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First published online May 1, 2006
Journal of Experimental Biology 209, 1827-1836 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02212
Common and specific inhibitory motor neurons innervate the intersegmental muscles in the locust thorax
1 Institut für Biologie II, Rheinisch-Westfälische Technische
Hochschule Aachen, Kopernikusstraße 16, D-52074 Aachen,
Germany
2 Abteilung Neurobiologie, Universität Ulm, Albert-Einstein-Allee 11,
D-89081 Ulm, Germany
* Author for correspondence (e-mail: harald.wolf{at}uni-ulm.de)
Accepted 16 March 2006
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Key words: insect, inhibitory motor neuron, neuroanatomy, electrophysiology, locust, Locusta migratoria
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) (see also Wolf,
1990). In keeping with this essential role, both limb (reviewed in
Wiens, 1989) and body wall
(Schmäh and Wolf, 2003a)
muscles receive inhibitory innervation, in addition to their standard
excitatory drive (e.g. Hoyle and Burrows,
1973). Inhibitory innervation is usually by common inhibitory
motor neurons (CIs), which supply several or even all muscles of an appendage
(e.g. Rathmayer and Erxleben,
1983). These common inhibitors function in the adjustment of
muscle performance to behavioural requirements, ranging from postural
maintenance to fast and forceful predatory strikes or escape movements
(Rathmayer, 1998). Such
adjustment is necessary due to the small number, small size and overlapping
composition of arthropod motor units, which are in turn dictated by the
limited potential of muscle fibres for miniaturisation. In fact, the diameters
of muscle fibres are not very different in elephants and fruit flies; adult
skeletal muscle fibres range from 5 to 100 µm in vertebrates, and actually
up to several 100 µm in invertebrates (e.g.
Dudel et al., 2001), endowing
the smaller invertebrates with correspondingly smaller numbers of muscle
fibres.
Contrasting with the ubiquitous occurrence of common inhibitors, specific
inhibitors that innervate just a single muscle are rare. In fact, just two
muscles in the distal leg segments of decapod crustaceans are supplied by
specific inhibitory motor neurons (in addition to their common inhibitory
supply). These are the propodite stretcher and the dactylopodite opener
muscles (Wiersma and Ripley,
1952; reviewed in Wiens,
1989), the muscles that move the two most distal leg segments
dorsally. Peculiarly, these two muscles share their only excitatory motor
neuron. Specific inhibitory innervation is needed, therefore, to uncouple
these two muscles' contractions, as elicited by discharges of the shared
excitor.
In the present study, we provide the first description of specific inhibitory innervation in an insect (the locust) as a representative of the second major arthropod group. It appears, in fact, that we have encountered a situation that may reflect an evolutionary transition from (vestigial) common inhibitory to specific inhibitory supply. The motor neuron that is a specific inhibitor in the first thoracic segment of the locust has segmental homologues in other body segments that are common inhibitors.
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Neuroanatomy and muscles
Nerves of interest were severed, their stumps isolated from the rest of the
preparation in small Vaseline® (Fluka, Buchs, Switzerland) vessels, and
immersed in 0.5–1.5% CoCl2 or NiCl2 (Fluka, Buchs,
Switzerland). After incubation for 18–36 h at 6°C, ganglia and
muscles were removed. Further histological treatment followed standard
procedures for cobalt staining and silver intensification (e.g.
Bräunig, 1997). In some
preparations, nerves were stained using biocytin (Molecular Probes, Eugene,
Oregon, USA) or neurobiotin (Vector Labs, Burlingame, California, USA)
following the procedures described
(Stevenson and Meuser, 1997).
Biocytin and neurobiotin stainings were visualised using CY3-tagged avidin
(Dianova, Hamburg, Germany) at dilutions ranging between 1:500 and 1:2000.
Both staining techniques yielded similar results.
Muscles and nerves are termed according to Snodgrass
(Snodgrass, 1929) and Campbell
(Campbell, 1961)
(Fig. 1). Campbell described
two dorsal longitudinal muscles, the large M81 and the much smaller M82. We
confirm the presence of a third small dorsal longitudinal muscle (see
Fig. 1, top right) described by
Wiesend and termed DLM3 (Wiesend,
1957). In Wiesend's terminology DLM1 corresponds to M81, DLM2 to
M82. DLM3 degenerates in the adult locust.
