OF NEUROPHYSIOLOGY Vol. 40, No. 2, March 1977. Printed
JOURNAL
in U.S.A.
Extrasynaptic Receptors on Cel1 Bod .ies of Neurons in Central Nervous System of the Leech PETER B. SARGENT,
KING-WA1
Department of Neurobiology, Department of Neurobiology, Stanford, California 94305
SUMMARY
AND
YAU,
AND
G. NICHOLLS
Harvard Medical School, Boston, Massachusetts Stanford University School of Medicine,
CONCLUSIONS
1. A systematic study has been made of the sensitivity of identified sensory and motoneurons in the leech central nervous system to chemical transmitter substances. 2. The following substances elicited responses from the cell bodies of individual neurons: acetylcholine, 5-hydroxytryptamine, yaminobutyric acid, glutamic acid, glycine, dopamine, and norepinephrine. Since the cell bodies of leech neurons are free of synapses, the receptors that give rise to these responses are extrasynaptic. 3. Sensory and motoneurons of different function had characteristic complements of extrasynaptic receptors. For example, mechanosensory cells responding to light touch, to pressure, and to noxious stimuli could be distinguished by their responses to iontophoretically applied compounds. For one of these modalities (nociceptive), neurons with different receptive fields but otherwise similar properties had markedly distinct extrasynaptic receptors. The possible significance of extrasynaptic receptors is discussed. INTRODUCTION
Recent electrophysiological and anatomical studies in the leech CNS have established many properties of specific sensory and motoneurons and their synaptic connections (15, 17, 19, 20, 24). Relatively little is known, however, about the chemical properties of these cells; in particular, which neurotransmitters they use. A considerable body of knowledge exists only for the Retzius cells, which contain 5hydroxytryptamine (5HT) (23) and which probably secrete 5-HT to activate mucous glands (12). It is clear that the transmitters used at specific synapses need to be identified in order to use the leech CNS as a model for studying problems concerning reflexes (16, 19) and long-term changes in synaptic transmission (8, 14). As a first step in apReceived for pu bkation
JOHN
May 20, 1976.
02115; and
proaching this problem we have examined the sensitivity of functionally identified sensory and motoneuronsto various transmitter substances. The following paper describes experiments designed to determine which transmitters are synthesized by the cell bodies of individual neurons. The sensory cells selectedfor study were the cutaneous mechanosensoryneurons which respond to light touch (T), pressure(P), and noxious stimuli (N cells) (17). These sensory neurons, in addition to making synapsesonto interneurons and motoneurons, receive synaptic input from other cells ( 1, 2, 8, 12). The motoneurons selected were those that raise the animal’s skin into ridges (AE cells) and those that shorten the animal (L cells) (24). A variety of transmitter compounds were iontophoretitally applied to the cell bodiesof these neurons: acetylcholine (ACh), 5-HT, y-aminobutyric acid (GABA), glutamic acid, glycine, dopamine, and norepinephrine. An initial aim was to determine whether the depolarizing effect of transmitter releasedonto AE and L motoneuronsby P or N sensory cells (16, 19) could be mimicked by application of compounds onto the motoneuron cell bodies. Although no synapsesoccur on the cell bodies(5), preliminary resultsindicated that extrasynaptic surfacesare receptive to transmitter substances.The presence of analogousreceptors in other invertebrates is well documented(seeref 7). An analysis of extrasynaptic receptors in leech might offer clues about the nature of subsynapticreceptors, which are inaccessiblein the neuropil. METHODS
Medicinal leeches(Hirudo medicinalis) were used. A general description of the nervous system and the techniquesfor recording intracellularly from leech neuronscan be found elsewhere (16, 17). All experiments were done at 2%23*C. The compositionof leech salinewas(mM) NaCI, 115; KCl, 4; CaCl,, 7.5 (except where noted); Tris-maleate, 10 (pH 7.4); glucose, 10.3. High
446
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EXTRASYNAPTIC
RECEPTORS
calcium concentration was used to improve the stability of recordings. Microelectrodes for recording membrane potential were filled with 4 M potassium acetate and had resistances of 50-100 Ma. Singlebarreled microelectrodes for iontophoresis were filled with 1-3 M solutions of transmitter substances (0.1 M for 5-HT). The filled microelectrodes had resistances of 20-150 Ma. All co.mpounds except glutamate were iontophore tically ejected as cations. Diffusion of compounds from the pipette tip was retarded by applying a bias current of l-10 nA. Figure 1 shows a segmental ganglion and the characteristic positions of the sensory and motoneurons examined (see also ref 17, 24). To apply transmitter substances to the surfaces of individual neuruns it was necessary to expose the nerve cell bodies, which are inside a capsule of connective tissue and surrounded by large “packet” glial cells (5). Small incisions were made through the capsule and into specific glial cells, thereby exposing a few neuronal cell bodies. The electrical properties of the neurons are unchanged by this procedure (11, 18). Identification of specific neurons was made by noting their positions in the ganglion and by recording intracellularly to determine the time course of their action potentials (17). The surfaces of ex-
anterior
connect
-
ives
FIG, 1. Line drawing of the ventral surface of a leech segmental ganglion. Typical positions of touch (T), pressure (P), and nsciceptive (N) mechanosensory neurons, and of annulus erector (AE) and longitudinal (L) excitatory motoneurons are shown. The L cells, indicated with a dotted line, are located on the dorsal surface of the ganglion. The large neurons in the center of the ganglion are the Retzius cells.
