Department of Biology, University of California San Diego, La Jolla, California 92093-0357
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ABSTRACT |
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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.
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INTRODUCTION |
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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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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).
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METHODS |
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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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Physiology
Intracellular recordings were made with 30-40 M 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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RESULTS |
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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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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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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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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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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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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
).
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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
shorteningit 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.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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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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REFERENCES |
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