Immunocytochemistry
Preparations consisted of the pro- and mesothoracic ganglion, the
peripheral branches of the intersegmental nerve (nerve 1) between the two
ganglia from one half of the body, and muscles innervated by these branches.
In 5th instar hoppers, the phragma, which provides the anterior attachment
surface for the main dorsal longitudinal muscle (M81), was cut from the
scutum, the muscle deflected posteriorly, and fixed in this position. In this
way it was possible to expose the underlying small larval muscles
(Wiesend, 1957;
Bernays, 1972).
After dissection, tissues were fixed in situ with GPA (1 part
glutaraldehyde, 25% aqueous solution; 3 parts picric acid, saturated aqueous
solution, and acetic acid to 0.5%) (Boer et
al., 1979) for at least 2 h at room temperature, or overnight at
6°C. In order to improve antibody penetration, the preparations were
dehydrated in an ascending series of isopropanol after fixation, immersed in
propylene oxide for 30 min, and rehydrated again in a descending series of
isopropanol. Subsequently, tissues were rinsed several times in
phosphate-buffered saline (in mmol l–1: NaCl 140, KCl 10,
CaCl2.2H2O 2,
NaH2PO4.H2O 4,
Na2HPO4.2H2O 6) containing 0.5% Triton X-100
(Merck, Darmstadt, Germany) (PBS-TX). Ganglia, nerves and muscles of interest
were removed by further careful dissection and transferred into small Petri
dishes for further processing. Care was taken during all subsequent
histological procedures not to disturb the natural orientation of nerves and
muscles.
To reduce non-specific background staining, tissues were incubated in
blocking medium (PBS-TX containing 2% normal goat serum) (Sigma, Taufkirchen,
Germany) and 5% bovine serum albumin (Jackson ImmunoResearch Lab. Inc., West
Grove, PA, USA) prior to incubation in primary antiserum for at least 1 h. Two
primary antisera were used in the present study, both yielding the same
results. Sera were donated by Sabine Kreissl and Werner Rathmayer, and also by
Hans Agricola (see Blechschmidt et al.,
1988). Primary antiserum was diluted in the same blocking medium
at 1:3000 v/v. Tissues were incubated in primary antiserum for 1–2 days
at 6°C under constant gentle irritation. Immunostainings were developed
using Vectastain® ABC kit (Vector Laboratories, Burlingame, CA, USA).
After thorough washing in PBS-TX (1 day at room temperature) tissues were
incubated overnight at 6°C in biotinylated secondary antibodies (dilution
according to Vectastain protocol). This was followed by a second washing
period of 1 day, this time using phosphate-buffered saline (PBS) without
detergent, and incubation in avidin–horseradish peroxidase complex
(overnight at 6°C, dilution according to Vectastain protocol). After
washing for 4 h in several changes of PBS at room temperature, tissues were
transferred into Tris-HCl-buffered diaminobenzidine solution (DAB; Sigma; 2.5
mg in 10 ml buffer, pH 7.6) for 1 h. After this pre-incubation period, tissues
were transferred into fresh DAB solution, now containing
H2O2 (1 µl per 10 ml buffer). Development was stopped
by washing in Tris-HCl buffer (pH 7.4). Subsequently, preparations were
dehydrated in an isopropanol series, cleared in methyl salicylate, and mounted
in Canada Balsam.
Combining immunocytochemistry and backfilling for double-labelling
The backfill and anti-GABA immunocytochemistry procedures were combined to
double-label those motor neurons in the intersegmental nerve that possess
GABA-immunoreactive cell bodies in the prothoracic ganglion. Neurobiotin was
used for backfilling in these cases, and the backfill was developed with
Cy3-coupled streptavidin (Sigma). Anti-GABA immunocytochemistry was performed
with Cy2-coupled secondary antibody (Dianova, Hamburg, Germany). All other
details were as stated above.
