Relative Roles of the S Cell Network and Parallel Interneuronal Pathways in the Whole-Body Shortening Reflex of the Medicinal Leech

Brian K. Shaw and William B. Kristan, Jr.

Department of Biology, University of California San Diego, La Jolla, California 92093-0357


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Shaw, Brian K. and William B. Kristan Jr.. Relative Roles of the S Cell Network and Parallel Interneuronal Pathways in the Whole-Body Shortening Reflex of the Medicinal Leech. J. Neurophysiol. 82: 1114-1123, 1999. The whole-body shortening reflex of the medicinal leech Hirudo medicinalis is a withdrawal response produced by anterior mechanical stimuli. The interneuronal pathways underlying this reflex consist of the S cell network (a chain of electrically coupled interneurons) and a set of other, parallel pathways. We used a variety of techniques to characterize these interneuronal pathways further, including intracellular stimulation of the S cell network, photoablation of the S cell axon, and selective lesions of particular connectives (the axon bundles that link adjacent ganglia in the leech nerve cord). These experiments demonstrated that the S cell network is neither sufficient nor necessary for the production of the shortening reflex. The axons of the parallel pathways were localized to the lateral connectives (whereas the S cell axon runs through the medial connective). We used physiological techniques to show that the axons of the parallel pathways have a larger diameter in the anterior connective and to demonstrate that the parallel pathways are activated selectively by anterior mechanosensory stimuli. We also presented correlative evidence that the parallel pathways, along with activating motor neurons during shortening, are responsible for inhibiting a higher-order "command-like" interneuron in the neuronal circuit for swimming, thus playing a role in the behavioral choice between swimming and shortening.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Especially large or giant axons are found in a variety of animals and often have been implicated in the production of fast withdrawal or escape responses (Eaton 1984). In systems in which closer inspections have been made, however, such axons have not always been found to be solely responsible for producing behavioral responses. In crayfish, for example, tail-flip escape responses can be mediated either by giant axons or by parallel nongiant pathways (Wine and Krasne 1972). Another well-studied case is the giant Mauthner cell of teleost fish. The Mauthner cell is sufficient by itself to cause a C start escape response, but a C start still can occur after the Mauthner cells are ablated bilaterally, demonstrating that they are not necessary for the response (Eaton et al. 1991). A recent study using in vivo calcium imaging in zebrafish showed that, along with the Mauthner cell, two of its serial homologues---MiD2cm and MiD3cm---are active during the stronger C starts elicited by head stimulation; whereas for weaker C starts elicited by tail stimulation, only the Mauthner cell is active (O'Malley et al. 1996). This indicates that the Mauthner cell and its serial homologues act in parallel in the production of escape responses. The probable reason for such parallel-pathway organization is that it allows for behavioral flexibility; an animal can modulate its response depending on the location of stimuli or the behavioral context.

Another case in which a parallel-pathway organization has been described to underlie a withdrawal response is for the whole-body shortening reflex of the medicinal leech Hirudo medicinalis. This reflex is induced by strong mechanical stimuli delivered to the anterior of the animal (Kristan et al. 1982). In a recent study of the neural basis of this behavior, we characterized its motor pattern, its sensory basis, and, to a first approximation, the interneuronal pathways that underlie it (Shaw and Kristan 1995). These interneuronal pathways consist of the S cell network, possessing the largest axon in the leech nerve cord, and other, slightly slower pathways, which act in parallel with the S cell network (Fig. 1). In the present study, with the goal of gaining general insights into this style of parallel-pathway organization, we further characterized these pathways and their relative roles in producing shortening. The present results also provide additional information on the neural mechanisms that underlie the behavioral choice between shortening and swimming, which we began to elucidate in a related study (Shaw and Kristan 1997).



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Fig. 1. Neural circuit for the whole-body shortening reflex (adapted from Shaw and Kristan 1995). T- and P-type mechanosensory neurons in the anterior nerve cord activate interneuronal pathways that exert their effects on longitudinal motor neurons throughout the body. L cell is a motor neuron that innervates both dorsal and ventral longitudinal muscle (Stuart 1970). Dorsal excitor (DE) and ventral excitor (VE) motor neurons innervate dorsal and ventral longitudinal muscle, respectively (Ort et al. 1974). perp , monosynaptic excitatory connections. +, excitatory effects that may not be direct. Resistance symbols indicate the electrical synapses between adjacent S cells.

