- Email: [email protected]
The synaptic basis of the monosynaptic stretch reflex
248 mal claw types suggests that the motor programs for their execution are always available to either claw, even though they are usually expressed asymmetrically. This conclusion necessarily restricts the sorts of neuronal rearrangements which may be used to bring about behavioral transformation as claw type changes. Furthermore, it suggests that the dramatic, rapid modifications in motor neuron cell body size are associated with vegetative changes in the target rather than with changes in the orchestration of behavior. The time course of modifications in cell size implies that motor neuron trophic activity is triggered rapidly after snapper nerve damage, in plenty of time to be responsible for subsequent growth and structural changes that eventuate in the peripheral targets.
Conclusion In alpheid shrimp and other crustaceans, a body of experimental data leads to the conclusion that claw transformation is triggered and controlled by elements within the limb nerves. Pincer transformation can be initiated simply by transecting both snapper limb nerves, while severing the corresponding pincer nerves after snapper removal suppresses transformation. Claw closer motor neurons are asymmetrical in anatomical detail, a feature which undergoes complete reversal early in the transformation process. This change is followed later by more gradual modification in the properties of transmitter release at the motor neuron terminals on the muscle. We have not yet identified the neural agents responsible for triggering and expressing transformation. But even if they were known, a variety of questions would remain unanswered. Compared to the asymmetry of lobster claws, that found in Alpheus is even more striking. The external morphology of the two claws is strikingly different and claw size differentials of 10× are not uncommon. Such disparate growth and form on the two sides raises important questions about the control of genetic expression. Arc different genes inw~lved, or is the larger claw simply the normal consequence of a more prolonged activation of certain parts of the genome? Other questions are suggested by the invariant association in transforming claws of changing external form, modification in muscle size and structure, and neuromuscular function. How are these separate events coordinated? It would be a curious twist if answers to these universally important but essentially developmental questions were arrived at from studies of an adult crustacean.
TINS - October 1981 Reading list 1 Bulter, A. J., Ecctes, J . C . and Eccles, R . M . (1960) J. Physiol. (London) 150, 417.-439 2 Govind, C, K. and Lang, F. ((198(I) Neurosei. Abst. 6, 370 3 Hazlen. B. A. and Winn, H . E . (1962) Crustaceana 4, 25-38 4 Herrick, F. H. (1911)Bull. Bur. Fish. Wash. 29. 149~-08 5 Lang, F,, Costello. W.J. and Govind, C.K. ( I ~1771 Biol. Bull. Wood~ Hole, Mass. 152, 75~q3 6 Lang, F.. Govind, C.K. and Costello, W.J. (1978)Scienee 201, 1037-1/t39 7 Lang, F., Govind, C. K. and She, J. (19771 Biol, Bull. Woods Hole, Mass. 152. 382-391 8 Lomo, T., Westgaard, R . H . and Dahl, H, A. (19741Proc. R. Soc. London. Ser. B. 187,99-103
c~ Mellon, DeF. and Stephens, P. J. (1~78)Nature (London) 2 7 2 , 2 4 6 - 2 4 8 10 Przibram, H. (1911!) Arch. Entwickh~ng~mech. Org. 11,321-345 11 Ritzmann. R. [ , (1t,~74),I, Comp. Physiol. t)5, 217-236 12 Sreler, F. A., Gcrgelcy, J., Salmons. S. and Romanul, F. ( 19731 Nature (London) New Biol. 24t, 17-1~,' 13 Srctcr. F. A., Luft. A. R, S. and (Jcrgclcy. ,I. ( 197513. Gen. Physiol. 66, 811 821 14 Stephen,, P. J. and Mellon, DeF. ( 197 t))]. Comp. Physiol. 132.97-1118 15 Wils¢m. F. B. (19113) Biol. Bull. Wood,$ tlole, Mav,s. 4, It)7-210
DeForest Melhm. Jr. is at the Department of Biology. University of Virginia, Charlottesville 22903, U.S.A.
The synapt.ic basis of monosynaptm stretch reflex Stephen Redmanand BruceWaimsley The synapses formed on motoneurones by sensory nerves from primary stretch receptors in muscle have been the model for studies on the mechanisms o f excitatory synaptic transmission in the CNS. Recent refinements in electrophysiological and anatomical techniques, and the application o f cable theory to motoneurone dendrites, have produced new results. These suggest that although transmission at these synapses involves the release o f a neurotransmitter, the mechanisms by which transmission is achieved at these synapses, and perhaps at CNS synapses in general, may be quite different from those which operate at the neuromuscular junction. The monosynaptic stretch reflex occurs when excitation of primary endings of muscle spindle stretch receptors in a muscle causes a reflex dischage, via a monosynaptic pathway, of motoneurones connected to that muscle and to other muscles which are functionally synergistic. This classical description of the monosynaptic stretch reflex must now be widened to include the effects of afferents from secondary endings of muscle spindle stretch receptors, which have been shown to make weak, but nonethe-less monosynaptic connection with motoneurones 11. The synapse between axons from primary endings and motoneurones has been the model for studies on excitatory synaptic transmission in the CNS, largely because of the relative ease of recording from spinal motoneurones, and also because of the ease with which the afferents from primary endings may be selectively excited. Most textbook accounts of transmission at this synapse describe a conventional chemical synapse where the synaptic current has a reversal potential of about 0 mV, quantal release of transmitter occurs with an average quantal content of 1-2. the synaptic delay is 200-300 p.s, and facilitated transmission is caused when an earlier condi-
tioning stimulus precedes the test stimulus by a short interval (2-10 ms). This account was developed from the pioneering efforts of Eccles e and his collaborators, together with Kuno's contributions TM on quantal analysis. There remained a number of gaps in the story. It had not been possible to show that the current causing the excitatory postsynaptic potential (e.p.s.p.) was ion specific, although the reversal potential suggested that Na +, K + and perhaps Ca 2+ were involved. The neurotransmitter was unidentified, although glutamate was the prime candidate. A subsequent phase of research on this synapse, which began about 15 years ago, took into account such factors as the location and pattern of termination of group la axons (from the primary endings) on the motoneurone surface. This introduced considerably more precision into the analysis of the e.p.s.p. For a period the chemical nature of transmission was challenged, and alternatives to quantal release mechanisms have been proposed. Recent reviews 2,19 provide a full description of this research.