Electrophysiology
Spike activity in nerves was recorded by en passant electrodes,
usually bipolar hook electrodes insulated with Vaseline®. Synaptic
potentials in muscle fibres, both EPSPs and IPSPs, were observed in
intracellular recordings performed with glass microcapillaries. The
microelectrodes were filled with 3 mol l–1 KCl and had tip
resistances of about 30 M
. Electrophysiological data were recorded with
an npi SEC-10L amplifier (Tamm, Germany) and stored on computer disc using a
biologic DRA-800 analogue-digital converter (CED Cambridge Electronic Devices,
Cambridge, UK). For evaluation of electrophysiological recordings, SPIKE2
software (CED Cambridge Electronic Devices) was used. Further details are
described elsewhere (Schmäh and Wolf,
2003a).
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) and for this reason, and to avoid inconsistencies in
the nomenclature, we decided to term the homologous branch innervating M60 and
M61 N1A, also, although in no case did it actually originate from N1. N1A divides further into two branches: N1A1, innervating M60, and N1A2, innervating M61. The GABA-immunoreactive axon proceeded into N1A1 and innervated M60 (N=11). In well-stained preparations the axon could be observed to subdivide further on the muscle and to form blebby terminal ramifications on the muscle surface (similar to the situation shown in Fig. 2C for M59, below). We never observed GABA-immunoreactive terminals on the fibres of M61, nor any stained axon or axon collateral in N1A2 (N=16).
The other two axons proceeded further in N6. After this nerve had fused with N1 of T2 (the two nerves forming the intersegmental nerve proper), both axons ran into N1B, which is also called the paramedian nerve (Fig. 2B). Nerve 1B may originate from N1 individually or it may form a common root with N1C (the nerve innervating the wing; the latter situation is depicted in Fig. 2B). N1B runs anteriorly, innervates M59 (usually with two distinct side branches), and proceeds further into the neck region. About half way between its origin from N1 and its side branches innervating M59, N1B forms an anastomosis with the transverse nerve. Within N1B and its side branches the two axons could be followed into M59 where they further divided and formed terminals on fibres of this muscle (Fig. 2C) (N=6).
In no case could we observe the immunoreactive axons in N1B to proceed
beyond M59 (N=16), which confirms the result of the
electrophysiological examination described below. These axons (and the
anterior segment of N1B) may thus be eliminated as potential sources of
inhibitory innervation to neck muscles
(Bräunig, 1997). Likewise,
the axons did not form axon collaterals to enter the transverse nerve that
innervates the spiracular muscles. One of the two GABA-immunoreactive fibres,
however, formed a collateral, which proceeded into nerve 1D
(Fig. 2B) (N=8). Nerve
1D innervates the dorsal longitudinal muscles (DLMs) (N1D1) and
sense organs associated with the wing hinge (N1D2). This axon
collateral was rather thin when compared to the main axons proceeding towards
M59 (Fig. 2B). In 5th instar
larvae, this collateral could be followed to proceed into N1D1 (and
not into the sensory N1D2) and into its side branches, which
innervate M81, the major dorsal longitudinal muscle, and M82. In a few
preparations, GABA-immunoreactive terminals could be observed on these
muscles. In no case was there any indication that DLM3 (see
Fig. 1)
(Wiesend, 1957), a larval
muscle that degenerates in the adult, was innervated by this axon.
Neuroanatomy
The results obtained by staining the intersegmental nervous system with
anti-GABA antisera, as described above, may be summarised as follows. Three
immunoreactive axons leave the prothoracic ganglion. One of these enters
N1A1 and innervates M60; the two remaining axons proceed into N1
and further distally into N1B to innervate M59. One of these two axons sends a
collateral towards the dorsal longitudinal muscles and innervates M81 and M82,
though not DLM3 (see summary in Fig.
6).
In order to scrutinise these results, individual motor nerves were stained using cobalt chloride, nickel chloride, biocytin or neurobiotin. In cases of common innervation, staining the motor nerve of one muscle should result in stained terminal ramifications on muscles innervated by the same (common) neuron. This backfilling technique can also reveal numbers and locations in the CNS of the motor neuron somata giving rise to these axons and collaterals.