The S cell network consists of a chain of electrically coupled interneurons that make up a "fast conducting pathway" running the length of the nerve cord (Bagnoli et al. 1975; Frank et al. 1975; Gardner-Medwin et al. 1973; Peterson 1984). The S cell has the largest-diameter axon in Hirudo, at least in midbody locations (Wilkinson and Coggeshall 1975). The electrical coupling between adjacent S cells is so strong that a spike initiated in any S cell is propagated to all S cells; thus the set of S cells truly functions as a unit. The S cell network is the fastest of the shortening pathways and accounts for the shortest-latency excitation of the L motor neurons (Shaw and Kristan 1995). However, there have been some suggestions that the S cell network by itself is neither sufficient nor necessary to produce the shortening reflex. On the sufficiency side, it has been reported that high-frequency stimulation of the S cell yields only weak muscle activation in an isolated body segment (Gardner-Medwin et al. 1973). On the necessity side, Sahley et al. (1994) reported that ablation of the S cell eliminated sensitization and reduced dishabituation of the shortening reflex but had no significant effect on the baseline magnitude of the reflex. However, because of the way these experiments were performed, it is likely that a portion of the S cell network was still functional even after the ablation in the body portion in which shortening behavior was measured (this problem is discussed in Shaw and Kristan 1995, p. 668). Thus these experiments did not furnish a definitive test of the necessity of the S cell network for the shortening reflex. In the present study, we sought to resolve the open question of how important a role the S cell network plays in shortening by testing its sufficiency and necessity for the reflex more directly than has been done previously.

Although the S cell network is understood at the level of single identified neurons, the other interneuronal pathways are not. How many interneurons make up these pathways, and where their cell bodies and axons are located, is not known. (In what follows, we will refer to these interneuronal pathways collectively as the "parallel" pathways, because they act in parallel with the S cell network.) Three connectives (distinct bundles of axons) link adjacent ganglia in the leech nerve cord. The two paired lateral connectives contain ~2,800 axons each; the smaller unpaired medial connective (also referred to as Faivre's nerve) contains ~100 axons, including that of the S cell (Wilkinson and Coggeshall 1975). The parallel pathways have conduction velocities that are quite fast, close to that of the S cell (Shaw and Kristan 1995), making it likely that the parallel pathways have relatively large axons. In an electron-microscopic study of Macrobdella decora, a leech closely related to Hirudo, Fernandez (1978) described a set of large-diameter axons found in the lateral connectives linking the more anterior ganglia of the nerve cord. In more posterior regions, the number and size of these large axons decreased, indicating that they either taper down or disappear as they run posterior. A similar set of large lateral connective axons is found in the more anterior nerve cord of Hirudo as well (J. Fernandez, personal communication). In the present study, we used several techniques to localize and investigate the axons of the parallel pathways, with an eye to evaluating whether they could be coextensive with these histologically identified large axons.

The shortening reflex is elicited for the most part by mechanical stimuli to the anterior of the animal; posterior stimuli tend to cause other behaviors, like swimming (Kristan et al. 1982; Misell et al. 1998). The S cell network, however, has symmetrical mechanosensory inputs: mechanical stimuli applied anywhere along the body activate the S cells more-or-less equally well (Laverack 1969; see Fig. 7). This led us to predict in our earlier study that the parallel interneuronal pathways would be activated selectively by anterior stimuli, unlike the S cell network (Shaw and Kristan 1995). In the present study, we test this hypothesis.

In examining the neuronal basis of the behavioral choice between swimming and shortening, we identified as an important factor a strong inhibition of the swim-gating cell 204 (a "command-like" neuron in the swim circuit) that occurs during shortening (Shaw and Kristan 1997). The latency of this inhibition is quite fast, comparable with the response latencies of motor neurons. This led us to suggest that some of the same interneuronal pathways that drive motor neurons during shortening also could be responsible for inhibiting cell 204. The S cell is not a candidate for this function, because it makes no connections with cell 204 (Weeks 1982); it is the parallel pathways that might play this role. In the present study, we provide further evidence for this hypothesis.

Some of this work has been published previously in abstract form (Shaw and Kristan 1994).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and procedure

Adult H. medicinalis were obtained from Leeches USA (Westbury, NY) or from a laboratory breeding colony. The general methods (dissection, recording, and cell identification techniques) and experimental procedure were as previously described (Kristan et al. 1974; Shaw and Kristan 1995). The leech nervous system consists of a head brain, a tail brain, and 21 midbody ganglia (MG1-MG21). Two types of semi-intact preparation were used in these experiments. One had intact anterior and posterior body portions, with the midbody cut away from segments 6-7 to segments 8-14. The other had an intact anterior portion (from the head to segments 6-9), with the nerve cord exposed posterior to this. In almost all of the preparations, the head and tail brains were removed, to reduce state-dependent variability in behavioral responses (Kristan et al. 1982; Shaw and Kristan 1995). The only exceptions to this were several semi-intact preparations (with anterior and posterior body portions) in which the head and tail brains were left intact. In these cases, the front and rear suckers were sutured closed to prevent attachment of the suckers.