Is it a chemical synapse? This question was asked in a reappraisal t [ I~cviCl/Norlh-llollandBiomedicalprc,s It~8I c>37~ ~4[ 2/81/OIH)I) (10110/$(}2~l)
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of junctional mechanisms at this synapse by Rail, et al. 1~. They found that the available evidence did not allow them to distinguish between chemical transmission and low resistance electrical coupling. This reexamination resulted from the difficulties they experienced in demonstrating a reversal potential for the e.p.s.p. Subsequently, other investigators had similar experiences, and electrical coupling, either alone, or jointly with chemical transmission, received more support. Even though the ultrastructure suggested a chemical synapse 5, the large area of apposition of the synaptic boutons with the motoneurone membrane led to the idea that some degree of electrical coupling could occur through the synaptic cleft. When this proposal was analysedl,.a, it was found that an unrealistically low value of resistivity of the synaptic cleft and the sub-synaptic membrane was required for significant electrical coupling to occur. The heat was taken out of this debate by a clear demonstration of reversed potentials by Engberg and Marshall*. Such a demonstration does not deny the presence of some degree of electrical coupling, but the accumulated evidence from other experimental approaches suggests that it does not exist. This evidence comes from measurements of synaptic delay, and from alterations in the extracellular concentration of divalent cations which normally suppress transmitter release. When Mn ~÷ and Co ~+ were electrophoresed outside motoneurones in which the Fig. la e.p.s.p, was being recorded, the peak of the e.p.s.p, was depressed and its rate of rise was decreased TM. An electrical component would normally contribute to the rising phase of the e.p.s.p., and it should not be affected by these procedures. Removal of Ca 2+ and the presence of Mn z÷ reversibly abolished the e.p.s.p, in isolated immature cat spinal cord 2~. Any electrical coupling would not be affected by these procedures. Together, these results suggest that electrical coupling does not contribute to the generation of the e.p.s.p. In contrast, both chemical and electrical modes of transmission occur at the synapses between primary afferents and motoneurones in the amphibian spinal cord '°. The synaptic delay which occurs in the monosynaptic reflex has been the subject of many disagreements. These date back to the early days of microelectrode recording, when Lloyd argued against Eccles for the existence of a monosynaptic inhibitory connection between muscle afferents and motoneurones. The main problem in determining synaptic delay at this synapse,
or anywhere in the CNS, is that it is difficult to determine the precise arrival time of the impulse in the presynaptic terminals. When the afferent input is restricted to impulses in a single fibre, averaging the resultant e.p.s.p, reveals a small (positive, negative) diphasic field potential prior to the e.p.s.p. The positive peak of this diphasic potential has been interpreted as indicating the arrival time of the impulse in the presynaptic terminals of the branch of the afferent fibre synapsing on that motoneurone TM. Accordingly, the positive potential has been the reference time for measurement of synaptic delay. The values obtained, after compensation for electrotonic delay, ranged from 170 to 440/,ts. The existence of such delays requires a chemical synapse. It remains to be established how reliably this field potential is derived solely from the action potential in the termination of the Ia axon on the penetrated motoneurone. The field potential is a temporal average resulting from a moving dipole in non-terminating and terminated axons, with different spatial weightings depending upon the size of the contributing axons and boutons, and their distance from the recording electrode. Major contributions to this potential could arise from large collaterals of the Ia axon in the vicinity of the penetrated motoneurone, and from nearby terminals other than those on the penetrated motoneurone. H R P studies 1.s have demonstrated that the branching pattern of Ia axons in the motoneurone pool is very extensive. Transmitter release
The manner in which transmitter is released from an assembly of boutons connecting an afferent fibre with a motoneurone is a matter of some debate. Fluctuations in the amount of transmitter released are normally reflected by fluctuations in the amplitude of the e.p.s.p. Kuno's investigationss of these fluctuations, using a classical quantal analysis approach, indicated that an average of one or two quanta are released per impulse from the collection of boutons involved. The unit synaptic potential was 100-200/zV in peak amplitude. However, neither Poisson nor binomial distributions could be used convincingly to describe the fluctuation pattern. An analysis of this type in a central neurone is at a disadvantage compared with quantal analysis of fluctuations at the neuromuscular junction. In neurones, spontaneous miniature potentials originating from the synapses under study cannot be distinguished from the entire population of spontaneous potentials, and so the unit
synaptic potential cannot be identified. Also, very poor signal to noise conditions often prevail. Because of these difficulties, Edwards et al. tried another approach 7, and these techniques have subsequently been refined in a recent series of experiments with Jack, Hirst and Wong (unpublished). The recorded noise (both neuronal and instrumentational) can be measured and described statistically. This noise adds to the actual fluctuations in the evoked e.p.s.p., and the recorded signal is the sum of two random variables. Provided the noise processes are statistically independent of the processes generating the fluctuations in the e.p.s.p., the fluctuations can be separated from the sum of these two random variables if the noise is adequately described. A computational procedure separates the histogram of the peak amplitude of the recorded e.p.s.p, from the distribution which represents the recording noise. The results of this procedure indicated that about 30% of single fibre potentials did not fluctuate in amplitude. For those potentials which did fluctuate, they did so between a number of discrete amplitudes. (An example is illustrated in Fig. 1C.) These discrete amplitudes were separated by a constant increment, or by multiples of this increment. The incremental voltage was about 100 p,V, which is similar to the value obtained by Kuno for the unit e.p.s.p. Occasionally, it could be shown that the time course of the e.p.s.p, was different when certain discrete amplitudes occurred. The discrete amplitudes had no detectable variability. The average amplitude of an e.p.s.p, could be increased by a prior tetanus, or by the administration of 4-aminopyridine. This increase occurred by a reduction in the probability of occurrence of smaller discrete amplitudes, and by an increase in the probability of occurrence of larger discrete amplitudes. We have not interpreted these fluctua-, tions as being a direct reflection of the release of a variable number of quanta at the collection of boutons involved. The constancy of the discrete amplit,:des, and the differences in time course for particular discrete amplitudes argue against this idea. Rather, we have suggested that at each bouton, impulse invasion results in either a failure to release transmitter, or the release of sufficient transmitter to saturate all available receptors. The various boutons in the termination on a single motoneurone can have different probabilities of failure. This hypothesis implies that each single fibre e.p.s.p, consists of a number of subunits, where each sub-unit results from
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PEAK ,,,,MP~.,TUOE I,V) Fig. 1. (A) This illustrates the connection formed by a single afferent fibre (solid line) and the dendrites of a motoneurone (dashed lines). This diagram was reconstructed from serial sections, and only that region o f the dendritic tree where synapses were found has been drawn in detail. The synaptic boutons are indicated by arrows. (B) The averaged e.p.s.p, recorded in the motoneurone shown in A, and evoked by impulses in the single afferent shown in A. (C) The fluctuation pattern o f the peak amplitudes o f the evoked e.p.s.p. This pattern was calculated from the histogram of recorded peak amplitudes o f the evoked e.p.s.p., and the probability distribution of the recording noise (Walmsleyand Redman, unpublished).
