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First published online April 18, 2008
Journal of Experimental Biology 211, 1434-1447 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016998
The pyloric neural circuit of the herbivorous crab Pugettia producta shows limited sensitivity to several neuromodulators that elicit robust effects in more opportunistically feeding decapods
1 Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME
04011, USA
2 Friday Harbor Laboratories, University of Washington, 620 University Road,
Friday Harbor, WA 98250, USA
3 Department of Chemistry, Bowdoin College, 6600 College Station, Brunswick, ME
04011, USA
4 Department of Biology, University of Washington, Box 351800, Seattle, WA
98195-1800, USA
5 Mount Desert Island Biological Laboratory, PO Box 35, Old Bar Harbor Road,
Salisbury Cove, ME 04672, USA
* Author for correspondence (e-mail: pdickins{at}bowdoin.edu)
Accepted 21 February 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: stomatogastric nervous system, neurohormone, neuropeptide, amine, feeding
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Harris-Warrick et al., 1992;
Nusbaum and Beenhakker, 2002;
Marder and Bucher, 2007).
Working in concert, these rhythmic movements allow food items to be ingested,
chewed, and ultimately filtered from this portion of the digestive tract
(Selverston and Moulins, 1987;
Harris-Warrick et al., 1992;
Nusbaum and Beenhakker, 2002;
Marder and Bucher, 2007). In
the decapod species examined thus far (i.e. the crab Cancer borealis,
the chelate lobsters Homarus americanus and Homarus gammarus
and the spiny lobster Panulirus interruptus), the STNS neural
circuits are extensively modulated both by locally released and circulating
substances (Selverston and Moulins,
1987; Harris-Warrick et al.,
1992; Nusbaum and Beenhakker,
2002; Marder and Bucher,
2007). The result of this modulation is the expression of a large
suite of distinct motor patterns that is hypothesized to allow these highly
opportunistic feeders to process multiple food types.
Unlike most decapod species, the Northern kelp crab Pugettia
producta is reported to be a dietary specialist, feeding almost
exclusively on kelp (Hines,
1982). If the extensive STNS modulation previously reported for
generalist feeders is an evolutionary consequence of a need to process highly
variable food types, then this system in P. producta may need less
modulatory control to process its relatively uniform diet. To test this
hypothesis, we investigated the distribution of a number of well-known and
highly conserved crustacean neuromodulators in the STNS and neuroendocrine
organs (i.e. the sinus gland and the pericardial organ) of P.
producta and tested their physiological actions on the pyloric motor
pattern of this species. Some of these data have appeared previously in
abstract form (Dickinson et al.,
2004).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Tissue collection
For the collection of tissues, crabs were anesthetized by packing in ice
for 30–60 min. After anesthetization, the dorsal carapace was removed
and the foregut was dissected free. The eyestalks and lateral walls of the
pericardial chamber were also isolated at this time. To obtain the STNS
(Fig. 1), which includes the
paired commissural ganglia (CoG), the esophageal ganglion (OG), the
stomatogastric ganglion (STG), as well as a number of interconnecting and the
motor nerves, the foregut was flattened by making a longitudinal cut from the
esophagus to the pylorus on its ventral side, followed by a pair of medial
cuts directed along the ossicles of the cardiac sac/gastric mill. The teeth of
the gastric mill were then removed and the flattened foregut was pinned,
inside down, in a wax- or Sylgard-lined Pyrex dish containing chilled
(10°C) physiological saline (composition in mmol l–1:
NaCl, 440.0; KCl, 11.0; CaCl2, 13.0; MgCl2, 26.0; Trizma
base, 12.0; maleic acid, 1.22; pH 7.4–7.5). The STNS was dissected free
in chilled physiological saline. To obtain the sinus gland, the carapace
encasing an eyestalk was split both dorsally and ventrally and one half of the
split shell was gently teased away from the other half. The remaining half of
the eyestalk was then pinned in a wax-lined Pyrex dish filled with chilled
physiological saline and the eyestalk ganglia, to which the sinus gland is
affixed, were subsequently isolated. To obtain the pericardial organ, the
isolated walls of the pericardial chamber were pinned in a wax-lined dish
filled with chilled physiological saline and the nerve roots constituting this
endocrine site were dissected free from the surrounding connective tissue.