The whole-body shortening reflex was elicited using stimulating electrodes implanted in the dorsal anterior skin, between segments 3 and 4. The electrodes and the method of implanting them were as described in Shaw and Kristan (1995). The stimulus was a 0.5-s, 10-Hz train of shocks (1-ms pulses, 8-V intensity), which mimics a mechanical stimulus (Kristan et al. 1982; Shaw and Kristan 1995). In a subset of preparations, stimulating electrodes also were implanted in the dorsal posterior skin, between segments 17 and 18, so that the effects of anterior and posterior stimuli could be compared. In these cases, the stimulus protocol used for anterior and posterior stimuli was identical.

Monitoring of behavior

Video recording was used to monitor shortening behavior in some of the preparations with intact anterior and posterior portions. Although the cut ends of the body portions in these preparations were pinned down and anchored, along with the nerve cord, they were otherwise free to behave. Videotaping was performed with a Sanyo VDC 3825 video camera and a Panasonic AG 1730 video cassette recorder. The camera was oriented to view the preparation from above. The videotapes were examined at a frame-by-frame level (at a rate of 30 frames/s) using the NIH Image program. Single frames were saved at a point just before the application of the stimulus (<1 s before stimulus onset) and at the point of maximal shortening (which occurred <1 s after stimulus onset). Approximate outlines of the preparations then were traced from these images, using Canvas (Deneba Systems). Results of this procedure are shown in Figs. 2 and 4A. From these images, the lengths of the body segments were quantified by measuring the length of a straight line drawn from the point where the nerve cord entered the body portion (which was fixed and constant) to the most distant point on the body outline. The normalized degree of shortening for a body portion was calculated as the change in length between the pre- and poststimulus traces divided by the prestimulus length.



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Fig. 2. Test of the sufficiency of the S cell network for shortening. Three successive trials from the same semi-intact preparation are shown. Approximate outlines of the preparation just before and after stimuli were applied were made from video recordings (see METHODS). As the outlines show, the preparation had intact anterior and posterior body portions with the midbody cut away and the nerve cord exposed from segment 7 to 11. The body portions were anchored at their cut ends but were otherwise free to behave. The pre- and poststimulus traces for each trial are overlaid; the shaded trace is the poststimulus one. In this and subsequent figures, the numbers in parentheses on physiological record labels give the midbody ganglion where the recording was made. In the first trial, the S cell was stimulated intracellularly at 30 Hz for 0.5 s. Stimulus consisted of a train of 20-ms pulses, each causing 1 spike in the cell and is indicated by the bars under the recording. In the second trial, the S cell was "overdriven" intracellularly with a continuous 0.5-s depolarizing pulse (indicated by the bar), which caused a mean spike frequency of 84 Hz in the cell. In the third trial, the standard anterior skin stimulus was given (see METHODS); this caused the S cell to fire at 42 Hz in the first 0.5 s after stimulus onset. In this and subsequent figures, this stimulus is indicated by the horizontal bar, with individual pulses indicated by the vertical tics. These trials were seperated from each other by >1.5 min. In the video recordings, the slight changes in the position of the anterior portion in the "S cell stimulus" trials appeared to be due to ongoing spontaneous movements of the animal, rather than to an effect of stimulating the S cell.

Physiology

Intracellular recordings were made with 30-40 MOmega electrodes filled with 3 M KAc. Extracellular recordings were made with suction electrodes. Depending on the experiment, extracellular recordings were made from the connectives (conn), the first two branches of the dorsal posterior nerve (DP:B1 and DP:B2), and/or the first two branches of the anterior root (A:B1 + B2). The recordings from the connectives were from the entire set, medial and lateral, performed en passant between two ganglia. The largest spike in connective recordings is that of the S cell (although see Fig. 7 and RESULTS), and the activity of the parallel interneuronal pathways also can be monitored in these recordings (Shaw and Kristan 1995). Recordings from nerves DP:B1 and DP:B2 allow the spikes of the L cell and cell 3 (a DE motor neuron) to be discriminated (Shaw and Kristan 1995). Nerves A:B1 + B2 contain the spikes of VE motor neurons (Ort et al. 1974; B. Shaw, personal observation). Physiological data were recorded and displayed as described in Shaw and Kristan (1995).