all-or-none transmission at a single bouton. Accordingly, the number of discrete amplitudes (excluding failures) that a single fibre e.p.s.p, can have should never exceed the number of boutons in the termination with the motoneurone in which the e.p.s.p. is recorded. We have examined this constraint in experiments in which a single fibre e.p.s.p, was recorded and subsequently both the afferent fibre and the motoneurone involved were identified using intraceUular injections of HRP. The results of one such experiment are shown in Fig. 1. The afferent fibre made four contacts with the motoneurone. The average e.p.s.p, evoked in that motoneurone by impulses in the afferent nerve is shown in Fig. lB. The fluctuation pattern of the e.p.s.p, is indicated in Fig. 1C. The peak amplitude of the e.p.s.p, was either 320 /xV, with a probability of 0.65, or 440/xV, with probability of 0.35. According to our hypothesis, transmission would always occur at the same three boutons, generating a 320/zV synaptic potential. At a fourth bouton, transmission occurs with a probability of 0.35, adding a further 120 p,V to the peak of the e.p.s.p. This result, and others in which various numbers of boutons were found, supports our suggestion that a single bouton either fails to
release transmitter, or releases sufficient to saturate the post-synaptic receptors. This result is also a clear demonstration that not all synapses in the one termination are equally effective in synaptic transmission, at least at the low stimulus rates employed in these experiments (3 Hz). We have proposed that failure to release transmitter follows adequate terminal depolarization by the afferent impulse. This hypothesis has been adopted rather than one which requires a failure of the impulse to invade the bouton, because we were unable to detect any significant latency variations in the onset time of the e.p.s.p. Such latency variations would be expected if the impulse spread through branch points and into terminal varicosities with marginal safety factors. However, latency variations in the onset of these potentials have been reported 4, and the hypothesis that the action potential does not always invade all boutons has been developed by Ltischer, Ruenzel and Henn e m a n a4 to account for their observations on post-tetanic potentiation.
The contribution of dendritic synapses to somatk membrane potential Group la axons terminate on both the soma and the dendrites of motoneurones.
The dendrites act as passive cables h~r tile spread of synaptic current towards the soma from the dendritic synapses. The potentials of dendritic origin become attenuated, and their time course prolonged, as they spread electrotonicatly towards the soma. As a result of theoretical analysis of current spread in dendrites it is possible to use the cable properties of the motoneurone, and the time course of the e.p.s.p, recorded at the soma, to calculate a site of origin for the e.p.s.p, on the dendrites 1°'1s, This distance is calculated in terms of an electrical space constant (X). These calculations have indicated that the most common location for synaptic termination is within 0.5;k from the soma. corresponding to a physical distance of about 250-400 p,m. But some synapses may occur at locations greater than one space constant from the soma. The H R P experiments described in connection with Fig. 1 have confirmed that the synaptic location calculated from the e.p.s.p, time course agrees with the actual synaptic location determined morphologically. The synaptie location calculated from the e.p.s.p, time course shown in Fig. 1B was 0.6,~. When the dendrites on which synapses were found were reconstructed into an equivalent dendritic cable, the synapses were at 0,62k and 0.65X from tile soma. It has often been suggested that synapses on distal dendrites are spatially disadvantaged in their ability to influence the membrane potential at the soma. This is not so for la synapses on motoneurones. The peak voltage of potentials at the soma shows no dependence on the distance to the site of generation of the e.p.s.p. 9 Also the average synaptie current which spreads to the soma (or alternatively the total synaptic charge reaching the soma) actually increases as .the site of origin of the e.p.s.p, becomes more distal. These are surprising results, especially when it is recognized that passive electrotonic spread towards the soma from the synaptic site must involve some loss of charge across the dendritic membrane, with consequent attentuation of peak voltage. In some way the 'gain' or 'sensitivity' of dendritic synapses compensates for their more distal location. Such compensation must occur through a greater synaptic current at distal synapses. This could result from a larger number of boutons/afferent fibre, or from a similar number of boutons covering a greater synaptie area, or from a more prolonged duration of synaptic current at distal synapses. Histology indicates that the synapses have similar coverage, and cable
251
TINS - October 1981 calculations indicate that the synaptic current is not prolonged. The first possibility will eventually be answered by HRP studies. However, our studies of fluctuations of dendritic potentials showed that the contribution to the e.p.s.p, from a single bouton did not alter as its location on the dendrites altered. For this reason we h a v e speculated that the answer lies in alterations of receptor density with synaptic location. As a final comment it should be pointed out that while we have confined this article to a discussion of results obtained in vivo, important contributions to our understanding of junctional mechanisms at synapses between dorsal root afferents and spinal neurons have come from studies using cultures of spinal neurones and dorsal root ganglion calls 18, and also from experiments using spinal cord slices. Some of the uncertainties alluded to in this article may be resolvable using these alternative preparations.