ON
LEECH
NEURONS
447
posed cell bodies are partially covered with glial membranes (10, 23). It is apparent that these afford a diffusional barrier to iontophoretically applied substances, for relatively high sensitivity (greater than 100 mV/nC) was often obtained only when the iontophoretic pipette was slightly disturbed mechanically after being placed so as to dimple the cell surface. The effect of drugs on the responses to iontophoretically applied transmitters and on synaptic potentials was tested by bath application. The effect of drugs on the input resistance of the cells was monitored by measuring the voltage deflections produced by constant pulses of hyperpolarizing current using a modified bridge circuit. RESULTS
The cell bodies of leech neurons responded to variety of transmitter substances. From these responses, summarized in Table 1, several conclusions can be drawn. Acetylcholine (ACh) affected each of the neurons examined, producing a depolarization, a hyperpolarization, or a composite, diphasic response. The other substances produced effects on fewer cells. While two cell types responded to ACh exclusively, it was more common to find cells which responded to two or three substances. One of the nociceptive sensory cells was depolarized by four different compounds (Fig. 2). The responses summarized in Table 1 were produced by interaction of the iontophore tically applied transmitters with receptors on the neuronal cell body and not by diffusion to distant in the neuropil. This was demreceptors onstrated by the fact that small movements of the iontophoretic pipette (5-10 pm) from the surface of the cell eliminated the response. Since the cell bodies of leech neurons are free of synapses (5), the receptors that underlie these responses are extrasynaptic. The sensitivity of leech neurons to transmitters was specific for cells of different function (Table 1). For example, the mechanosensory cells responding to light touch, to pressure, and to noxious stimulation showed characteristic responses to transmitter substances. This distinction also extended to the two nociceptive (N) cells (see Fig. 1). The N cell situated laterally in the ganglion was depolarized by ACh, 5-HT, GABA, and glycine (Fig. 2). In contrast, the medially situated N cell was depolarized only by ACh and was hyperpolarized by dopamine and norepinephrine. These two cells have the same sensory modality and make similar synaptic connections onto the AE and L motoneurons (16, 19). They do, however, differ in their receptive fields: the laterally situated cell in the ganglion innervates the ventral half of skin on the ipsilateral side of the segment, and the medially a
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SARGENT,
448 TABLE
1.
YAU,
AND
NICHOLLS
Responses of leech neuronal cell bodies to trunsmitter substances Cell Sensory
-~ T
N
P Medial
Lateral
AE
L
D D
D
D D D
H H
D, H
H
H H
ComDound Ace tylc hoIine 5Hydroxytryptamine y-Aminobutyric acid L-Glutamic acid Glycine Dopamine Noradrenaline
Motor
D, H
D
The abbreviations for the responses are II, depolarization; H, hyperpolarization; D, H, diphasic response consisting of a depolarization followed by a hyperpolarization; -, no apparent response. The abbreviations for the cells are given in the legend to Fig. 1, No differences in response were detected between members of a bilaterally symmetrical pair of cells. The sensitivity to each compound was tested an average of 10 times for each cell. L-Glutamic acid, ineffective when applied to any of these neurons, hyperpolarized a functionally unidentified pair of Iarge cells located immediately anterior to the AE motoneurons (see Fig. 1).
situated cell innervates dorsal skin (17). The three T cells and the two P cells, which also have different receptive fields but otherwise similar properties, could not be distinguished by their complementsof extrasynaptic receptors. Acetylcholine
receptors on L cell body
In order to learn more &out the properties of extrasynaptic receptors, the response of the L
motoneuron to ACh was studied in greater detail. The diphasic responseof the L cell to ACh consistedof a depolarization followed by a hyperpolarization (Fig. 3A). Either phase of this responsecould be obtained in isolation. Thus, repeated application of ACh at short intervals resulted in a selective loss through desensitization of the depolarizing phase (Fig. 3B); conversely, low dosesof ACh elicited a depolariza-
GABA
ACh
CI l
I
glycine
50 nA 1 r
5 mV
J
I
1000
nA
1000
nA
I
5HT
50 msec
I *5
I
500
nA
-pL
I
FIG. 2. Depolarization of the lateral N sensory cell by ACh, GABA, glycine, and 5HT. The lower trace of each of the four records indicates the current applied to the iontophoretic pipette and the upper trace, the membrane potential. The sensitivity of this cell is markedly higher to ACh than to any of the other three compounds. No consistent differences were noted in the time course of the four responses, although a careful investigation was not made.