Photoaxotomy

In experiments to test the necessity of the S cell network for shortening, the S cell axon was photoablated by injecting the cell with Lucifer yellow and then irradiating it with blue light (Miller and Selverston 1979). Electrode tips were filled with 8% Lucifer yellow and the remainder of the electrode filled with 0.1 M LiCl. The dye was iontophoresed into the soma of the S cell for ~30 min. For ablation, the preparation was moved to a Zeiss Standard compound microscope equipped with a 50-W mercury arc lamp. The S cell axon in the connective, just anterior or posterior to the ganglion where the cell had been filled, was irradiated with blue light (Zeiss filter BP436) through a ×40 objective (water immersion, NA 0.75) for 5-20 min. The segment of the axon that was irradiated was limited by contracting the iris diaphragm. The axon usually exhibited visible signs of distress during irradiation: it sometimes twisted and curled (e.g., Fig. 3D), became swollen, or showed discrete bleaching. The following physiological checks were performed afterwards to ensure that the S cell network had been severed functionally. Recordings were made from the connective anterior and posterior to the photoablation site (usually simultaneously), while the S cell network was driven by skin stimulation and light flashes delivered to the anterior and posterior body portions (e.g., Fig. 3E). In all cases, it was confirmed that the S cell spikes anterior and posterior to the ablation site were decoupled. Furthermore it was confirmed that S cell spikes anterior to the ablation site were caused only by stimuli to the anterior body portion, whereas S cell spikes posterior to the ablation site were caused only by stimuli to the posterior portion.

Connective lesions

In some experiments, selective lesions were performed on particular connectives, either the medial connective or both of the lateral connectives. These lesions were accomplished by using two pairs of fine forceps to pull the targeted connective apart (Weeks 1981). Transections of the lateral connectives were confirmed visually. It was sometimes more difficult to detect visually that the medial connective had been cut, so these lesions were checked further by confirming that S cell spikes no longer crossed the lesion site. In the lateral connective lesions, the two connectives were transected at a slight distance from one another (see the diagrams of Fig. 6) so as not to put too much strain on the remaining medial connective.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

S cell network is neither sufficient nor necessary for the shortening reflex

To test the sufficiency of the S cell network for shortening, the network was stimulated intracellularly and the effect on behavior assessed. An example of an experiment of this type is shown in Fig. 2. In the first trial of this experiment, the S cell was stimulated at 30 Hz, a firing rate close to that it shows to stimuli that produce shortening (Shaw and Kristan 1995). This resulted in essentially no movement of the preparation. In the second trial, the S cell was driven at a rate well above its physiological frequency; still, the preparation showed little to no movement. In the third trial, an anterior skin stimulus was given; this stimulus produced strong shortening, demonstrating that the preparation was viable and capable of behaving. This general result---that stimulating the S cell intracellularly, even at unphysiologically high frequencies, yielded very little or no behavior in the animal---was seen in five preparations. The effect of S cell stimulation on motor neurons was tested in four preparations. Stimulating the S cell at 30 Hz for 0.5 s produced a mean spike frequency of 6.0 ± 4.3 Hz in the L cell (mean ± SD; calculated for the 0.5-s period of S cell stimulation). This is below the L cell firing rate observed during shortening (Shaw and Kristan 1995). S cell stimulation did not activate cell 3, a DE motor neuron.

The preceding experiments were performed in preparations in which the head and tail brains had been removed. Removal of the brains has been used routinely in previous studies of shortening and other behaviors to reduce state-dependent variability in responses (Kristan et al. 1982), and the features of the shortening motor pattern have been shown to be the same in debrained preparations and preparations with the brains intact (Shaw and Kristan 1995). However, to thoroughly ensure that the presence of the brains has no effect on the efficacy of the S cell network, we performed similar S cell stimulation experiments in two preparations in which the head and tail brains were left intact (and the front and rear suckers had been sutured closed). As was the case with the debrained preparations, intracellular stimulation of the S cell network, even at unphysiologically high rates, produced little to no shortening in these preparations. Thus the S cell network is not sufficient by itself to produce shortening, regardless of whether or not the head and tail brains are present. With this issue settled, all the remaining experiments presented in this study were performed in preparations in which the brains had been removed.

To test the necessity of the S cell network for whole-body shortening, the axon of the S cell was photoablated, thus functionally severing the network. Shortening behavior was assessed before and after photoaxotomy. The photoaxotomy technique is illustrated in Fig. 3. Experiments testing the behavioral effect of S cell photoaxotomy are shown in Fig. 4. After photoaxotomy, the stimulus still activated the S cell in these preparations, but the S cell spikes no longer crossed the ablation site and entered the posterior body portion. Thus the key point in the experiments of Fig. 4 is to compare the amount of shortening that occurred in the posterior portion of the preparations before and after photoaxotomy. In fact, as can be seen, the posterior portions continued to show appreciable shortening after photoaxotomy (Fig. 4A). S cell ablation did not appear to cause a consistent reduction in the degree of posterior shortening (Fig. 4B). It remains quite possible that a more sensitive measure of behavior would pick up some subtle effect on shortening due to interrupting the S cell network, but any such effect would clearly be minor at best. The outstanding result of these experiments is that strong shortening still occurred after removing the contribution of the S cell network.