Backfills of the motor nerve to M59
In backfills of the motor nerve that supplies M59 (branches of N1B), one
axon collateral was seen clearly in nerve 1D (N=49), and in
well-stained preparations (N=9) this collateral could be followed to
its terminal ramifications on M81 and M82
(Fig. 3B). Vice versa,
when staining N1D (N=4), an axon collateral was observed to innervate
M59 via N1B.
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). In six
preparations this DUM neuron, termed DUM1B, with its soma located in the
mesothoracic ganglion, stained exceptionally well such that its ramifications
could be followed individually. These backfills confirmed the peripheral
branching pattern as revealed previously by intracellular staining
(Bräunig, 1997): DUM1B
innervates M60, M61 and M59 but it does not project into N1D.
In the present backfills of the motor nerve to M59 (N=34) at least
four axons were discernible, with diameters much larger than DUM1B. Two of
these entered N1 of the mesothoracic ganglion (together with the axon of
DUM1B), another two entered N6 of the prothoracic ganglion. The fibres
entering N1 could be followed to two mesothoracic somata and presumably belong
to the excitatory motor neurons to M59; they are clearly discernible by their
anterior and contralateral soma location
(Steffens and Kutsch, 1995).
The axons in N6 originated from two somata in the prothoracic ganglion. These
cell bodies occupied a conspicuous posterior ventral position, just
contralateral to the ganglion midline. They exhibited looping primary neurites
in preparations where internal structures of the ganglia were discernible
(Fig. 3A; arrowheads indicate
primary neurites). One of the two axons in N6 formed a small-diameter
collateral entering N1A1 (Fig.
3C), which projected to M60 and formed terminal ramifications in
the anterior medial region of this muscle. In contrast to the less intensely
labelled axons of the DUM1B neuron this collateral did not project into
N1A2 and to M61.
Backfills of the M60 motor nerve
Backfills of the nerve supplying M60 (N=13) often (N=9)
labelled a collateral axon in N1B and the nerve branches innervating M59. This
confirms the common innervation of muscles M59 and M60 observed in the
backfills of M59. The common axon appeared distinctly thicker than that of
DUM1B described above. Occasionally, an additional much smaller axon was
observed (N=3). In these preparations a thin axon proceeding through
mesothoracic N1 and/or a posterior medial soma in the mesothoracic ganglion
was also present, indicating that this thin axon belonged to DUM1B.
We did not observe axon collaterals in the nerve supply of M61, other than a very thin axon (N=4), to be interpreted as a DUM1B collateral according to the criteria mentioned above.
Six to nine axons were counted in N6 of the prothoracic ganglion in backfills of the M60 motor nerve. This variability was apparently produced by a number of small-diameter axons, presumably belonging to a corresponding number of similarly small somata in the prothoracic ganglion, that were sometimes labelled, and sometimes not. The larger cell bodies (30–45 µm diameter) were labelled reliably (N=13). These were four somata in the ipsilateral ventral soma cortex, which presumably belong to the excitatory motor neurons of M60, and two posterior somata near the ganglion midline, which gave rise to looping primary neurites where discernible (compare Fig. 3A, arrowheads). Three smaller cell bodies (20 µm or even smaller) located ventrally on the ganglion midline were stained occasionally. In addition, a DUM soma was labelled in the mesothoracic ganglion of well-stained preparations. According to the above description this was DUM1B.
Backfills of the M61 motor nerve
The motor supply of M61 was backfilled to scrutinise the results reported
above, in particular, the lack of common innervation with muscles 59 and 60,
except for DUM1B. These backfills (N=21) corroborated the above data.
Two axons were reliably observed in the nerve supply of M61, running through
N1A2, prothoracic N6, and mesothoracic N1 into the mesothoracic
ganglion to connect to two cell bodies located anteriorly and contralaterally.
These two somata were reminiscent of the excitatory motor neurons that supply
M59, and they presumably represent the excitatory motor neurons to M61. In a
few (N=9) well-stained preparations DUM1B was also labelled, with the
characteristics outlined above, namely, a very thin axon supplying M59 and
occasionally M60.
