell from Haemopis which has been filled with Procion Yellow. The major axonal branching of this cell can be clearly seen by this figure. Therefore, the four N cells found in these three species of leech are very probably homologues as they have similar cell sizes, positions and branches; and they demonstrate slow. tonic impulses which increase in response to “painful”

Action potential (mV) X (range)

-41 (35545)

76 (6%82)

-42 -40

79 (68-82) 75 (68-79)

(3347) (33-42)

Duration (msec) X (range)

R;

3 (3.5 4.2)

17

4 (3.X 4.01 3.9 (3X 4 I)

1x IX

Neuronal homologies in the leech Pressure Medlal

291

ceils Latera

H. medicinalis

Nocloceptlve

I

Medlol

cells Lateral

H. medicinalis

H. marmorata H. marmorata -I_

M. decora

AL-

&I M. decora

Fig. 6. Simultaneous intracellular and extracellular recordings from the P cell son-rata and the anterior, dorsal and posterior roots (top to bottom). For all three species, these data were obtained in 20mM Mg2+ saline. The units in the roots of all three species occur at invariant latencies following intracellular P action potentials induced by current injection. The medial P cells have axons in the dorsal root (the extra unit in the posterior root of Hirudo was spurious). The lateral P cells have axons in the anterior and posterior roots. Calibrations: 50 mV, 200 pV, 10 msec.

AL

=?=I

Fig. 8. Simultaneous intra- and extracellular recordings from N cell bodies and the anterior, dorsal and posterior roots of all three species. The medial N cell has an axonal unit in the dorsal root and the lateral N cell has axonal units in all three roots. These fixed latency impulses were generated with intracellular current injection in preparations bathed in 20mM Mg2+ saline. Calibrations: 50 mV. 200 pV. IO msec.

Fig. 7. A fluorescence photomicrograph of a lateral P cell which has been injected with Procion Yellow (whole mount, Haemopis mnrmorata). This cell sends one axon directly into the anterior root and another curves through the neuropile and then enters the posterior root. The morphology of this Hnemopis P cell is very like the lateral P cell in Hirudo. Calibration: 50pm.

KENT

292 Table 4. The

electrophysiological

Resting

Haemopis

Hirudo Macrohdella

KEYSER AND CHARLESM. LENT

parameters of nociceptive cells in the three is the number of ceils sampled

potential X (range)

-41

T.

(mV)

Actlon

(3348)

- 40 (33-46) - 40 (32.-43)

stimulation. The N cells also have similar receptive fields across these species and these will now be discussed. D. Receptiue fields of mechanosensorv cells. A typical midbody segment of the Hirudcnid leeches is divided into five annuli, the middle one of these bears several light sensitive sensillae (Kretz et al., 1976). The segmental ganglion is situated upon this central annulus. Both Hirudo and Macrohdella have colorful markings upon the skin which reliably delineate the ventral, lateral and dorsal surfaces. Haemopis has no reliable landmarks save their sensillae. Thus, for Haemopis receptive fields, the first two sensillae going dorsally from the ventral midline are considered to be on the ventral surface. The next two are considered lateral and the next three, dorsal. The primary receptive fieldsfor the mechanosensory cells of Macrohdella are shown by Fig. 10, and these fields are indistinguishable from those we find in Haemopis and those reported for Hirudo (Nicholls & Baylor, 1968). The field for which each cell responds to mechanical stimulation consists of an oval

potential X (range)

(mV)

85 (75m98)

85 (74 100) x3 (73 -95)

spec~cs 01 l~chc~

Duration (m\ccr X (ranger J (3.X 44:

31 fJ’l3-1) J I (3.X WI

\

.\ IT

20 I (1

area of skin which always includes the central annulus and 2--3 annuli on each side of it. The fields of homologous cells in adjacent ganglia overlap at their coincident field edges somewhat ;ts do cells of like modality within the Same segment. The most lateral T cells have receptive tields LI~OII the dorsal surface of the skin. The medial T cells have receptive fields on the lateral skin surf~c and the mid T cells on the ventral skin The lateral P and N cells each have receptive fields on the venter covering part of the lateral surface. The medial P and N cells respond to stimulation of the dorsum and the remaining

lateral

skin. The sensitivity

of any

receptive

field is greatest at its center and decreases towards its margins. We also compared the subdlvlsions of the receptive fields of the mechanosensorq cells which send axons out more than one lateral root. For all three species. the central annulus and the more :lntcrior skin arc innervated by the axons within the Ltnterior root. The more posterior skin is innervated hy mechansosensory cell axons within the posterior- root. Thus. the

Fig. 9. A fluorescence micrograph of a lateral N cell which has been injected with Proclon Yellow (whole mount, Haemopis marmorata).The initial segment sends a process into the antertor connective and branches into two major axons that enter the anterior and posterior roots. A process emerges from the latter axon and enters the posterior connective. The morphology of these cells is very similar to homologous N cells in Hirudo. Calibration: 50 pm.