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Fig. 3. Photoablation of the S cell axon. A: transmitted-light photomicrograph of a midbody ganglion in which the S cell had been filled with Lucifer yellow. In this and subsequent micrographs, anterior is to the right. B: flourescent photomicrograph of the same ganglion, showing the large axon of the S cell (indicated by arrows). C: higher-resolution photomicrograph of the S cell axon in the same preparation, taken just anterior to the ganglion shown in A and B before ablation. D: same axon after ~5 min. of irradiation with blue light. Physiological checks confirmed that the axon had been ablated. Scale bar is 100 µm in A and B, 40 µm in C and D. E: example of a physiological test to confirm that the S cell network had been severed after photoaxotomy. S cell axon had been photoablated just posterior to MG9 before this test. Large spikes in the connective recordings are those of the S cell. S cell spikes in the recordings anterior to the photoablation site (the extracellular connective recording between MG6 and MG7 and the intracellular recording from the S cell in MG8) are matched to each other but are decoupled from the S cell spikes recorded posterior to the ablation site (from the extracellular connective recording between MG10 and MG11).



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Fig. 4. Tests of the necessity of the S cell network for shortening. Traces of video images from 3 different semi-intact preparations are shown in A, 1-3; a quantitative summary for the same 3 preparations is shown in B. Preparations had intact anterior and posterior body portions with the midbody cut away and the nerve cord exposed from segment 7 to 11 in A, 1 and 2, and from segment 6 to 8 in A3. In each preparation, the shortening reflex was tested before and after the S cell network was photoaxotomized in the exposed nerve cord linking the body portions (2 "Pre" trials were performed before photoaxotomy and 2 "Post" trials after it). Shortening was elicited with the standard anterior skin stimulus. S cell (in MG7 or MG9) was injected with Lucifer yellow before the Pre trials; after the Pre trials the axon was irradiated. Pre and Post trials were seperated by ~20-30 min. Physiological checks (described in METHODS) performed after photoaxotomy in each of these preparations confirmed that the S cell network had been severed. Also, extracellular recordings from the connective made posterior to the site of photoaxotomy during the Post trials verified that S cell spikes were no longer seen. A: traces made from video recordings are presented as in Fig. 2; the pre- and poststimulus traces for each trial are overlaid, with the poststimulus trace shaded. Trials shown are those that occurred just before and after photoaxotomy. Crossed-out S symbol next to the nerve cord in the traces from Post trials is to indicate that axotomy has taken place. B: degree of shortening of the posterior portion before and after S cell photoaxotomy in the 3 preparations. Normalized degree of shortening was calculated from the traced images as described in METHODS. Preparation 1 corresponds to that of A1, 2 to A2, and 3 to A3. Trials 2 and 3 are those shown in A.

The results of this section demonstrate directly that the S cell network is neither sufficient nor necessary for the production of the shortening reflex. An implication of this is that the other, parallel, interneuronal pathways, taken as a set, must be both sufficient and necessary for shortening. Their role appears to be the more important one. We now turn to experiments intended to elucidate aspects of these parallel pathways.

Localization of the axons of the parallel interneuronal pathways

To localize the axons of the parallel pathways, selective lesions were performed on particular connectives. Figure 5 illustrates the results of selectively transecting the medial connective while leaving the lateral connectives intact. After the medial connective lesion, the spikes of the S cell dropped out of the connective recording, as expected, but connective activity produced by the parallel pathways (Shaw and Kristan 1995) was still observed (Fig. 5A). L cells posterior to the lesion site still were activated but with a consistently greater latency than before the lesion (Fig. 5, B and C). Also, the initial rise of the L cell excitatory postsynaptic potential (EPSP) was not as sharp as before; in many cases, this resulted in a longer latency to the first spike in the L cell. These findings confirm directly our earlier suppositions that the S cell network accounts for the shortest-latency excitation of the L cells and that the L cells receive input from pathways besides the S cell (Shaw and Kristan 1995). The spike frequencies of the L cell and cell 3 were measured before and after medial connective lesion in three preparations (Fig. 5D). There was a substantial decrease in the L cell spike frequency, whereas the spike frequency of cell 3 showed only a small reduction.



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Fig. 5. Selective lesions of the medial connective. These experiments were peformed in semi-intact preparations with an intact anterior portion and the nerve cord exposed in the midbody and posterior. A: pattern of connective activity before and after transection of the medial connective. Diagrams of the nerve cord show that the medial connective was cut between MG11 and MG12. Large spikes in the Pre recording are those of the S cell; they are not seen in the Post recording, after the lesion. B: L cell response before and after transection of the medial connective. Pairwise recordings from L cells in MG10 and MG13 were performed before and after the lesion. - - -, latencies of the L cell excitatory postsynaptic potentials (EPSPs). , action potentials in the cells (intracellularly recorded spikes in the L cell tend to be small, but in these cases extracellular recordings were made simultaneously from the appropriate DP: B1; by matching the 2 recordings spikes could be confidently distinguished). C: L cell EPSP latencies before and after transection of the medial connective. Latencies were calculated from the onset of the stimulus, which was delivered between segments 3-4. Data from 2 preparations are shown; top: same as that of B. D: effect of transecting the medial connective on motor neuron spike frequencies. Data are from 3 preparations. Medial connective was cut between MG11 and MG12. Stimulus was delivered between segments 3-4. To count L cell and cell 3 spikes, simultaneous extracellular recordings were made from DP: B1 and DP: B2. Spike frequencies were measured in the first 0.5 s after stimulus onset. Means and SDs are shown.