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). As a general marker for regions of synaptic neuropil,
a mouse monoclonal antibody generated against a glutathione S-transferase
fusion protein that included a portion of a Drosophila synapsin
homolog (code SYNORF; kindly provided by Dr E. Buchner, Universität
Würzburg, Würzburg, Germany)
(Klagges et al., 1996) was
used at a final dilution of 1:100. To assay tissues for the presence of
Cancer borealis tachykinin-related peptide I (CabTRP I), a rat
monoclonal antibody generated against substance P (clone NC1/34 HL; Abcam
Incorporated, Cambridge, MA, USA; catalog no. ab6338)
(Cuello et al., 1979) was used
at a final dilution of 1:300. To assay tissues for the presence of crustacean
cardioactive peptide (CCAP), a rabbit polyclonal antibody generated against
this peptide (Dircksen and Keller,
1988; Stangier et al.,
1988) (kindly provided by Dr H. Dircksen, Stockholm University,
Stockholm, Sweden) was used at a final dilution of 1:500. To assay tissues for
the presence of the peptide proctolin, a rabbit polyclonal antibody to
proctolin (code K9832/13; kindly provided by Dr D. Nässel, Stockholm
University) (Johnson et al.,
2003) was used at a final dilution of 1:500. To assay tissues for
the presence of red pigment concentrating hormone (RPCH), a rabbit polyclonal
antibody generated against this peptide
(Madsen et al., 1985) (kindly
provided by Dr R. Elde, University of Minnesota, Minneapolis, MN, USA) was
used at a final dilution of 1:300. To assay tissues for the amine dopamine, a
mouse monoclonal antibody generated against tyrosine hydroxylase (Immunostar
Inc., Hudson, WI, USA; catalog no. 22941), the biosynthetic enzyme of dopamine
was used at a final dilution of 1:1000. The secondary antibodies used in our
experiments were donkey anti-rabbit IgG conjugated to Alexa Fluor 488
(Molecular Probes, Eugene, OR, USA; catalog no. A-21206) or Alexa Fluor 594
(Molecular Probes; catalog no. A-21207); donkey anti-mouse IgG conjugated to
Alexa Fluor 488 (Molecular Probes; catalog no. A-21202) or Alexa Fluor 594
(Molecular Probes; catalog no. A-21203) or donkey anti-rat IgG conjugated to
Alexa Fluor 488 (Molecular Probes; catalog no. A-21208) or Alexa Fluor 594
(Molecular Probes; catalog no. A-21209).
Confocal and epifluorescence microscopy
After immunolabeling, preparations were viewed and data collected using one
of two Bio-Rad MRC 600 laser scanning confocal microscopes (Bio-Rad
Microscience Ltd, Hemel Hempstead, UK), a Bio-Rad Radiance 2000 laser scanning
confocal microscope or a Nikon Eclipse E600 epifluorescence microscope.
Descriptions of the hardware and software used for imaging on these systems
have been extensively described in previous publications
(Christie et al., 1997a;
Messinger et al., 2005;
Christie et al., 2007).
Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry
For direct tissue matrix-assisted laser desorption/ionization-Fourier
transform mass spectrometry (MALDI-FTMS), STGs and sinus glands were analyzed
as freshly dissected tissue samples; pericardial organs were stored in
acidified water and frozen prior to analysis. STGs or sinus glands were
isolated as described earlier, removed from the saline with fine forceps,
rinsed sequentially in two 25 µl droplets of 0.75 mol l–1
fructose (Sigma-Aldrich, St Louis, MO, USA) and then placed on a face of a
ten-faceted probe tip, minimizing co-transfers of solution. STGs were left
intact (with the exception of removal of the ganglionic sheath), as were sinus
glands. Pericardial organs were thawed, rinsed with fructose, and cut into
pieces before being applied to one face of a ten-faceted probe. Once on the
probe, the tissue was sliced 10–20 times with a 0.2 mm needle; the
macerated tissue was then gathered together and covered with a 0.5 µl
droplet of 1.0 mol l–1 2,5-dihydroxybenzoic acid (DHB;
SigmaAldrich; 98%, sublimed prior to use), prepared in 1:1 acetonitrile
(Fisher Scientific, Pittsburgh, PA, USA; HPLC grade) and water containing 0.1%
(v/v) trifluoroacetic acid (SigmaAldrich, 99%). All samples were analyzed
using a HiResMALDI Fourier transform mass spectrometer (IonSpec, Lake Forest,
CA, USA) equipped with a 4.7 Tesla actively shielded superconducting magnet
(Cryomagnetics, Oak Ridge, TN, USA) as described previously
(Christie et al., 2006).
Electrophysiology
For physiological recordings, the STNS was dissected and pinned out in a
Sylgard-lined Petri dish as described above, with the motor nerves and the
nerves interconnecting the ganglia left intact. In addition, the sheath over
the STG was removed to provide access to the cell bodies of neurons contained
within the ganglion, and a section of sheath around the stomatogastric nerve
(stn) was removed so that action potential conduction could be
reversibly blocked using isotonic (750 mmol l–1) sucrose in a
petroleum jelly well surrounding this desheathed area of nerve. This sucrose
block eliminated all modulatory inputs to the STG, since the stn is
the only nerve that carries inputs from the CoGs and OG to the STG. During
recordings, the dish containing the STNS was constantly superfused with
chilled (10–12°C) physiological saline at a rate of 2–3 ml
min–1. It should be noted that a number of different saline
compositions, based on those used in other species, were tested in preliminary
experiments. The saline used here was based on Cancer borealis saline
(Hooper et al., 1986), and was
chosen because it gave the most robust activity and was the one in which
recovery from stn block was quickest and most complete. Modulators
were made up immediately before use, and were added via a manual
switching port to the superfusion system. The peptides CabTRP I
(APSGFLGMRamide; synthesized by the Cancer Research Center of the University
of Pennsylvania School of Medicine and kindly provided by Dr M. Nusbaum,
Department of Neuroscience, University of Pennsylvania School of Medicine,
Philadelphia, PA, USA), CCAP (PFCNAFTGCamide; Bachem AG, King of Prussia, PA,
USA; catalog no. H-6745), proctolin (RYLPT; Sigma-Aldrich; catalog no. P4280)
and RPCH [pELNFSPGWamide; Bachem Biosciences, Inc., King of Prussia, PA, USA;
catalog no. H-6750 (dissolved first in 7% dimethyl sulfoxide)] were each
reconstituted and stored frozen as stock solutions at 10–3
mol l–1, then diluted. Oxotremorine (Sigma-Aldrich; catalog
no. O-9126) and dopamine (Sigma-Aldrich; catalog no. H-8502) were dissolved
directly in the saline.
Neuronal activity was recorded extracellularly using standard electrophysiological techniques. Specifically, activity on the motor nerves was recorded via A-M Systems Model 1700AC amplifiers (A-M Systems, Inc., Carlsborg, WA, USA) using stainless steel pin electrodes, which were isolated from the bath with petroleum jelly wells. All electrical activity was further processed with a Brownlee 410 instrumentation amplifier (Brownlee Precision Co., San Jose, CA, USA) and recorded directly onto a PC computer via a Micro 1401 board and Spike 2 software (Cambridge Electronic Design, Cambridge, UK). Data were processed using Spike 2 and further analyzed with Prism4 (GraphPad Software, Inc., San Diego, CA, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Skiebe and
Wollenschlager, 2002). Using this antibody, we mapped the
distribution of putative synaptic neuropil in the STNS of P. producta
and found it to be essentially identical to that reported previously for other
brachyuran species (Skiebe and Ganeshina,
2000; Skiebe and
Wollenschlager, 2002). Specifically, synapsin labeling was
routinely seen in the CoGs and the STG, but not in the OG. In the CoGs and
STG, all staining was confined to the neuropil, with little or no label seen
in or around the intrinsic somata. In the CoGs, the synapsin label appeared to
fill the entire volume of the neuropilar region with only a few sites of
apparent avoidance. Within the STG, synapsin staining was confined primarily
to the peripheral portion of the neuropil, with the central core of the
structure relatively devoid of immunoreactivity. Extraganglionic patches of
synapsin labeling were also present in each superior esophageal nerve
(son; near the junction of the dorso-posterior esophageal nerve;
dpon), at the junction of the sons, esophageal nerve
(on) and the stomatogastric nerve (stn), as well as in the
anterior portion of the stn proper. Regardless of location, the
labeling was found in small blob-like varicosities. The distribution of
synapsin-like labeling in the P. producta STNS is shown schematically
in Fig. 1.