Neuronal homologies in the leech Receptive

P

293

fields

ceils

87,

Dorsal

i I

N cells

Med

.~ Lot

Fig. 10. The receptive

longitudinal

motor

muro~I

are for

(L)

segmental ganglia possess a pair of neurons upon their dorsal lateral surface, and the efferent impulse activity of these cells chemically excite the longitudinal muscles to contract (Stuart, 1970). Each L cell has 5510 mV action potentials, projects to the contralateral musculature through all three roots, and is strongly coupled to the other L cell by means of an electrotonic junction. Such central coupling assures a simultaneous shortening of both halves of the leech when the L cells fire impulses. We find similarly positioned cells in the ganglia of Haemopis and Macrohdella, and when these neurons are impaled, their impulses cause the contralateral longitudinal muscles to contract. These neurons have small action potentials, and in Haemopis are also electrotonically coupled (Fig. 1 I). Hirudo

Fig. Il.

Simultaneous

N

fields of the T, P and N cells from a mid-body

receptive fields and their patterns of innervations consistent across species as they were reported Hirudo (Nicholls 8~ Baylor, 1968). Large

N

intracellular

recordings

segment

of Macrohdefla

decora

Further, these cells always send their axons out all three contralateral roots. Therefore, a motor neuron whose impulse activity governs the shortening reflex of the leech appears to be homologously conserved by all three of the leeches examined in this study. Other rleurons A pair of heart excitor motor neurons (HE) are found in the anterior regions of the ganglia in Hirudo (Thompson & Stent, 1976). These cells fire small impulses at tonic high frequencies which are periodically interrupted by large chemical IPSPs. The two HE cells receive their IPSP barrages in antiphase, and this pattern of inhibitory input is responsible for their role in exciting the pair of lateral tubular hearts into rhythmic, antiphasic contractions. We find that similarly positioned cells in the ganglia of Haemopis fire similar impulses and are periodically inhibited by a chemical IPSP (Fig. 12). A large (5-8 pm) unpaired axon is found within the longitudinal connectives of Hirudo and has a cell

from the paired

L cells in Haemopis marmorata. Passage Calibrations: 10 mV. 5 sec.

of current into either cell (underlined) produces a d.c. shift in the other.

Fig. 12. A recording from a heart excitor motor neuron in a Haemopis marmorata ganglion, periodic hyperpolarizations are blocked by Mg’+. can be hyperpolarized beyond their inhibitory librium potential, and are therefore 1.P.S.P.s. Calibrations: 10 mV, 5 sec.

c B.P.(A) 58/3-

F

The equi-

hix~ T. KEYSEK AND CHARLES M. LEVI

294

1 Fig. 13. Simultaneous recordmgs from the S cell body (top) and large axon in the longitudinal connective of Hamopis. The axonal spikes and intracellular impulses have a I: I correspondence both 111 the first, spontaneous burst and in the second burst induced by intracellular current injection. This relationship persists in 20 mM Mg2+ saline and the axon conducts equally well in either direction. Calibrations: 50 mV. 700 /tV, I sec.

body within each segmental ganglion. This cell appears to be an interneuron and is uniquely-identifiable by its large extracellular spike. It is termed the S cell because of this feature (Frank c’t al., 1975). In Huemopis, we find a cell body in the same position (near the RZ) which has an axon in the longitudinal connective which generates an extracellular impulse 5-10 times larger than other units in the connective. Impulses travel equally well in either direction along this axon as they do in Hirudo (Fig. 13). Thus, in these two most distantly-related species, the S cell appears to be homologous by virtue of its cell body and axonal sizes and positions, and impulse properties. The segmental ganglia of the medicinal leech have a small population of neurons which contain biogenic amines (Rude, 1969). These include the serotonergic RZ and a pair of dopamine cells located in the anterior roots. In addition S or 7 small monoamine cells (MA) are found in most segmental ganglia at characteristic sites. These cells are selectively stained by the vital dye. Neutral Red (Stuart et (I/.. 1974). Hamopis has precisely the same number of stainable MA cells of very similar sizes and ganglionic locations (Fig. 14). In both species, these small MA cells have 5-l 5 mV action potentials and are electrotonicallycoupled one to another (Lent & Frarer. unpublished). Thus. the same numbers of monoamine neurons are found in both these species and have very similar anatomical and physiological properties. These neurons are very probably homologous to one another in the two distantly related genera of this study.