The results from these experiments suggested that a large portion of the parallel pathways must pass through the lateral connectives because so much motor activation remained after lesioning the medial connective. To further resolve this, we performed the complementary experiment of transecting the lateral connectives, leaving just the medial connective intact. These experiments are presented in Fig. 6. After transection of the lateral connectives, the pattern of activity seen in connective recordings posterior to the cut changed: while S cell spikes were still present, the other activity was much reduced (compare the connective recordings in Fig. 6A and the "Pre" trial of Fig. 5A). The response of the L cells changed considerably also: in two preparations in which intracellular recordings were made from L cells after the lateral connectives were cut, the L cell response was made up entirely of unitary EPSPs, which could be matched one-to-one with the spikes of the S cell (e.g., Fig. 6A). Unitary EPSPs could not be detected in the L cell response when all the connectives were intact; under these conditions the response of the L cell is a complex compound EPSP (e.g., Fig. 5B). Cell 3, as well as VE motor neurons, were no longer activated following lesion of the lateral connectives (data not shown).



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Fig. 6. Selective lesions of the lateral connectives. These experiments were peformed in semi-intact preparations with an intact anterior portion and the nerve cord exposed in the midbody and posterior. A: response of the L cell after transection of the lateral connectives. Diagram of the nerve cord indicates that the lateral connectives had been cut between MG11 and MG12. Unitary EPSPs in the L cell were linked in a 1-to-1 fashion with the spikes of the S cell, which are the large spikes in the connective recording. , spikes in the L cell. B: experiment to corroborate that the S cell accounts for the entire L cell response after transection of the lateral connectives. Two successive trials from the same preparation are shown. In the first trial, the standard anterior skin stimulus was given, and the response of the S cell was recorded. In the following trial, the S cell was stimulated intracellularly to reproduce the response that it gave in the 1st trial (this stimulus is indicated by bars under the S cell recording). C: overlay of L cell responses from 3 successive trials in the experiment shown in B (the 2 trials displayed in B and a further "S cell stimulus" trial). Three traces are positioned relative to each other such that they are aligned just before the start of the EPSP.

These results, taken together, are consistent with the S cell being the only shortening pathway that runs through the medial connective, implying that the other, parallel, pathways all run through the lateral connectives. To further support this, we performed the experiment shown in Fig. 6, B and C. In a preparation in which the lateral connectives had been cut, a standard anterior skin stimulus was given and the responses of the S cell and the L cell were recorded. In subsequent trials, the S cell was stimulated intracellularly to match its response to skin stimulation. As can be seen in Fig. 6C, the L cell responses to the skin stimulus and the S cell stimulus were virtually indistinguishable, indicating that the S cell accounts for the entire input that the L cell receives after lateral connective lesion.

An implication of the S cell network being the only shortening pathway in the medial connective is that transecting this connective or axotomizing the S cell network should be functionally equivalent in terms of their effect on shortening. In support of this, in two preparations with intact anterior and posterior portions in which the medial connective was cut, we observed that the posterior portions still showed strong shortening after the lesion. Hence medial connective lesions can legitimately be used as a method for selectively removing the contribution of the S cell to shortening (e.g., Modney et al. 1997)---a point that was not clear before the present study.

Further investigations of the parallel pathways

The finding that all of the parallel pathways for shortening are located in the lateral connectives signifies that they resemble the large-diameter axons identified histologically by Fernandez (1978) in at least this respect. Because of their large diameter in the more anterior nerve cord, these axons would be expected to produce large-amplitude spikes in the more anterior connectives. To determine if this is true of the parallel pathways, we recorded from the connective at more anterior and posterior locations in preparations in which it was possible to apply anterior and posterior skin stimuli in alternation. An experiment of this type is shown in Fig. 7. Both anterior and posterior stimuli caused activation of the S cell network and also drove other activity in the connective, which could be seen in the extracellular recordings. The key feature of the results is that numerous large-amplitude spikes occurred in the more anterior connective in response to the anterior stimulus, which were so large that they obscured the spikes of the S cell in this recording, whereas the S cell spikes stood out in all the other recordings. This large-amplitude activity in the anterior connective was not seen when the posterior stimulus was applied. The implication is that anterior stimuli activate axons that have larger diameter---and therefore produce larger-amplitude spikes---in the more anterior connective. These axons are selectively activated by anterior, but not posterior, stimuli. Connective response patterns of this nature were observed in three preparations tested in this way. Thus these results further implicate the histologically identified large lateral connective axons as part of the parallel shortening pathways. The results also confirm our hypothesis that the parallel pathways, unlike the S cell network, are selectively activated by anterior stimuli.