Double-labelling by combined backfills and immunocytochemistry
The cell bodies of putative inhibitory motor neurons were identified by
combining anti-GABA immunochemistry with backfills of (motor) nerves
(N=13). Backfills of prothoracic nerve 6 labelled the cell bodies of
all neurons noted above, except those located in the mesothoracic ganglion
(i.e. the excitors of M59 and M61, and DUM1B). Processing such backfill
preparations for anti-GABA immunocytochemistry produced a double label in
those neurons that have an axon in nerve 6 and are GABA-immunoreactive, and
thus are presumptive inhibitory motor neurons. This is illustrated in
Fig. 4, which presents an area
of the prothoracic ganglion just contralateral to the ganglion midline, with
regard to the filled nerve. Four somata in the centre of the image show red
fluorescence and thus have axons in N6
(Fig. 4Ai; in a sample cross
section of the same ganglion area, though from a different animal, only two of
these somata are visible, Fig.
4Aii). Three of the four cell bodies are also marked by anti-GABA
immunocytochemistry, that is, green fluorescence
(Fig. 4Bi,Ci; cross section in
Fig. 4Bii,Cii). These are the
three presumptive inhibitory motor neurons described above, namely, the cells
with looping primary neurites and somata contralateral to the ganglion midline
and posterior ventral soma locations. The fourth cell body located in close
proximity to these three inhibitors was regularly marked in backfill
preparations. It never stained for the inhibitory transmitter GABA,
however.
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All backfilled cells were visible, of course, in the whole-mount preparations (Fig. 4Ai,Bi,Ci). Comparison with sectioned ganglia revealed double-labelling more accurately (Fig. 4Aii,Bii,Cii) in cases where superpositions like in Fig. 4Ci appeared ambiguous. For instance, the most posterior backfilled cell body in Fig. 4Ai is not labelled by the GABA antiserum (marked by asterisk). This is discernible by a detailed comparison of soma locations between Fig. 4Bi and 4Ci (see arrowheads). And it holds true despite an overlay of green fluorescence with the red backfill label, with the two labels occupying different depths (Fig. 4Ci). In cross sections no such ambiguity is present (the same is true of optical sections in the confocal microscope, of course; not shown).
Electrophysiology
The results described above show that, in the intersegmental nervous
system, there are neurons that innervate more than one muscle, in addition to
the DUM1B neuron [and possible axon collaterals of an
allatostatin-immunoreactive cell (Kreissl
et al., 1999); we never encountered any indication for those in
our preparations]. One neuron clearly innervates M59, M81 and M82. It was not
unequivocally clear, however, whether this neuron also forms the collateral
innervating M60, or whether this innervation originates from the second
prothoracic cell, which stains after filling the M59 motor nerve. To resolve
this matter conclusively, we further examined the innervation of muscles 59,
60, 61 and 81 using electrophysiological techniques. These experiments also
elaborated and scrutinised the other results obtained with the
immunocytochemical and neuroanatomical techniques.
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In some preparations, the inhibitory units were spontaneously active (cf.
Schmäh and Wolf, 2003a).
In such cases (N=2), two units were recorded from the motor nerve
supplying M59. Both were followed in a 1:1 fashion by IPSPs of distinct shapes
and amplitudes in the muscle fibres (see also
Fig. 5B,D; see figure legend
for details of the experimental situation). Only one of these units could also
be recorded in N1D1, the motor nerve to the DLMs
(Fig. 5A). This was the unit
producing the larger-amplitude IPSPs in M59. These results indicate that two
inhibitory units innervate M59. One of these also innervates the DLMs but not
M60, since stimulation of N1D never elicited phase-locked spikes in the motor
nerve to M60 (see above).
In a corresponding set of experiments, we observed common inhibitory innervation of muscles 59 and 60 (N=5) (Fig. 5C). By inference from the above data, this had to be the unit not common to muscles 59 and 81/82. Common innervation of M59 and M60 was most clearly illustrated by paired intracellular recordings from fibres of these two muscles. IPSPs occurred in 1:1 relationship in both recordings (Fig. 5D; dotted reference lines). In keeping with the above data, this (common) inhibitory innervation to M59 gave rise to the smaller-amplitude IPSPs (while the common innervation to M59 and M81 generated the larger-amplitude IPSPs in M59, above; and note arrowheads). Stimulating the motor nerve to M59 corroborated these findings. IPSPs occurred in M60 in a 1:1 fashion with regard to the stimulus (Fig. 5C). It is particularly noteworthy in the present context that IPSPs of different amplitude, and not related to the stimulus on the motor supply of M59, often occurred in the intracellular recordings from M60 fibres (arrowhead in Fig. 5C). This is strongly indicative of double inhibitory innervation of M60, with only one of these inhibitory motor neurons being common to M60 and M59.