DISCUSSlO>

Leech segmental ganglia have 350-400 neurons enveloped by six glial packets, a condition which produces superficially-similar ganglia in all members of the class Hirudinea. To determine whether the neurons of leeches were homologous at the cell level. we have examined several morphological, physiological, and functional properties of about 30 different identifiable neurons in three species of leeches

(Fig. 15). These large leeches are all in the Same family; however, Hirudo mrdicinulis and Mucrohdrllu drcora are in a different sub-family from Haewwpis marmorata. Our findings on the 14 mechanosensory cells, the pair of muco-effector Retzius cells. and the pair of L motor neurons lead to the inescapable conclusion that these neurons are indeed homologous to one another in these three species of leech. These neurons are virtually invariant across the three species (genera) with respect to cell size and location within the glial packets, axonal branching patterns, and fields of contact with the periphery. The sensory and mucoeffector cells have membrane properties which are indistinguishable between the species: resting potential magnitudes. action potential amplitudes and durations, degree of tonic and phasic firing, and relative conduction velocities by peripheral axons. These neurons conserve many of their central connections and functional roles across the species. The RZ arc coupled and control mucus secretion, the T. P and N cells respond to mechanical stimulation. and the electrotonically coupled L cells excite the longitudinal muscles. At every level of organization which we have examined. from membrane to functional anatomical properties, there appears to be no more variabilit) in any of these neurons across species than exists from ganglion to ganglion within any individual of any of the species. Our data on the S interneuron. the HE motor neurons, and the monoamine neurons were derived only from Hurnu~pis and Hirrrdo. These two species have the greatest taxonomic distance between them however, and it is probably valid to conclude that if they have common properties. these would bc present in Mucrohdellu as well. The elcctrophysiological properties and projections of the S cell and its large axon are very similar in both species. The positions and central inputs of large IPSPs to the Heart Excitor motor neurons are indistinguishable between these two leech genera. These two leeches are also conservative in their ganglion distribution of MA cells. The MA cells have identical numbers. sires and locations in both species. Further. their electrophysio-

Fig. 14. A dark-field photomicrograph of the ventral aspects of living Hirudo (A) and Haemopis (B) segmental ganglia after their monoamine (MA) neurons have been stained with Neutral Red (0.01%). The cells which are stained include the RZ, a pair of ventro-lateral cells and an unpaired medial cell. A pair of lateral cells can be seen on the dorsal aspect of the ganglion. The first three segmental ganglia of both species have an extra pair of cells near the RZ. Both species have a soma in each of the anterior roots. The cross species similarities in these chemically similar cells in terms of sizes, positions and numbers is evident. Calibration: 250 pm.

KI NT T. KEYS~R AND CHARLES M.

Anterior

Lrik.1

connective

Fig. 15. A diagram of a leech ganglion showing the homologous neurons which we studied. In the three leeches: Haemopis, Hirudo, and Macrohdelln we believe these cells are homologous: RZ, T. P. N and L. For Haemopis and Hirudo: HE, S and the MA cells (stippled: visible in certain ganglia or on dorsal surface).

logical nections

properties are

and central

electrotonic

intercon-

indistinguishable.

In no instance have we seen a morphological, physiological or functional difference in these identifiable neurons in the CNS of these leech genera. Even though it is impossible to demonstrate a common ancestor within the fossil record, we must conclude that these neurons are homologous in the nervous systems of these leeches. The primary criterion for this conclusion is the extreme stereotypy among these identifiable neurons. The neurons we have examined are involved in some important taxon-wide behaviors: the secretion of mucus. rhythmic heart activity, and shortening of the body wall, a behavior which is utilized both for crawling and for the withdrawal from sources of tactile stimulation. A behavior which is common to many species of leeches is swimming, and the patterned neuronal activity which generates this behavior appears to be very similar in all three of the species of this study (Kristan, personal communication). Thus. leech species have many homologous neurons and are fruitful for comparative neuiophysiological investigations. Leeches are protostomous and have a rigidly-fixed cell lineage during development. Thus, it is possible that each neuron in a leech ganglion might have a homologue in the ganglia of all other leech species.

The interesting question which arises from this study showing such a high degree of neuronal conservatism among leeches is this: At what level of neuronal organization does species-specific behavior emerge? Leeches often have distinctive feeding habits requiring prey recognition, and they are usually specific in their choices of partners for copulation. We assume that such species-specific behaviors are controlled either by connectivity within their brains. by small, yet-unidentified neurons. or by species differences in the connectivity of identifiable neurons. Acknowledgements~We thank Jeffrey Demian and Bryan Frazer for the use of some of their unpublished data and acknowledge the competent assistance of Joyce Roe with some of the figures. This work was begun at the Department of Zoology of the University of Oklahoma and we appreciate its support. Supported in part by N S.F grants GB-39614 and BMS 75-0464 to C.M.L.

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