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Fig. 7. Comparison of the connective response patterns produced by anterior and posterior skin stimuli. Two successive trials from the same semi-intact preparation are shown. Preparation had intact anterior and posterior body portions and had stimulating electrodes implanted in both the anterior and posterior skin so that alternating anterior and posterior stimuli could be presented. , S cell spikes in each of the recordings. Spikes of the S cell were clearly distinct in all the connective recordings except for the recording between MG6 and MG7 in the "Anterior stimulus" trial; in this case other large-amplitude activity tended to obscure the S cell spikes. S cell spikes in this recording could be identified only (with a fair degree of confidence) because the S cell also was recorded intracellularly and because the S cell conduction times between the recording sites were known. Some of the S cell spikes in the MG6-MG7 connective recording in the "Anterior stimulus" trial were considerably larger than any seen in this recording in the "Posterior stimulus" trial; this is probably because the S spikes summated with other large action potentials. Two trials were seperated by >3 min. All the recordings were maintained constantly throughout.

Role of the parallel pathways in behavioral choice

To test whether the parallel pathways could be responsible for the inhibition of cell 204 during shortening, we employed preparations like those described in the preceding section, in which anterior and posterior stimuli could be applied in alternation while recording from the same cell. Our logic was as follows: if the parallel pathways are selectively activated by anterior stimuli and if some of the parallel pathways inhibit cell 204, then cell 204 should be inhibited selectively by anterior stimuli. In fact, this was the case: anterior stimuli inhibited cell 204, as we have already shown (Shaw and Kristan 1997), but posterior stimuli had the opposite effect, exciting the cell (Fig. 8). This was observed consistently in three preparations in which cell 204 was recorded while anterior and posterior stimuli were presented in alternation. This result provides further (albeit indirect) support for the proposal that some of the parallel pathways may inhibit cell 204 as well as driving motor neurons during shortening. Another piece of evidence for this proposal is that cell 204 inhibition remains after medial connective lesion but is eliminated by lesioning the lateral connectives (data not shown).



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Fig. 8. Comparison of the response of cell 204 to anterior and posterior skin stimuli. Shown are 2 successive trials from the same semi-intact preparation, which had intact anterior and posterior body portions and had stimulating electrodes implanted in both the anterior and posterior skin. Anterior stimulus caused inhibition in cell 204, whereas the posterior stimulus caused excitation. Two trials were seperated by >2 min.

The response latencies of cell 204 to anterior stimuli in these experiments were short, <100 ms from stimulus onset, in agreement with our earlier findings (Shaw and Kristan 1997). The latencies of cell 204 to posterior stimuli, in contrast, were slower, in the range of 131-205 ms (for trials in which precise latencies could be determined). This is in accord with the notion that the inhibitory signals from the anterior are carried by larger-diameter axons. The excitation of cell 204 caused by posterior stimuli could be fairly persistent, lasting <= 10-15 s; this excitation of cell 204 is likely to play a role in the tendency for posterior stimuli to cause swimming in intact animals (Kristan et al. 1982).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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From this study, we have gained a more detailed understanding of the nature and functional roles of the interneuronal pathways that mediate the shortening reflex. While the S cell network participates in shortening---it contributes to the excitation of the L cells, and accounts for the shortest-latency excitation of those motor neurons---its contribution is neither sufficient nor necessary for the reflex to occur. This emphasizes the importance of the other, parallel, pathways. The axons of these parallel pathways are located in the lateral connectives, and their physiological properties indicate that the axons have larger diameter in the more anterior connective. These findings make it likely that these pathways are coextensive, at least in part, with the large axons identified histologically by Fernandez (1978). The parallel pathways are selectively activated by stimulating the anterior of the animal, unlike the S cell network; this is likely to account for the observation that the whole-body shortening reflex is generally elicited by anterior stimuli. Our observation of large-diameter interneuronal axons selectively activated by anterior stimuli may be related to the report that the intersegmental pathways that link more anterior P sensory neurons to the serotonergic Retzius neurons have faster conduction velocities in the anterior region of the nerve cord than do the pathways that link posterior P cells to the Retzius cells (Szczupak and Kristan 1995).