IPSPs were never recorded in fibres of muscle 61, despite probing several muscle fibres in each of the 6 preparations examined.
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All the three techniques that we applied – immunocytochemistry,
neuroanatomy, electrophysiology – alone and in combination, support this
interpretation (summarised in Fig.
6). Backfills of the prothoracic part of the intersegmental nerve
(nerve 6), apart from numerous ispsilateral somata, which presumably belong to
excitatory motor neurons (cf. Kutsch and
Heckmann, 1995), marked four somata, which are located ventrally
in the ganglion near the midline, but contralateral to the filled nerve. The
primary neurites leave these somata in characteristic loops, which had already
been noted in previous reports (Kutsch and
Heckmann, 1995; Schmäh
and Wolf, 2003a). Three of the four contralateral somata are
relatively small with diameters ranging from 20 to 25 µm. In
double-labelling experiments these three neurons exhibit GABA-like
immunoreactivity. The fourth soma is somewhat larger (close to 30 µm
diameter) and does not stain with GABA antisera. It appears to supply M60
(data not shown) (cf. Kutsch and Heckmann,
1995).
The existence of three GABA-immunoreactive cells is also demonstrated by
immunocytochemistry in the periphery. Three axons are visible in N6; one of
these innervates M60, the other two proceed further into N1B to innervate M59.
One of the latter two axons branches into N1D to supply the DLMs. The other
axon of the two should form a collateral innervating M60, according to the
neuroanatomical and electrophysiological data. However, this was never
observed with immunocytochemical staining. This additional collateral showed
up only in backfills and here it was distinctly smaller than the main axon in
N6 (Fig. 3C). It has previously
been reported that very small axons show variable staining in peripheral
nerves with anti-GABA immunocytochemistry
(Watson and Pflüger,
1987).
Not all muscles innervated by branches of the intersegmental nerve receive GABA-immunoreactive inhibitory innervation. One example is DLM3, and the other exception is M61. In the case of DLM3, not one of the more than 50 backfills from M59 revealed labelled axons in its specific motor branch (a small side branch of N1D1; see Fig. 1), nor terminals on the muscle. It is very unlikely that this is due to a failure of the backfill technique since axon collaterals and terminals were clearly stained in the case of M82, which is located much more dorsally than DLM3, that is, farther away from the site of the nerve fill.
The case of M61 is less clear. A previous study of the intersegmental
muscles plainly shows a prothoracic neuron, resembling one of the inhibitors
described here, stained in a backfill from the motor nerve to M61
(Kutsch and Heckmann, 1995).
By contrast, we could not find any indication for an inhibitory innervation of
this muscle in the present study. There are several possible explanations for
this discrepancy. First, the innervation pattern might differ between various
locust strains (compare e.g. Goodman et
al., 1974) for differences in neuronal arborisations between
breeding strains). Second, there might be differences between individuals.
That is, occasionally M61 does receive an axon collateral from one of the two
inhibitors that innervate M60, and sometimes it does not. The fact that we
never observed inhibitory innervation of M61 in 70 preparations would seem to
argue against this interpretation. Third, there might be problems caused by
the staining technique, for instance, dyes entering axons damaged in the
course of dissection. In this respect, researchers often tend to interpret
backfill data with a certain bias, regarding preparations that exhibit the
maximum number of neurons as being the most representative. This is based on
the assumption that in all the other preparations particular neurons have
failed to stain (which may indeed easily happen, depending on the nerve
studied). This interpretation, however, is problematic when axons are exposed
for prolonged periods to cytotoxic staining solutions such as heavy metal
salts.