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EXTRASYNAPTIC
RECEPTORS
ON LEECH
NEURONS
449
the relationship illustrated in Fig. 4A was obtained. This curve characteristically had three regions: 1) a “foot” at low dosesof ACh, 2) an approximately linear region, and 3) a nonlinear region at high doses.The antagonistsusedin this study (tubocurarine, atropine, nicotine, and neostigmine)reversibly reduced the slope of the linear region (e.g., Fig. 4B), By determining the antagonist concentration necessary to reduce this slope by 50%, a quantitative measure of antagonist potency independent of ACh dose B was obtained. The results of these measurements(Table 2) indicate that all four antagonists blocked the depolarizing ACh responseof the L cell. Tubocurarine and nicotine reduced the slope of the dose-responsecurve by half at concentrations on the order of 0.1 PM; neostigmine and atropine were about 10 and IO0 times less effective, respectively. At these concentrations, 5 mV none of these compounds except neostigmine had antagonistic effects on the subsynaptic L 100 nA cell receptors, as measured on stimulation of the lateral N sensory cell. In fact, tubocurarine and nicotine were ineffective in blocking this synaptic potential at concentrations two orders of magnitudeabove the levels required to completely abolish the extrasynaptic response, All the antagonistsexcept neostigminereduced the input resistance of the L cell by up to 50% at concentrations required to reduce to half the FIG. 3. Responses of the L cell body to ACh. In synaptic potential (Table 2). This precluded deeach record the lower trace records the current applied termining the specifrty of action. It is not surpristo the ACh pipetteandthe uppertrace, the membrane ing that these compounds had nonspecific efpotential. In A, a diphasic response is recorded. In B, only the hyperpolarizing phase of the response is re- fects at such high concentrations. That they decreasedinput resistancemay indicate that their corded following desensitization of the depolarizing phase. In C, a pure depolarizing response to low doses effects are not limited to antagonismof synaptic of ACh is recorded. The three traces were obtained in potentials which arisefrom increasesin conducdifferent experiments. tance. The experiments designed to measure antagonist potency on synaptic potentials were tion without the subsequenthyperpolarization done at only one calcium concentration (7.5 (Fig. 3C). These observations suggestthat dif- mM). Under these conditions subsynaptic referent classes of receptors underlie the de- ceptors might be saturated by the natural transpolarizing and hyperpolarizing phasesof the re- mitter, and competitive antagonistsmight appear sponse.No further attempt was made to differ- spuriously ineffective. Experiments were thereentiate between the two receptors, e.g., by fore repeated for tubocurarine at normal (1.8 pharmacological means. mM) calcium concentration. The lateral N cellIt was natural to wonder if the depolarizing to-L cell synaptic potential is 3-5 times smaller receptors on the L cell body resemblethe sub- at 1.8 mM than at 7.5 mM calcium, yet it was synaptic receptors that interact with excitatory neverthelessreduced only slightly by 0.14 mM transmitter liberated by P or N sensory cells tubocurarine. This suggests,at least for tubo(19). To investigate this a quantitative compari- curarine, that receptor saturation cannot exson was made between the pharmacological plain the insensitivity of the N cell to L cell properties of the extrasynaptic and subsynaptic synaptic potential to cholinergic antagonists. receptors as determined by introduction of anThe results summarizedin Table 2 imply that tagonists to the bathing fluid. Ideally, such a ACh can be a candidate for the sensory transcomparison should be made so that antagonist mitter only if synaptic and extrasynaptic receppotency is assayedindependent of agonist con- tors have distinct pharmacological properties. centration. This proved possible,at least for the The samekind of conclusion can be drawn from extrasynaptic receptors. When the ACh re- noting that another motoneuron, the AE cell, sponsesof the L cell were plotted againstdose, although depolarized by sensory transmitter, is
J
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450
SARGENT,
20
YAU,
AND
NICHOLLS
hyperpolarized when ACh is applied to its cell body (Table 1, Fig. 5). Thus, either ACh is not the sensory transmitter, or the complement of receptors on the cell body and in the neuropil are different (or both), This second possibility has received support from recent experiments in collaboration with S. Miyazaki (unpublished results), in which ACh has been found to depolarize the AE cell when applied to its process close to the neuropil.
IS
DISCUSSION
The receptors that underlie the responses produced by iontophoresis onto leech neurons 0 are extrasynaptic. Their functional significance m is at present unknown. They might represent l imperfect localization of receptors to the subsynaptic surfacesin the neuropil; they might be an intermediate stagein transport from their presumed site of synthesis, the cell body, to the 5 subsynaptic membrane; alternatively, they l might serve a function unrelated to synaptic 0 transmission,perhaps as hormone receptors. l In Aplysia clear similarities exist between extrasynaptic and subsynaptic receptors. Blankenship, Wachtel, and Kandel (4) have demonI 1 1 1 0.10 0.15 0.20 0.25 strated an equivalence of ionic mechanisms nC for extra- and subsynaptic ACh receptors in an instance where independent chemical evidence suggeststhat the synapses under study are cholinergic. In other studieson Aplysia, Kehoe (9) has found both ionic and pharmacological similarities between extrasynaptic ACh receptors and subsynaptic receptors that are assumed to be cholinergic by virtue of these similarities. Similar results have been obtained in insectsby Pitman and Kerkut (21). In the leech CNS, however, the relation between extrasynaptic and subsynaptic receptors is less well established. The pharmacological comparisonmadebetween extra- and subsynaptic receptors of the L motoneuron(Table 2) indicates that both receptors are blocked by neostigmine,but that the extrasynaptic receptors are considerably more sensitive to tubocurarine, atropine, and nicotine. The discrepancy in sensitivity is apparently not explained by saturation of subsynapticreceptors by the natural transmitFIG. 4. A: relation between the L cell response and the dose of ACh. The L cell response was mea- ter nor by limited accessof these compoundsto the neuropil since a) ions such as Mg*+ can afsured as peak amplitude (mV), and the dose of ACh was measured as the charge (nC) applied to the ion- fect synaptic transmission within seconds of tophoretic pipette. Stimulus pulses were 4 ms in duratheir addition to the bathing medium, and 6) tion. The slope of the linear portion of this relation is other compounds,such as neostigmine,do have 125 mV/nC. B: effect of atropine on the ACh dose- accessto the neuropil. The magnitudeof these response relation of the L cell. The relation between pharmacological differences and the insensitivpeak voltage response (mV) and ACh dose (nC) was ity of the N cell-to-L cell synaptic potential to obtained first in saline (circles), then in 29 PM atatropine and tubocurarine suggestthat this senropine (squares), and again in saline (triangles): In this experiment atropine reversibly lowered the slope of sorimotor synapseis not cholinergic or, alternatively, that there are profound pharmacological the linear region of the curve from 130 to 55 mV/nC. The shift of the curve to the right followingreturn to differencesbetween extra- and subsynaptic ACh saline was not usually observed. receptors on the L cell. In mammalianskeletal mv IO
l
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EXTRASYNAPTIC
RECEPTORS
ON LEECH
2. Concentrations of compounds prodbcing synaptic potentials of the L cell
TABLE
Compound
NEURONS
a 50% reduction
451
in ACh and
ACh Potential PM
Synaptic Potential PM
Discrepancy
< 0.14 14 0.075 1.6-3.3
1,400” 290* 31* 6.6
> 10,000 20 400 2-4
Tubocurarine Atropine Nicotine Neostigmine
Estimates of these “half-bIock” concentrations were made in 6-17 experiments for each drug. The synaptic potential used was the depolarization produced by stimulation of the lateral N sensory cell. The data in this table indicate that the ACh potential elicited from the cell body is considerably more sensitive to tubocurarine, *Input resistance lowered by 2&W%. atropine, and nicotine than is the N to L cell synaptic potential.