Reading 1 Brown, A. G. and Fyffe, R. E. W. (1978) J. Physiol. (London) 274, 111-127 2 Burke, R. E. and Rudomin, P. (1977) in The
Handbook of Physiology, Section 1: The Nervous System (Kandel, E. R., ed.), Vol. 1, Part 2, pp. 877-944, American Physiological Society, Bethesda 3 Burke, R. E., Walmsley.B. and Hodgson,J. A. (1979) in Integration in the Nervous System (Asanuma,H. and Wilson, V. J., eds), pp. 27-45. Igaku-Shoin,New York 4 CoUatos,T. C., Niechaj, A. J., Nelson, S. G. and Mendell, L. M. (1979) Brain Res. 160, 514-518 5 Conradi, S. (1969) Acta Physiol. Scand. Suppl. 332, 85-115 6 Eccles, J. C. (1961) Ergeb. Physiol. Biol. Chem. Exp. Pharmakol. 51,299-430 7 Edwards,F. R., Redman, S. J. and Walmsley,B. (1976) J. Physiol. (London) 259, 665-688 8 Engberg,I. and Marshall, K. C. (1979) Neuroscience 4, 1583-1591 9 Iansek, R. and Redman, S. J. (1973)J. Physiol. (London) 234,665-688 10 Jack, J. J. B., Miller, S., Porter, R. and Redman, S. J. (1971)J. Physiol. (London) 215. 353-380 l 1 Kirkwood,P. A. and Sears, T. A. (1974)Nature (London) 252,242-244 12 Krnjevic, K., Lamour, Y., Macdonald, J. F. and
Cell lineage in the development of the leech nervous system Gunther S. Stent and David A. Weisblat The developmental lines o f descent o f leeches neurons have been ascertained by injection o f tracers into identified cells o f early embryos. One o f these tracers is a fluorescent molecule consisting o f rhodamine coupled to a synthetic dodecapeptide and the other is horseradish peroxidase. These studies have shown that the neurons on either side o f a segmental ganglion fall into four distinct classes, representing the descendants from each o f the four ipsilateral ectodermal teloblasts N, O. P and Q. However, the embryological origin o f the cells o f the supraesophageal ganglion is entirely different from that o f the cells o f the segmental ganglia. One key aspect of neuronal development is cell lineage, i.e. the embryonic lines of descent of various types of neurons. The importance of cell lineage for understanding developmental processes was first realized in the 1880s by C. O. Whitman ~'14. From his studies on leech development, he proposed that each cell of the early embryo, and the clone of its descendant cells, plays a specific role in later development. We have continued and extended Whitman's studies placing particular emphasis on the developmental origins of the leech nervous system. For this purpose we have used the embryos of two leech species belonging to the family of Glossiphonidae: Helobdella triserialis and Haementeria ghilianii 3.
Leech nervous system The nervous system of the leech consists of a chain of 32 interconnected segmental ganglia. Each ganglion contains about 200 bilaterally symmetrical neuron pairs, as well as a few unpaired neurons. The foremost rostral segmental ganglia are fused to form the subesophageal ganglion, which, at its anterior edge, is connected to the dorsal supraesophageal ganglion. Studies carried out since the mid-1960s have shown that the anatomy of the segmental ganglia is sufficiently stereotyped from segment to segment, and sufficiently invariant from specimen to specimen, to enable a large fraction of their neurons to be reliably identified s. Moreover, it has been possible to establish the connection pattern of
Nistri, A. (1979) Neuroscience4, 1331-i 339 13 Kuno,M. (1974)J. Physiol. (London) 175,81-99 14 Luscher, H.-R., Ruenzel, P. and Henneman, E. (1979) Nature(London). 282,859-861 15 Munson,J. B. and Sypert,G. W. (1979)J. Physiol. (London) 296, 329-342 16 Rail, W. (1977) in The Handbook of Physiology. Section 1; The Nervous System (Kendell, E, R., ed.). Vol. l, Part 1, pp. 39-97, American Physiological Society, Bethesda 17 Rail,W., Burke, R. E., Smith,T. G., Nelson,P. G. and Frank. K. (1967) J. Neurophysiol. 30, 1167-1193 18 Ransom.B. R., Christian, C. N.. Bullock, P. N. and Nelson, P. G. (1977) J. Neurophysiol. 40. 1151-1162 19 Redman,S. J. (1979)Prog. Neurobiol. 12, 33-83 20 Shapovalov, A. 1. and Shiriaev. B. I. (1980) J. Physiol. (London) 306, 1-15 21 Shapovalov,A. I., Shiriaev, B. I. and Tamarova. Z. A. (1979) Brain Res. 160, 524-528
Stephen Redman is a Reader in the Departments of Physiology and ElectricalEngineeringat Monash University, Wellington Road, Clayton Victoria 3168. Australia and Bruce Walmsleyis a ResearchFellowin the Experimental Neurology Unit at the John Curtin School of Medical Research,A ustralian National University.A.C.T., Australia.