To a degree, the functional organization of the leech shortening circuit resembles that of the network underlying the C start escape response of teleost fish, in which the giant Mauthner cell is activated both by head and tail stimulation, while the serial homologues of the Mauthner cell are selectively activated by head stimulation only (O'Malley et al. 1996). For the fish, this organization presumably allows the strength of the behavioral response to be appropriately matched to the location of the stimulus: head stimuli call for strong C-bends to turn the fish away from the stimulus, whereas tail stimuli require weaker C-bends (Foreman and Eaton 1993). For the leech, it is likely that the parallel-pathway organization of the shortening circuit also allows for behavioral flexibility. In line with this is the observation that the shortening reflex generally is elicited by anterior mechanical stimuli, which selectively activate the parallel pathways, whereas posterior stimuli tend to cause other behavioral responses (Kristan et al. 1982).

Several questions remain to be addressed. Although the axons of the parallel pathways have been localized, the location of their cell bodies is still unknown. This is an important issue because intracellular recording in the leech generally is performed in the soma, and intracellular techniques will be needed to further advance our understanding of these pathways. Related to this is the as yet unanswered question of whether the parallel pathways are made of up of segmentally iterated interneurons that are connected to their homologues in neighboring ganglia, like the S cell network, or whether they consist of long, continuous axons with just one associated cell body at some location in the nerve cord. Another issue concerns behavioral responses to posterior versus anterior stimuli. While the overriding tendency is for posterior stimuli to elicit behaviors other than shortening, there have been reports of posterior stimuli causing some degree of shortening, for example just before the occurrence of swimming (Kristan et al. 1982) or as the initial part of a crawling step (Misell et al. 1998). We would predict that such shortening to posterior stimuli would be mediated by pathways that are distinct, at least in part, from those described in the present study: whereas the S cell network could be involved in such responses, the parallel pathways identified here would not because they are selectively activated by anterior stimuli.

Correlative evidence has been presented that supports the hypothesis that some of the parallel pathways could be responsible for inhibiting the swim-gating cell 204 during shortening as well as driving motor neurons. If this proves to be true, it would indicate that the same neurons that cause one behavior also suppress an incompatible behavior, thus playing a role in behavioral choice. There are several examples of this in another system (Huang and Satterlie 1990; Norekian and Satterlie 1996). To prove the hypothesis conclusively, however, probably will require intracellular identification of the parallel pathway interneurons.

Notably, there does exist a conflict between some of our results and those of a previous study. Baader and Kristan (1995) tested the effects of chronically performed connective lesions on various behaviors and found a strong effect of transecting the medial connective on the shortening reflex: the shortening response largely was interrupted at the site where the medial connective was cut. Currently we are unable to account for this discrepancy. One possibility is that in the previous study, the connective lesions, which were performed under difficult conditions in the whole animal, resulted in damage to the lateral connectives as well as to the medial connective.

Behavioral role of the S cell network

Because of its outstandingly large spike in the connective, the S cell was one of the first interneurons to be identified and studied in the leech (Bagnoli et al. 1975; Frank et al. 1975; Gardner-Medwin et al. 1973). Its part in producing behavior, however, has turned out to be a remarkably small one. It is active during the shortening reflex and contributes to the excitation of the L cells, but this contribution is not necessary for shortening to occur. It is not sufficient to produce shortening by itself even when driven at unphysiologically high rates. On the face of it, it is surprising that the S cell network, which is the closest thing the leech has to a giant fiber system, has so little apparent behavioral importance. One proposed solution to this conundrum is that the function of the S cell is to mediate behavioral plasticity rather than to simply cause behavior (Modney et al. 1997; Sahley et al. 1994). The notion derived from these studies is that the S cell plays a specialized role as a neuron that mediates simple forms of learning.

Another perspective on this issue is provided by a comparative study of Haementeria ghilianii, a leech distantly related to Hirudo. Kramer (1981) reported that Haementeria has an S cell network (homologous to that of Hirudo) that is sufficient by itself to cause whole-body shortening. This raises the interesting possibility that the behavioral efficacy of the S cell network has undergone changes in the evolution of different lines of leeches. Perhaps in the primitive condition the network was a true "command" system for shortening, both sufficient and necessary for the behavior, but as behavioral repertoires changed in evolution the S cell network assumed a less significant role in some leeches.


    ACKNOWLEDGMENTS

We thank R. Satterlie for comments on an earlier version of this manuscript.

This work was supported by a National Science Foundation predoctoral fellowship (B. K. Shaw), National Institutes of Health Training Grant GM-08107 (B.K.S.), and National Institute of Mental Health Research Grant MH-43396 (W. B. Kristan).


    FOOTNOTES

Present address and address for reprint requests: B. K. Shaw, The Neurosciences Institute, 10640 John Jay Hopkins Dr., San Diego, CA 92121.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 July 1998; accepted in final form 27 April 1999.


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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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