muscle clear but less marked pharmacological differences do exist between junctional receptors and the extrajunctional receptors that appear following denervation (3) Leech neurons of different function can be distinguished by their complements of extrasynaptic receptors (Table 1). Differences were found between excitatory motoneurons that innervate different muscles. Differences were also found among the three kinds of mechanosensoryneurons. Most surprisingwere the differencesbetween the two N sensorycells, which both relay information about noxious stimulation of the skin but which have different receptive fields. The presence of extrasynaptic l
receptors
on primary
sensory
neurons
invites
The significanceof extrasynaptic receptors on leech neuronsremains unclear. If extrasynaptic receptors have correlates in the neuropil, the results of Table I generate specific predictions. For example, one would expect to find inhibitory synaptic input onto the AE motoneuron, but not the L motoneuron, from serotonergic neurons. One might also expect to find serotonergic, excitatory synaptic input onto the lateral, but not the medial, nociceptive sensory neuron. Primary candidatesfor these presynaptic cells are the neuronswhose cell bodies contain 5-HT (6, 13, 22). A test of these predictions may reveal what relationship, if any, exists between extrasynaptic and subsynaptic receptors in the leech.
comment. Touch sensory cells receive both excitatory
and inhibitory
chemical
synaptic
poten-
ACKNOWLEDGMENTS
tials on stimulation of pressuresensorycells, but We thank Zach Hall for advice and encouragement. these pathways are polysynaptic and the This work was supported by Public Health Service neurons directly presynaptic to the touch cells Training Grant No. MH 07084 and Public Health Serhave not been identified (1). Chemically vice Grant NS 11544. mediated synaptic potentials are elicited from touch and other sensory cells on stimulation of Present address of P, B. Sargent: Dept. of Physroots and connectives (2, 14), but again, none of iology, School of Medicine, University of California, the neurons directly presynaptic to the sensory San Francisco, California 94 143. Please address incells has been identified. quiries to him,
II
I
5mV
50 nA
100 msec 5. Hyperpolarization of the AE cell body by ACh. The lower trace records the current appIied to the ACh pipette and the upper trace, the membrane potential FIG.
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452
SARGENT,
YAU,
AND NICHOLLS
REFERENCES 1. BAYLOR, D. A. AND NICHOLLS, J. G. Chemical and electrical synaptic connexions between cutaneous mechanoreceptor neurones in the central nervous system of the leech. J. Physiol., Loadon 203: 591-609, 1969. 2. BAYLOR, D. A. AND NICHOLLS,J.G. Patternsof regeneration between individual nerve cells in the central nervous system of the leech. Nature 232: 268-269, 1971. 3. BE~~NEK, R. AND VYSKO&L, F. The action of tubocurarine and atropine on the normal and denervated rat diaphragm. J. Physiol., London 188: 53-66, 1967. 4. BLANKENSHIP, J. E., WACHTEL, H., AND KANDEL, E. R. Ionic mechanisms of excitatory, inhibitory, and dual synaptic actions mediated by an identified interneuron in abdominal ganglion of Aplysia. J. Neurophysiol. 34: 7692, 1971. 5. COGGESHALL, R. E. AND FAWCETT, D. W. The fine structure of the central nervous system of the leech, Hirudo medicinalis. J. Newophysiul. 27: 229-289, 1964. 6. EHINGER, B., FALCK, B., ANDMYHRBERG, H. E. Biogenic monoamines in Hirudo medicinalis. Histochemie 15: 140-149, 1968. 7. GERSCHENFELD, H. M. Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Physiol. Rev. 53: I-1 19, 1973. 8. JANSEN, J. K. S., MULLER, K. J., AND NICHOLLS, J. G. Persistent modification of synaptic interactions between sensory and motor nerve cells following discrete lesions in the central nervous system of the leech. J. Physiul., London 242: 289-305, 1974. 9. &HOE, J. Three acetylcholine receptors on Aplysia neurones. J. Physiol., London 225: 115-146, 1972. 10. KUFFLER,~. W. ANDNICHOLLS, J.G. Thephysiology of neuroglial cells. Ergeb, Physiol. Biol. Chem. Exptl. Pharmakol. 57: l-90, 1966. 11, KUFFLER,~. W. AND POTTER, D.D.Gliainthe leech central nervous system: physiological properties and neuron-glia relationship. J. Neurophysiol. 27: 290-320, 1964. 12. LENT, C. M. Retzius cells: neuroeffectors controlling mucus release by the leech. Science 179: 693496, 1973. 13. MARSDEN, C. A. AND KERKUT, G. A. Fluorescent microscopy of the 5-HT and catecholaminecontaining cells in the central nervous system of
the leech Hirudo medicinalis. Comp. Biochem. 31: 851-862, 1969. 14. MIYAZAKI, &NICHOLLS, J. G., AND WALLACE, B. G. Modification and regeneration of synaptic connections in cultured leech ganglia. In: The Physiol.
Synapse.
Cold Spring
Harbor
Symp.
Quant.
Biul.
40: 483-493, 1976. 15, MULLER, K. J. AND MCMAHAN, U. J. The shapes of sensory and motor neurones and the distribution of their synapses in ganglia of the leech: a study using intracellular injection of horseradish peroxidase. Proc. Roy. Sot., London, Ser. B 194: 481-499, 1976. properties of synapses between a single sensory neurone and two different motor cells in the leech C.N.S.