sensory-, motor- and inter-neurons and thus account not only for some simple acts of reflex behavior4, such as the shortening of the leech body in response to tactile stimulation, but also for some moderately complex integrated movements, such as the heartbeaP. The most complex behavioral routine of the leech described so far in terms of its neural substrates, is swimmingL Consequently, the leech nervous system provides a model system for examining neurodevelopmental processes. Leech development
At the outset of its development the leech egg cleaves into two cells, AB and CD (Fig. 1). The second cleavage gives rise to four cells, A, B. C and D. Cell D then cleaves to yield two cells, one designated DNOPQ, which lies more dorsally, and another designated DM, which lies more ventrally. At this stage, separation of the embryo into the three germinal tissue layers has been accomplished: the progeny cells of A, B and C will give rise to endoderm, the progeny of D N O P Q to ectoderm, and the progeny of DM to mesoderm. The next two cleavages establish the bilateral symmetry of the embryo: DM divides to yield a pair.of right and left M cells, and D N O P Q divides to yield a pair of NOPQ cells, lying on either side of the future midline. Three further cleavages of the N O P Q cell pair produce four bilateral cell pairs designated as N, O, P and Q, which, together with the M cells are referred to as teloblasts. As soon as each teloElsevier/North-HollandBiomedicalPressIt)81 I)378
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Conclusion In alpheid shrimp and other crustaceans, a body of experimental data leads to the conclusion that claw transformation is triggered and controlled by elements within the limb nerves. Pincer transformation can be initiated simply by transecting both snapper limb nerves, while severing the corresponding pincer nerves after snapper removal suppresses transformation. Claw closer motor neurons are asymmetrical in anatomical detail, a feature which undergoes complete reversal early in the transformation process. This change is followed later by more gradual modification in the properties of transmitter release at the motor neuron terminals on the muscle. We have not yet identified the neural agents responsible for triggering and expressing transformation. But even if they were known, a variety of questions would remain unanswered. Compared to the asymmetry of lobster claws, that found in Alpheus is even more striking. The external morphology of the two claws is strikingly different and claw size differentials of 10× are not uncommon. Such disparate growth and form on the two sides raises important questions about the control of genetic expression. Arc different genes inw~lved, or is the larger claw simply the normal consequence of a more prolonged activation of certain parts of the genome? Other questions are suggested by the invariant association in transforming claws of changing external form, modification in muscle size and structure, and neuromuscular function. How are these separate events coordinated? It would be a curious twist if answers to these universally important but essentially developmental questions were arrived at from studies of an adult crustacean.
TINS - October 1981 Reading list 1 Bulter, A. J., Ecctes, J . C . and Eccles, R . M . (1960) J. Physiol. (London) 150, 417.-439 2 Govind, C, K. and Lang, F. ((198(I) Neurosei. Abst. 6, 370 3 Hazlen. B. A. and Winn, H . E . (1962) Crustaceana 4, 25-38 4 Herrick, F. H. (1911)Bull. Bur. Fish. Wash. 29. 149~-08 5 Lang, F,, Costello. W.J. and Govind, C.K. ( I ~1771 Biol. Bull. Wood~ Hole, Mass. 152, 75~q3 6 Lang, F.. Govind, C.K. and Costello, W.J. (1978)Scienee 201, 1037-1/t39 7 Lang, F., Govind, C. K. and She, J. (19771 Biol, Bull. Woods Hole, Mass. 152. 382-391 8 Lomo, T., Westgaard, R . H . and Dahl, H, A. (19741Proc. R. Soc. London. Ser. B. 187,99-103
c~ Mellon, DeF. and Stephens, P. J. (1~78)Nature (London) 2 7 2 , 2 4 6 - 2 4 8 10 Przibram, H. (1911!) Arch. Entwickh~ng~mech. Org. 11,321-345 11 Ritzmann. R. [ , (1t,~74),I, Comp. Physiol. t)5, 217-236 12 Sreler, F. A., Gcrgelcy, J., Salmons. S. and Romanul, F. ( 19731 Nature (London) New Biol. 24t, 17-1~,' 13 Srctcr. F. A., Luft. A. R, S. and (Jcrgclcy. ,I. ( 197513. Gen. Physiol. 66, 811 821 14 Stephen,, P. J. and Mellon, DeF. ( 197 t))]. Comp. Physiol. 132.97-1118 15 Wils¢m. F. B. (19113) Biol. Bull. Wood,$ tlole, Mav,s. 4, It)7-210
DeForest Melhm. Jr. is at the Department of Biology. University of Virginia, Charlottesville 22903, U.S.A.
The synapt.ic basis of monosynaptm stretch reflex Stephen Redmanand BruceWaimsley The synapses formed on motoneurones by sensory nerves from primary stretch receptors in muscle have been the model for studies on the mechanisms o f excitatory synaptic transmission in the CNS. Recent refinements in electrophysiological and anatomical techniques, and the application o f cable theory to motoneurone dendrites, have produced new results. These suggest that although transmission at these synapses involves the release o f a neurotransmitter, the mechanisms by which transmission is achieved at these synapses, and perhaps at CNS synapses in general, may be quite different from those which operate at the neuromuscular junction. The monosynaptic stretch reflex occurs when excitation of primary endings of muscle spindle stretch receptors in a muscle causes a reflex dischage, via a monosynaptic pathway, of motoneurones connected to that muscle and to other muscles which are functionally synergistic. This classical description of the monosynaptic stretch reflex must now be widened to include the effects of afferents from secondary endings of muscle spindle stretch receptors, which have been shown to make weak, but nonethe-less monosynaptic connection with motoneurones 11. The synapse between axons from primary endings and motoneurones has been the model for studies on excitatory synaptic transmission in the CNS, largely because of the relative ease of recording from spinal motoneurones, and also because of the ease with which the afferents from primary endings may be selectively excited. Most textbook accounts of transmission at this synapse describe a conventional chemical synapse where the synaptic current has a reversal potential of about 0 mV, quantal release of transmitter occurs with an average quantal content of 1-2. the synaptic delay is 200-300 p.s, and facilitated transmission is caused when an earlier condi-
tioning stimulus precedes the test stimulus by a short interval (2-10 ms). This account was developed from the pioneering efforts of Eccles e and his collaborators, together with Kuno's contributions TM on quantal analysis. There remained a number of gaps in the story. It had not been possible to show that the current causing the excitatory postsynaptic potential (e.p.s.p.) was ion specific, although the reversal potential suggested that Na +, K + and perhaps Ca 2+ were involved. The neurotransmitter was unidentified, although glutamate was the prime candidate. A subsequent phase of research on this synapse, which began about 15 years ago, took into account such factors as the location and pattern of termination of group la axons (from the primary endings) on the motoneurone surface. This introduced considerably more precision into the analysis of the e.p.s.p. For a period the chemical nature of transmission was challenged, and alternatives to quantal release mechanisms have been proposed. Recent reviews 2,19 provide a full description of this research.