Assuming that there is indeed an occasional innervation of M61 by one of the inhibitors, the question arises which neuron sends axons or axon collaterals into N1A in the respective cases. There are two possible candidates: the common inhibitor CI59/81/82, and the specific inhibitor, SI60. In the latter case, the specific inhibitor would turn into an occasional third common inhibitory neuron. This raises the interesting possibility that this first specific inhibitor ever found in insects might represent a common inhibitor, too, the number of its targets being in the process of reduction to just one muscle, for unknown reasons.
In the remaining body segments of the locust, there are just two inhibitory
motor neurons that supply the body wall muscles
(Schmäh and Wolf, 2003a).
What are the segmental correspondencies between these neurons and the three
prothoracic inhibitors? That is, which are the segmental homologues, if any,
of CI59/60, CI59/81/82 and SI60? The
morphologies of all three inhibitory motor neurons in the prothoracic ganglion
are rather similar (although SI60 appears to possess the smallest
cell body of the three inhibitors), thus offering few clues for specific
homologisation. Their morphologies also agree closely with those of the
previously described body wall inhibitors in other body segments: `loop cell'
(Kutsch and Heckmann, 1995),
corresponding to CIa, and `common contralateral A-type cell'
(Steffens and Kutsch, 1995),
corresponding to CIb. That is, they feature small somata, compared
to those of other motor neurons, located in the posterior half of the ventral
soma cortex close to the ganglion midline and just contralateral with regard
to their axons; and the primary neurites exhibit characteristic loops before
crossing the midline (see Schmäh and
Wolf, 2003a).
Thus, it is the inhibitors' innervation patterns which provide the main
characteristics that one may employ for segmental comparison. According to
these criteria, CI59/81/82 corresponds to CIa. This
homology appears safe to infer, based in particular on the (common)
innervation of the dorsal longitudinal muscles, which are the most clearly
similar and homologous between different body segments. By the same token,
when considering the (common) innervation of ventral body wall muscles,
CI59/60 may correspond to CIb. Due to the more derived
structure of M59 and M60, however, this inference is more tentative. For one
of these muscles, M60, segmental homology with ventral longitudinal muscles in
the more posterior segments is safe to assume
(Steffens and Kutsch, 1995),
supporting homology of the inhibitory innervation. However, M60 is supplied
not only by CI59/60 but also by SI60. This makes the
assignment of homology problematic, although the common innervation exhibited
by CI59/60 argues for correspondence to CIb.
SI60, thus, has no corresponding neuron in the other body segments,
at least in the locust.
Under an evolutionary perspective, the plesiomorphic condition appears to
be the presence of three body wall inhibitors per segment. The prothoracic
ganglion is the only ganglion of the locust in support of this hypothesis. The
abdominal ganglia of the dragonfly, by contrast, regularly possess three body
wall inhibitors per segment (Schmäh
and Wolf, 2003b; M.S. and H.W., unpublished data). Three body wall
inhibitors also seem to be present in the abdomen of bushcrickets and crickets
(Consoulas and Theophilidis,
1992; Consoulas et al.,
1993). Perhaps this holds even for the cricket thorax
(Böser, 1999).
As indicated above, specific inhibitors may originate from common
inhibitory motor neurons by restriction of their innervation field to just a
single muscle. The latter may occur due to the absence of target muscles, for
example, when muscles are lost in the course of segment fusion
(tagmatisation). The sturdy thoracic tagma of locusts has quite obviously
entailed such muscle loss; compare, for instance, the thoracic muscle
endowment of locusts (Snodgrass,
1929) and crickets with their much more moveable thorax
(Du Porte, 1920). The apparent
presence of three body wall inhibitors per segment in the cricket thorax
(Böser, 1999; M.S. and
H.W., unpublished data) seems to point in this direction. Perhaps the most
plausible explanation for a possible occasional common innervation of M61
– as discussed above – would be an axon collateral of
SI60. That is, SI60 would not just innervate M60 but
send an occasional, and perhaps vestigial, collateral through N1A2.
This situation would represent the ongoing loss of inhibitory innervation in a
very small muscle (M61) that spans between fused sclerites that can no longer
be moved relative to each other (spine of spinasternum and sternal apodeme).
This scenario appears more parsimonious than an attempt to infer some
functional necessity for a specific inhibitory neuron in just one of the
numerous longitudinal intersegmental muscles.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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