J. Physiol.,
London
238: 357-369,
1974.
17. NICHOLLS, J. G. AND BAYLOR, D. A. Specific modalities and receptive fields of sensory neurons in CNS of the leech. J. Neurophysiol. 31: 74& 756, 1968. 18. NICHOLLS, J.G. AND KUFFLER, S. W. Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neurophysiol. 27: 64547 1, 1964. 19. NICHOLLS, J.G. AND PURVES, D. Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech. J. Physiol., London 209: 647667, 1970. 20. ORT,C. A., KRISTAN, W. B., ANDSTENT, G.S. Neuronal control of swimming in the medicinal leech. II. Identification and connections of motor neurons. J. Comp. Physiol. 94: 121-154, 1974. 21. PITMAN, R. M. AND KERKUT, G. A. Comparison of the actions of iontophoretically applied acetylcholine and gamma-aminobutyric acid with the EPSP and IPSP in cockroach central neurons. Comp. Gen. Pharmacol. 1: 221-230, 1970. 22. RUDE, S., Monoamine-containing neurons in the central nervous system and peripheral nerves of the leech, Hirudo medicinalis. J. Comp. Neural. 136: 349-371, 1969. 23. RUDE, S., COGGESHALL, R. E., AND VAN ORDEN, L. S. Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neuron. J. Cell Biol. 41: 832-854, 1969. 24. STUART, A. E. Physiological and morphological properties of motoneurones in the central nervous system of the leech. J. Physiol., London 209: 627-646, 1970.
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JOURNAL
in U.S.A.
Extrasynaptic Receptors on Cel1 Bod .ies of Neurons in Central Nervous System of the Leech PETER B. SARGENT,
KING-WA1
Department of Neurobiology, Department of Neurobiology, Stanford, California 94305
SUMMARY
AND
YAU,
AND
G. NICHOLLS
Harvard Medical School, Boston, Massachusetts Stanford University School of Medicine,
CONCLUSIONS
1. A systematic study has been made of the sensitivity of identified sensory and motoneurons in the leech central nervous system to chemical transmitter substances. 2. The following substances elicited responses from the cell bodies of individual neurons: acetylcholine, 5-hydroxytryptamine, yaminobutyric acid, glutamic acid, glycine, dopamine, and norepinephrine. Since the cell bodies of leech neurons are free of synapses, the receptors that give rise to these responses are extrasynaptic. 3. Sensory and motoneurons of different function had characteristic complements of extrasynaptic receptors. For example, mechanosensory cells responding to light touch, to pressure, and to noxious stimuli could be distinguished by their responses to iontophoretically applied compounds. For one of these modalities (nociceptive), neurons with different receptive fields but otherwise similar properties had markedly distinct extrasynaptic receptors. The possible significance of extrasynaptic receptors is discussed. INTRODUCTION
Recent electrophysiological and anatomical studies in the leech CNS have established many properties of specific sensory and motoneurons and their synaptic connections (15, 17, 19, 20, 24). Relatively little is known, however, about the chemical properties of these cells; in particular, which neurotransmitters they use. A considerable body of knowledge exists only for the Retzius cells, which contain 5hydroxytryptamine (5HT) (23) and which probably secrete 5-HT to activate mucous glands (12). It is clear that the transmitters used at specific synapses need to be identified in order to use the leech CNS as a model for studying problems concerning reflexes (16, 19) and long-term changes in synaptic transmission (8, 14). As a first step in apReceived for pu bkation
JOHN
May 20, 1976.
02115; and
proaching this problem we have examined the sensitivity of functionally identified sensory and motoneuronsto various transmitter substances. The following paper describes experiments designed to determine which transmitters are synthesized by the cell bodies of individual neurons. The sensory cells selectedfor study were the cutaneous mechanosensoryneurons which respond to light touch (T), pressure(P), and noxious stimuli (N cells) (17). These sensory neurons, in addition to making synapsesonto interneurons and motoneurons, receive synaptic input from other cells ( 1, 2, 8, 12). The motoneurons selected were those that raise the animal’s skin into ridges (AE cells) and those that shorten the animal (L cells) (24). A variety of transmitter compounds were iontophoretitally applied to the cell bodiesof these neurons: acetylcholine (ACh), 5-HT, y-aminobutyric acid (GABA), glutamic acid, glycine, dopamine, and norepinephrine. An initial aim was to determine whether the depolarizing effect of transmitter releasedonto AE and L motoneuronsby P or N sensory cells (16, 19) could be mimicked by application of compounds onto the motoneuron cell bodies. Although no synapsesoccur on the cell bodies(5), preliminary resultsindicated that extrasynaptic surfacesare receptive to transmitter substances.The presence of analogousreceptors in other invertebrates is well documented(seeref 7). An analysis of extrasynaptic receptors in leech might offer clues about the nature of subsynapticreceptors, which are inaccessiblein the neuropil. METHODS
Medicinal leeches(Hirudo medicinalis) were used. A general description of the nervous system and the techniquesfor recording intracellularly from leech neuronscan be found elsewhere (16, 17). All experiments were done at 2%23*C. The compositionof leech salinewas(mM) NaCI, 115; KCl, 4; CaCl,, 7.5 (except where noted); Tris-maleate, 10 (pH 7.4); glucose, 10.3. High
446
Downloaded from www.physiology.org/journal/jn by ${individualUser.givenNames} ${individualUser.surname} (131.170.021.110) on January 1, 2019.