Is it a chemical synapse? This question was asked in a reappraisal t [ I~cviCl/Norlh-llollandBiomedicalprc,s It~8I c>37~ ~4[ 2/81/OIH)I) (10110/$(}2~l)
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of junctional mechanisms at this synapse by Rail, et al. 1~. They found that the available evidence did not allow them to distinguish between chemical transmission and low resistance electrical coupling. This reexamination resulted from the difficulties they experienced in demonstrating a reversal potential for the e.p.s.p. Subsequently, other investigators had similar experiences, and electrical coupling, either alone, or jointly with chemical transmission, received more support. Even though the ultrastructure suggested a chemical synapse 5, the large area of apposition of the synaptic boutons with the motoneurone membrane led to the idea that some degree of electrical coupling could occur through the synaptic cleft. When this proposal was analysedl,.a, it was found that an unrealistically low value of resistivity of the synaptic cleft and the sub-synaptic membrane was required for significant electrical coupling to occur. The heat was taken out of this debate by a clear demonstration of reversed potentials by Engberg and Marshall*. Such a demonstration does not deny the presence of some degree of electrical coupling, but the accumulated evidence from other experimental approaches suggests that it does not exist. This evidence comes from measurements of synaptic delay, and from alterations in the extracellular concentration of divalent cations which normally suppress transmitter release. When Mn ~÷ and Co ~+ were electrophoresed outside motoneurones in which the Fig. la e.p.s.p, was being recorded, the peak of the e.p.s.p, was depressed and its rate of rise was decreased TM. An electrical component would normally contribute to the rising phase of the e.p.s.p., and it should not be affected by these procedures. Removal of Ca 2+ and the presence of Mn z÷ reversibly abolished the e.p.s.p, in isolated immature cat spinal cord 2~. Any electrical coupling would not be affected by these procedures. Together, these results suggest that electrical coupling does not contribute to the generation of the e.p.s.p. In contrast, both chemical and electrical modes of transmission occur at the synapses between primary afferents and motoneurones in the amphibian spinal cord '°. The synaptic delay which occurs in the monosynaptic reflex has been the subject of many disagreements. These date back to the early days of microelectrode recording, when Lloyd argued against Eccles for the existence of a monosynaptic inhibitory connection between muscle afferents and motoneurones. The main problem in determining synaptic delay at this synapse,
or anywhere in the CNS, is that it is difficult to determine the precise arrival time of the impulse in the presynaptic terminals. When the afferent input is restricted to impulses in a single fibre, averaging the resultant e.p.s.p, reveals a small (positive, negative) diphasic field potential prior to the e.p.s.p. The positive peak of this diphasic potential has been interpreted as indicating the arrival time of the impulse in the presynaptic terminals of the branch of the afferent fibre synapsing on that motoneurone TM. Accordingly, the positive potential has been the reference time for measurement of synaptic delay. The values obtained, after compensation for electrotonic delay, ranged from 170 to 440/,ts. The existence of such delays requires a chemical synapse. It remains to be established how reliably this field potential is derived solely from the action potential in the termination of the Ia axon on the penetrated motoneurone. The field potential is a temporal average resulting from a moving dipole in non-terminating and terminated axons, with different spatial weightings depending upon the size of the contributing axons and boutons, and their distance from the recording electrode. Major contributions to this potential could arise from large collaterals of the Ia axon in the vicinity of the penetrated motoneurone, and from nearby terminals other than those on the penetrated motoneurone. H R P studies 1.s have demonstrated that the branching pattern of Ia axons in the motoneurone pool is very extensive. Transmitter release
The manner in which transmitter is released from an assembly of boutons connecting an afferent fibre with a motoneurone is a matter of some debate. Fluctuations in the amount of transmitter released are normally reflected by fluctuations in the amplitude of the e.p.s.p. Kuno's investigationss of these fluctuations, using a classical quantal analysis approach, indicated that an average of one or two quanta are released per impulse from the collection of boutons involved. The unit synaptic potential was 100-200/zV in peak amplitude. However, neither Poisson nor binomial distributions could be used convincingly to describe the fluctuation pattern. An analysis of this type in a central neurone is at a disadvantage compared with quantal analysis of fluctuations at the neuromuscular junction. In neurones, spontaneous miniature potentials originating from the synapses under study cannot be distinguished from the entire population of spontaneous potentials, and so the unit
synaptic potential cannot be identified. Also, very poor signal to noise conditions often prevail. Because of these difficulties, Edwards et al. tried another approach 7, and these techniques have subsequently been refined in a recent series of experiments with Jack, Hirst and Wong (unpublished). The recorded noise (both neuronal and instrumentational) can be measured and described statistically. This noise adds to the actual fluctuations in the evoked e.p.s.p., and the recorded signal is the sum of two random variables. Provided the noise processes are statistically independent of the processes generating the fluctuations in the e.p.s.p., the fluctuations can be separated from the sum of these two random variables if the noise is adequately described. A computational procedure separates the histogram of the peak amplitude of the recorded e.p.s.p, from the distribution which represents the recording noise. The results of this procedure indicated that about 30% of single fibre potentials did not fluctuate in amplitude. For those potentials which did fluctuate, they did so between a number of discrete amplitudes. (An example is illustrated in Fig. 1C.) These discrete amplitudes were separated by a constant increment, or by multiples of this increment. The incremental voltage was about 100 p,V, which is similar to the value obtained by Kuno for the unit e.p.s.p. Occasionally, it could be shown that the time course of the e.p.s.p, was different when certain discrete amplitudes occurred. The discrete amplitudes had no detectable variability. The average amplitude of an e.p.s.p, could be increased by a prior tetanus, or by the administration of 4-aminopyridine. This increase occurred by a reduction in the probability of occurrence of smaller discrete amplitudes, and by an increase in the probability of occurrence of larger discrete amplitudes. We have not interpreted these fluctua-, tions as being a direct reflection of the release of a variable number of quanta at the collection of boutons involved. The constancy of the discrete amplit,:des, and the differences in time course for particular discrete amplitudes argue against this idea. Rather, we have suggested that at each bouton, impulse invasion results in either a failure to release transmitter, or the release of sufficient transmitter to saturate all available receptors. The various boutons in the termination on a single motoneurone can have different probabilities of failure. This hypothesis implies that each single fibre e.p.s.p, consists of a number of subunits, where each sub-unit results from
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PEAK ,,,,MP~.,TUOE I,V) Fig. 1. (A) This illustrates the connection formed by a single afferent fibre (solid line) and the dendrites of a motoneurone (dashed lines). This diagram was reconstructed from serial sections, and only that region o f the dendritic tree where synapses were found has been drawn in detail. The synaptic boutons are indicated by arrows. (B) The averaged e.p.s.p, recorded in the motoneurone shown in A, and evoked by impulses in the single afferent shown in A. (C) The fluctuation pattern o f the peak amplitudes o f the evoked e.p.s.p. This pattern was calculated from the histogram of recorded peak amplitudes o f the evoked e.p.s.p., and the probability distribution of the recording noise (Walmsleyand Redman, unpublished).