EXTRASYNAPTIC
RECEPTORS
calcium concentration was used to improve the stability of recordings. Microelectrodes for recording membrane potential were filled with 4 M potassium acetate and had resistances of 50-100 Ma. Singlebarreled microelectrodes for iontophoresis were filled with 1-3 M solutions of transmitter substances (0.1 M for 5-HT). The filled microelectrodes had resistances of 20-150 Ma. All co.mpounds except glutamate were iontophore tically ejected as cations. Diffusion of compounds from the pipette tip was retarded by applying a bias current of l-10 nA. Figure 1 shows a segmental ganglion and the characteristic positions of the sensory and motoneurons examined (see also ref 17, 24). To apply transmitter substances to the surfaces of individual neuruns it was necessary to expose the nerve cell bodies, which are inside a capsule of connective tissue and surrounded by large “packet” glial cells (5). Small incisions were made through the capsule and into specific glial cells, thereby exposing a few neuronal cell bodies. The electrical properties of the neurons are unchanged by this procedure (11, 18). Identification of specific neurons was made by noting their positions in the ganglion and by recording intracellularly to determine the time course of their action potentials (17). The surfaces of ex-
anterior
connect
-
ives
FIG, 1. Line drawing of the ventral surface of a leech segmental ganglion. Typical positions of touch (T), pressure (P), and nsciceptive (N) mechanosensory neurons, and of annulus erector (AE) and longitudinal (L) excitatory motoneurons are shown. The L cells, indicated with a dotted line, are located on the dorsal surface of the ganglion. The large neurons in the center of the ganglion are the Retzius cells.
ON
LEECH
NEURONS
447
posed cell bodies are partially covered with glial membranes (10, 23). It is apparent that these afford a diffusional barrier to iontophoretically applied substances, for relatively high sensitivity (greater than 100 mV/nC) was often obtained only when the iontophoretic pipette was slightly disturbed mechanically after being placed so as to dimple the cell surface. The effect of drugs on the responses to iontophoretically applied transmitters and on synaptic potentials was tested by bath application. The effect of drugs on the input resistance of the cells was monitored by measuring the voltage deflections produced by constant pulses of hyperpolarizing current using a modified bridge circuit. RESULTS
The cell bodies of leech neurons responded to variety of transmitter substances. From these responses, summarized in Table 1, several conclusions can be drawn. Acetylcholine (ACh) affected each of the neurons examined, producing a depolarization, a hyperpolarization, or a composite, diphasic response. The other substances produced effects on fewer cells. While two cell types responded to ACh exclusively, it was more common to find cells which responded to two or three substances. One of the nociceptive sensory cells was depolarized by four different compounds (Fig. 2). The responses summarized in Table 1 were produced by interaction of the iontophore tically applied transmitters with receptors on the neuronal cell body and not by diffusion to distant in the neuropil. This was demreceptors onstrated by the fact that small movements of the iontophoretic pipette (5-10 pm) from the surface of the cell eliminated the response. Since the cell bodies of leech neurons are free of synapses (5), the receptors that underlie these responses are extrasynaptic. The sensitivity of leech neurons to transmitters was specific for cells of different function (Table 1). For example, the mechanosensory cells responding to light touch, to pressure, and to noxious stimulation showed characteristic responses to transmitter substances. This distinction also extended to the two nociceptive (N) cells (see Fig. 1). The N cell situated laterally in the ganglion was depolarized by ACh, 5-HT, GABA, and glycine (Fig. 2). In contrast, the medially situated N cell was depolarized only by ACh and was hyperpolarized by dopamine and norepinephrine. These two cells have the same sensory modality and make similar synaptic connections onto the AE and L motoneurons (16, 19). They do, however, differ in their receptive fields: the laterally situated cell in the ganglion innervates the ventral half of skin on the ipsilateral side of the segment, and the medially a
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SARGENT,
448 TABLE
1.
YAU,
AND
NICHOLLS
Responses of leech neuronal cell bodies to trunsmitter substances Cell Sensory
-~ T
N
P Medial
Lateral
AE
L
D D
D
D D D
H H
D, H
H
H H
ComDound Ace tylc hoIine 5Hydroxytryptamine y-Aminobutyric acid L-Glutamic acid Glycine Dopamine Noradrenaline
Motor
D, H
D
The abbreviations for the responses are II, depolarization; H, hyperpolarization; D, H, diphasic response consisting of a depolarization followed by a hyperpolarization; -, no apparent response. The abbreviations for the cells are given in the legend to Fig. 1, No differences in response were detected between members of a bilaterally symmetrical pair of cells. The sensitivity to each compound was tested an average of 10 times for each cell. L-Glutamic acid, ineffective when applied to any of these neurons, hyperpolarized a functionally unidentified pair of Iarge cells located immediately anterior to the AE motoneurons (see Fig. 1).
situated cell innervates dorsal skin (17). The three T cells and the two P cells, which also have different receptive fields but otherwise similar properties, could not be distinguished by their complementsof extrasynaptic receptors. Acetylcholine
receptors on L cell body
In order to learn more &out the properties of extrasynaptic receptors, the response of the L
motoneuron to ACh was studied in greater detail. The diphasic responseof the L cell to ACh consistedof a depolarization followed by a hyperpolarization (Fig. 3A). Either phase of this responsecould be obtained in isolation. Thus, repeated application of ACh at short intervals resulted in a selective loss through desensitization of the depolarizing phase (Fig. 3B); conversely, low dosesof ACh elicited a depolariza-
GABA
ACh
CI l
I
glycine
50 nA 1 r
5 mV
J
I
1000
nA
1000
nA
I
5HT
50 msec
I *5
I
500
nA
-pL
I
FIG. 2. Depolarization of the lateral N sensory cell by ACh, GABA, glycine, and 5HT. The lower trace of each of the four records indicates the current applied to the iontophoretic pipette and the upper trace, the membrane potential. The sensitivity of this cell is markedly higher to ACh than to any of the other three compounds. No consistent differences were noted in the time course of the four responses, although a careful investigation was not made.
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EXTRASYNAPTIC
RECEPTORS
ON LEECH
NEURONS
449
the relationship illustrated in Fig. 4A was obtained. This curve characteristically had three regions: 1) a “foot” at low dosesof ACh, 2) an approximately linear region, and 3) a nonlinear region at high doses.The antagonistsusedin this study (tubocurarine, atropine, nicotine, and neostigmine)reversibly reduced the slope of the linear region (e.g., Fig. 4B), By determining the antagonist concentration necessary to reduce this slope by 50%, a quantitative measure of antagonist potency independent of ACh dose B was obtained. The results of these measurements(Table 2) indicate that all four antagonists blocked the depolarizing ACh responseof the L cell. Tubocurarine and nicotine reduced the slope of the dose-responsecurve by half at concentrations on the order of 0.1 PM; neostigmine and atropine were about 10 and IO0 times less effective, respectively. At these concentrations, 5 mV none of these compounds except neostigmine had antagonistic effects on the subsynaptic L 100 nA cell receptors, as measured on stimulation of the lateral N sensory cell. In fact, tubocurarine and nicotine were ineffective in blocking this synaptic potential at concentrations two orders of magnitudeabove the levels required to completely abolish the extrasynaptic response, All the antagonistsexcept neostigminereduced the input resistance of the L cell by up to 50% at concentrations required to reduce to half the FIG. 3. Responses of the L cell body to ACh. In synaptic potential (Table 2). This precluded deeach record the lower trace records the current applied termining the specifrty of action. It is not surpristo the ACh pipetteandthe uppertrace, the membrane ing that these compounds had nonspecific efpotential. In A, a diphasic response is recorded. In B, only the hyperpolarizing phase of the response is re- fects at such high concentrations. That they decreasedinput resistancemay indicate that their corded following desensitization of the depolarizing phase. In C, a pure depolarizing response to low doses effects are not limited to antagonismof synaptic of ACh is recorded. The three traces were obtained in potentials which arisefrom increasesin conducdifferent experiments. tance. The experiments designed to measure antagonist potency on synaptic potentials were tion without the subsequenthyperpolarization done at only one calcium concentration (7.5 (Fig. 3C). These observations suggestthat dif- mM). Under these conditions subsynaptic referent classes of receptors underlie the de- ceptors might be saturated by the natural transpolarizing and hyperpolarizing phasesof the re- mitter, and competitive antagonistsmight appear sponse.No further attempt was made to differ- spuriously ineffective. Experiments were thereentiate between the two receptors, e.g., by fore repeated for tubocurarine at normal (1.8 pharmacological means. mM) calcium concentration. The lateral N cellIt was natural to wonder if the depolarizing to-L cell synaptic potential is 3-5 times smaller receptors on the L cell body resemblethe sub- at 1.8 mM than at 7.5 mM calcium, yet it was synaptic receptors that interact with excitatory neverthelessreduced only slightly by 0.14 mM transmitter liberated by P or N sensory cells tubocurarine. This suggests,at least for tubo(19). To investigate this a quantitative compari- curarine, that receptor saturation cannot exson was made between the pharmacological plain the insensitivity of the N cell to L cell properties of the extrasynaptic and subsynaptic synaptic potential to cholinergic antagonists. receptors as determined by introduction of anThe results summarizedin Table 2 imply that tagonists to the bathing fluid. Ideally, such a ACh can be a candidate for the sensory transcomparison should be made so that antagonist mitter only if synaptic and extrasynaptic receppotency is assayedindependent of agonist con- tors have distinct pharmacological properties. centration. This proved possible,at least for the The samekind of conclusion can be drawn from extrasynaptic receptors. When the ACh re- noting that another motoneuron, the AE cell, sponsesof the L cell were plotted againstdose, although depolarized by sensory transmitter, is
J
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450
SARGENT,
20
YAU,
AND
NICHOLLS
hyperpolarized when ACh is applied to its cell body (Table 1, Fig. 5). Thus, either ACh is not the sensory transmitter, or the complement of receptors on the cell body and in the neuropil are different (or both), This second possibility has received support from recent experiments in collaboration with S. Miyazaki (unpublished results), in which ACh has been found to depolarize the AE cell when applied to its process close to the neuropil.