all-or-none transmission at a single bouton. Accordingly, the number of discrete amplitudes (excluding failures) that a single fibre e.p.s.p, can have should never exceed the number of boutons in the termination with the motoneurone in which the e.p.s.p. is recorded. We have examined this constraint in experiments in which a single fibre e.p.s.p, was recorded and subsequently both the afferent fibre and the motoneurone involved were identified using intraceUular injections of HRP. The results of one such experiment are shown in Fig. 1. The afferent fibre made four contacts with the motoneurone. The average e.p.s.p, evoked in that motoneurone by impulses in the afferent nerve is shown in Fig. lB. The fluctuation pattern of the e.p.s.p, is indicated in Fig. 1C. The peak amplitude of the e.p.s.p, was either 320 /xV, with a probability of 0.65, or 440/xV, with probability of 0.35. According to our hypothesis, transmission would always occur at the same three boutons, generating a 320/zV synaptic potential. At a fourth bouton, transmission occurs with a probability of 0.35, adding a further 120 p,V to the peak of the e.p.s.p. This result, and others in which various numbers of boutons were found, supports our suggestion that a single bouton either fails to
release transmitter, or releases sufficient to saturate the post-synaptic receptors. This result is also a clear demonstration that not all synapses in the one termination are equally effective in synaptic transmission, at least at the low stimulus rates employed in these experiments (3 Hz). We have proposed that failure to release transmitter follows adequate terminal depolarization by the afferent impulse. This hypothesis has been adopted rather than one which requires a failure of the impulse to invade the bouton, because we were unable to detect any significant latency variations in the onset time of the e.p.s.p. Such latency variations would be expected if the impulse spread through branch points and into terminal varicosities with marginal safety factors. However, latency variations in the onset of these potentials have been reported 4, and the hypothesis that the action potential does not always invade all boutons has been developed by Ltischer, Ruenzel and Henn e m a n a4 to account for their observations on post-tetanic potentiation.
The contribution of dendritic synapses to somatk membrane potential Group la axons terminate on both the soma and the dendrites of motoneurones.
The dendrites act as passive cables h~r tile spread of synaptic current towards the soma from the dendritic synapses. The potentials of dendritic origin become attenuated, and their time course prolonged, as they spread electrotonicatly towards the soma. As a result of theoretical analysis of current spread in dendrites it is possible to use the cable properties of the motoneurone, and the time course of the e.p.s.p, recorded at the soma, to calculate a site of origin for the e.p.s.p, on the dendrites 1°'1s, This distance is calculated in terms of an electrical space constant (X). These calculations have indicated that the most common location for synaptic termination is within 0.5;k from the soma. corresponding to a physical distance of about 250-400 p,m. But some synapses may occur at locations greater than one space constant from the soma. The H R P experiments described in connection with Fig. 1 have confirmed that the synaptic location calculated from the e.p.s.p, time course agrees with the actual synaptic location determined morphologically. The synaptie location calculated from the e.p.s.p, time course shown in Fig. 1B was 0.6,~. When the dendrites on which synapses were found were reconstructed into an equivalent dendritic cable, the synapses were at 0,62k and 0.65X from tile soma. It has often been suggested that synapses on distal dendrites are spatially disadvantaged in their ability to influence the membrane potential at the soma. This is not so for la synapses on motoneurones. The peak voltage of potentials at the soma shows no dependence on the distance to the site of generation of the e.p.s.p. 9 Also the average synaptie current which spreads to the soma (or alternatively the total synaptic charge reaching the soma) actually increases as .the site of origin of the e.p.s.p, becomes more distal. These are surprising results, especially when it is recognized that passive electrotonic spread towards the soma from the synaptic site must involve some loss of charge across the dendritic membrane, with consequent attentuation of peak voltage. In some way the 'gain' or 'sensitivity' of dendritic synapses compensates for their more distal location. Such compensation must occur through a greater synaptic current at distal synapses. This could result from a larger number of boutons/afferent fibre, or from a similar number of boutons covering a greater synaptie area, or from a more prolonged duration of synaptic current at distal synapses. Histology indicates that the synapses have similar coverage, and cable
251
TINS - October 1981 calculations indicate that the synaptic current is not prolonged. The first possibility will eventually be answered by HRP studies. However, our studies of fluctuations of dendritic potentials showed that the contribution to the e.p.s.p, from a single bouton did not alter as its location on the dendrites altered. For this reason we h a v e speculated that the answer lies in alterations of receptor density with synaptic location. As a final comment it should be pointed out that while we have confined this article to a discussion of results obtained in vivo, important contributions to our understanding of junctional mechanisms at synapses between dorsal root afferents and spinal neurons have come from studies using cultures of spinal neurones and dorsal root ganglion calls 18, and also from experiments using spinal cord slices. Some of the uncertainties alluded to in this article may be resolvable using these alternative preparations.