IS
DISCUSSION
The receptors that underlie the responses produced by iontophoresis onto leech neurons 0 are extrasynaptic. Their functional significance m is at present unknown. They might represent l imperfect localization of receptors to the subsynaptic surfacesin the neuropil; they might be an intermediate stagein transport from their presumed site of synthesis, the cell body, to the 5 subsynaptic membrane; alternatively, they l might serve a function unrelated to synaptic 0 transmission,perhaps as hormone receptors. l In Aplysia clear similarities exist between extrasynaptic and subsynaptic receptors. Blankenship, Wachtel, and Kandel (4) have demonI 1 1 1 0.10 0.15 0.20 0.25 strated an equivalence of ionic mechanisms nC for extra- and subsynaptic ACh receptors in an instance where independent chemical evidence suggeststhat the synapses under study are cholinergic. In other studieson Aplysia, Kehoe (9) has found both ionic and pharmacological similarities between extrasynaptic ACh receptors and subsynaptic receptors that are assumed to be cholinergic by virtue of these similarities. Similar results have been obtained in insectsby Pitman and Kerkut (21). In the leech CNS, however, the relation between extrasynaptic and subsynaptic receptors is less well established. The pharmacological comparisonmadebetween extra- and subsynaptic receptors of the L motoneuron(Table 2) indicates that both receptors are blocked by neostigmine,but that the extrasynaptic receptors are considerably more sensitive to tubocurarine, atropine, and nicotine. The discrepancy in sensitivity is apparently not explained by saturation of subsynapticreceptors by the natural transmitFIG. 4. A: relation between the L cell response and the dose of ACh. The L cell response was mea- ter nor by limited accessof these compoundsto the neuropil since a) ions such as Mg*+ can afsured as peak amplitude (mV), and the dose of ACh was measured as the charge (nC) applied to the ion- fect synaptic transmission within seconds of tophoretic pipette. Stimulus pulses were 4 ms in duratheir addition to the bathing medium, and 6) tion. The slope of the linear portion of this relation is other compounds,such as neostigmine,do have 125 mV/nC. B: effect of atropine on the ACh dose- accessto the neuropil. The magnitudeof these response relation of the L cell. The relation between pharmacological differences and the insensitivpeak voltage response (mV) and ACh dose (nC) was ity of the N cell-to-L cell synaptic potential to obtained first in saline (circles), then in 29 PM atatropine and tubocurarine suggestthat this senropine (squares), and again in saline (triangles): In this experiment atropine reversibly lowered the slope of sorimotor synapseis not cholinergic or, alternatively, that there are profound pharmacological the linear region of the curve from 130 to 55 mV/nC. The shift of the curve to the right followingreturn to differencesbetween extra- and subsynaptic ACh saline was not usually observed. receptors on the L cell. In mammalianskeletal mv IO
l
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EXTRASYNAPTIC
RECEPTORS
ON LEECH
2. Concentrations of compounds prodbcing synaptic potentials of the L cell
TABLE
Compound
NEURONS
a 50% reduction
451
in ACh and
ACh Potential PM
Synaptic Potential PM
Discrepancy
< 0.14 14 0.075 1.6-3.3
1,400” 290* 31* 6.6
> 10,000 20 400 2-4
Tubocurarine Atropine Nicotine Neostigmine
Estimates of these “half-bIock” concentrations were made in 6-17 experiments for each drug. The synaptic potential used was the depolarization produced by stimulation of the lateral N sensory cell. The data in this table indicate that the ACh potential elicited from the cell body is considerably more sensitive to tubocurarine, *Input resistance lowered by 2&W%. atropine, and nicotine than is the N to L cell synaptic potential.
muscle clear but less marked pharmacological differences do exist between junctional receptors and the extrajunctional receptors that appear following denervation (3) Leech neurons of different function can be distinguished by their complements of extrasynaptic receptors (Table 1). Differences were found between excitatory motoneurons that innervate different muscles. Differences were also found among the three kinds of mechanosensoryneurons. Most surprisingwere the differencesbetween the two N sensorycells, which both relay information about noxious stimulation of the skin but which have different receptive fields. The presence of extrasynaptic l
receptors
on primary
sensory
neurons
invites
The significanceof extrasynaptic receptors on leech neuronsremains unclear. If extrasynaptic receptors have correlates in the neuropil, the results of Table I generate specific predictions. For example, one would expect to find inhibitory synaptic input onto the AE motoneuron, but not the L motoneuron, from serotonergic neurons. One might also expect to find serotonergic, excitatory synaptic input onto the lateral, but not the medial, nociceptive sensory neuron. Primary candidatesfor these presynaptic cells are the neuronswhose cell bodies contain 5-HT (6, 13, 22). A test of these predictions may reveal what relationship, if any, exists between extrasynaptic and subsynaptic receptors in the leech.
comment. Touch sensory cells receive both excitatory
and inhibitory
chemical
synaptic
poten-
ACKNOWLEDGMENTS
tials on stimulation of pressuresensorycells, but We thank Zach Hall for advice and encouragement. these pathways are polysynaptic and the This work was supported by Public Health Service neurons directly presynaptic to the touch cells Training Grant No. MH 07084 and Public Health Serhave not been identified (1). Chemically vice Grant NS 11544. mediated synaptic potentials are elicited from touch and other sensory cells on stimulation of Present address of P, B. Sargent: Dept. of Physroots and connectives (2, 14), but again, none of iology, School of Medicine, University of California, the neurons directly presynaptic to the sensory San Francisco, California 94 143. Please address incells has been identified. quiries to him,
II
I
5mV
50 nA
100 msec 5. Hyperpolarization of the AE cell body by ACh. The lower trace records the current appIied to the ACh pipette and the upper trace, the membrane potential FIG.
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452
SARGENT,
YAU,
AND NICHOLLS
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17. NICHOLLS, J. G. AND BAYLOR, D. A. Specific modalities and receptive fields of sensory neurons in CNS of the leech. J. Neurophysiol. 31: 74& 756, 1968. 18. NICHOLLS, J.G. AND KUFFLER, S. W. Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. J. Neurophysiol. 27: 64547 1, 1964. 19. NICHOLLS, J.G. AND PURVES, D. Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech. J. Physiol., London 209: 647667, 1970. 20. ORT,C. A., KRISTAN, W. B., ANDSTENT, G.S. Neuronal control of swimming in the medicinal leech. II. Identification and connections of motor neurons. J. Comp. Physiol. 94: 121-154, 1974. 21. PITMAN, R. M. AND KERKUT, G. A. Comparison of the actions of iontophoretically applied acetylcholine and gamma-aminobutyric acid with the EPSP and IPSP in cockroach central neurons. Comp. Gen. Pharmacol. 1: 221-230, 1970. 22. RUDE, S., Monoamine-containing neurons in the central nervous system and peripheral nerves of the leech, Hirudo medicinalis. J. Comp. Neural. 136: 349-371, 1969. 23. RUDE, S., COGGESHALL, R. E., AND VAN ORDEN, L. S. Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neuron. J. Cell Biol. 41: 832-854, 1969. 24. STUART, A. E. Physiological and morphological properties of motoneurones in the central nervous system of the leech. J. Physiol., London 209: 627-646, 1970.
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