Reading 1 Brown, A. G. and Fyffe, R. E. W. (1978) J. Physiol. (London) 274, 111-127 2 Burke, R. E. and Rudomin, P. (1977) in The
Handbook of Physiology, Section 1: The Nervous System (Kandel, E. R., ed.), Vol. 1, Part 2, pp. 877-944, American Physiological Society, Bethesda 3 Burke, R. E., Walmsley.B. and Hodgson,J. A. (1979) in Integration in the Nervous System (Asanuma,H. and Wilson, V. J., eds), pp. 27-45. Igaku-Shoin,New York 4 CoUatos,T. C., Niechaj, A. J., Nelson, S. G. and Mendell, L. M. (1979) Brain Res. 160, 514-518 5 Conradi, S. (1969) Acta Physiol. Scand. Suppl. 332, 85-115 6 Eccles, J. C. (1961) Ergeb. Physiol. Biol. Chem. Exp. Pharmakol. 51,299-430 7 Edwards,F. R., Redman, S. J. and Walmsley,B. (1976) J. Physiol. (London) 259, 665-688 8 Engberg,I. and Marshall, K. C. (1979) Neuroscience 4, 1583-1591 9 Iansek, R. and Redman, S. J. (1973)J. Physiol. (London) 234,665-688 10 Jack, J. J. B., Miller, S., Porter, R. and Redman, S. J. (1971)J. Physiol. (London) 215. 353-380 l 1 Kirkwood,P. A. and Sears, T. A. (1974)Nature (London) 252,242-244 12 Krnjevic, K., Lamour, Y., Macdonald, J. F. and
Cell lineage in the development of the leech nervous system Gunther S. Stent and David A. Weisblat The developmental lines o f descent o f leeches neurons have been ascertained by injection o f tracers into identified cells o f early embryos. One o f these tracers is a fluorescent molecule consisting o f rhodamine coupled to a synthetic dodecapeptide and the other is horseradish peroxidase. These studies have shown that the neurons on either side o f a segmental ganglion fall into four distinct classes, representing the descendants from each o f the four ipsilateral ectodermal teloblasts N, O. P and Q. However, the embryological origin o f the cells o f the supraesophageal ganglion is entirely different from that o f the cells o f the segmental ganglia. One key aspect of neuronal development is cell lineage, i.e. the embryonic lines of descent of various types of neurons. The importance of cell lineage for understanding developmental processes was first realized in the 1880s by C. O. Whitman ~'14. From his studies on leech development, he proposed that each cell of the early embryo, and the clone of its descendant cells, plays a specific role in later development. We have continued and extended Whitman's studies placing particular emphasis on the developmental origins of the leech nervous system. For this purpose we have used the embryos of two leech species belonging to the family of Glossiphonidae: Helobdella triserialis and Haementeria ghilianii 3.
Leech nervous system The nervous system of the leech consists of a chain of 32 interconnected segmental ganglia. Each ganglion contains about 200 bilaterally symmetrical neuron pairs, as well as a few unpaired neurons. The foremost rostral segmental ganglia are fused to form the subesophageal ganglion, which, at its anterior edge, is connected to the dorsal supraesophageal ganglion. Studies carried out since the mid-1960s have shown that the anatomy of the segmental ganglia is sufficiently stereotyped from segment to segment, and sufficiently invariant from specimen to specimen, to enable a large fraction of their neurons to be reliably identified s. Moreover, it has been possible to establish the connection pattern of
Nistri, A. (1979) Neuroscience4, 1331-i 339 13 Kuno,M. (1974)J. Physiol. (London) 175,81-99 14 Luscher, H.-R., Ruenzel, P. and Henneman, E. (1979) Nature(London). 282,859-861 15 Munson,J. B. and Sypert,G. W. (1979)J. Physiol. (London) 296, 329-342 16 Rail, W. (1977) in The Handbook of Physiology. Section 1; The Nervous System (Kendell, E, R., ed.). Vol. l, Part 1, pp. 39-97, American Physiological Society, Bethesda 17 Rail,W., Burke, R. E., Smith,T. G., Nelson,P. G. and Frank. K. (1967) J. Neurophysiol. 30, 1167-1193 18 Ransom.B. R., Christian, C. N.. Bullock, P. N. and Nelson, P. G. (1977) J. Neurophysiol. 40. 1151-1162 19 Redman,S. J. (1979)Prog. Neurobiol. 12, 33-83 20 Shapovalov, A. 1. and Shiriaev. B. I. (1980) J. Physiol. (London) 306, 1-15 21 Shapovalov,A. I., Shiriaev, B. I. and Tamarova. Z. A. (1979) Brain Res. 160, 524-528
Stephen Redman is a Reader in the Departments of Physiology and ElectricalEngineeringat Monash University, Wellington Road, Clayton Victoria 3168. Australia and Bruce Walmsleyis a ResearchFellowin the Experimental Neurology Unit at the John Curtin School of Medical Research,A ustralian National University.A.C.T., Australia.
sensory-, motor- and inter-neurons and thus account not only for some simple acts of reflex behavior4, such as the shortening of the leech body in response to tactile stimulation, but also for some moderately complex integrated movements, such as the heartbeaP. The most complex behavioral routine of the leech described so far in terms of its neural substrates, is swimmingL Consequently, the leech nervous system provides a model system for examining neurodevelopmental processes. Leech development
At the outset of its development the leech egg cleaves into two cells, AB and CD (Fig. 1). The second cleavage gives rise to four cells, A, B. C and D. Cell D then cleaves to yield two cells, one designated DNOPQ, which lies more dorsally, and another designated DM, which lies more ventrally. At this stage, separation of the embryo into the three germinal tissue layers has been accomplished: the progeny cells of A, B and C will give rise to endoderm, the progeny of D N O P Q to ectoderm, and the progeny of DM to mesoderm. The next two cleavages establish the bilateral symmetry of the embryo: DM divides to yield a pair.of right and left M cells, and D N O P Q divides to yield a pair of NOPQ cells, lying on either side of the future midline. Three further cleavages of the N O P Q cell pair produce four bilateral cell pairs designated as N, O, P and Q, which, together with the M cells are referred to as teloblasts. As soon as each teloElsevier/North-HollandBiomedicalPressIt)81 I)378
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