Immunohistochemical survey for putative neuromodulators in the stomatogastric ganglion and neuroendocrine organs
The stomatogastric ganglion
The results of our synapsin immunostaining showed that among the putative
synaptic neuropils of the P. producta STNS is one located within the
STG. In other decapod species, local release of substances from the STG
neuropil modulates the output of the pyloric motor pattern, which is produced
by a neural circuit contained within the ganglion
(Selverston and Moulins, 1987;
Harris-Warrick et al., 1992;
Nusbaum and Beenhakker, 2002;
Marder and Bucher, 2007). To
determine if several well-known and highly conserved neuromodulators were
present in the STG of P. producta (i.e. the amine dopamine and the
peptides CabTRP I, CCAP, proctolin and RPCH), we immunolabeled the STNS of
this species with antibodies to these substances or to their biosynthetic
enzyme. Although each of these antibodies produced labeling within the STNS,
only those used to detect CabTRP I, proctolin and RPCH labeled the STG
neuropil (Table 1). For each of
these antibodies, labeling in the ganglion appeared to originate from input
axons descending from the anterior ganglia (i.e. the CoGs and/or OG).
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Neuroendocrine organs
In addition to locally released neuroactive substances, the output of
circuits within the STG is also known to be modulated by hormones released
from several neuroendocrine organs located outside the STNS, specifically the
sinus gland of the eyestalk and the pericardial organ that surrounds the heart
(Christie et al., 1995;
Marder et al., 1995;
Skiebe, 2001). To determine
whether any of the above mentioned compounds might reach the P.
producta STG neuropil via a hormonal route, we immunolabeled
both the sinus gland and the pericardial organ of this species for each of the
substances. In the sinus gland, only the proctolin and the RPCH antibodies
stained putative endocrine release terminals
(Table 1). Within the
pericardial organ, the CCAP, proctolin, RPCH and TH antibodies each
immunolabeled an extensive set of release sites
(Table 1). No CabTRP I-like
labeling was found in either the sinus gland or pericardial organ
(Table 1).
Mass spectrometric analysis of native peptides
The results of our immunohistochemical surveys suggested the presence of
CabTRP I, proctolin and RPCH in the STG, proctolin and RPCH in the sinus
gland, and CCAP, proctolin and RPCH in the pericardial organ. To confirm the
presence of authentic peptide in these tissues, we conducted direct tissue
MALDI-FTMS analyses on isolated tissue samples from each of these structures.
We found peaks corresponding to the [M+Na]+ ion of authentic RPCH
(Stemmler et al., 2006) in the
sinus gland, as well as [M+H]+ ions corresponding to authentic
CabTRP I and authentic CCAP in the STG and the pericardial organ, respectively
(Fig. 2;
Table 2). By contrast, and in
spite of the fact that we saw proctolin-like immunoreactivity in the STG,
sinus gland and pericardial organ, we did not find a peak corresponding to the
mass of authentic proctolin in any of the tissue samples we tested from those
regions.
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Expression of the pyloric motor pattern is dependent on modulatory input from anteriorly located sources
The pyloric motor pattern in P. producta strongly resembled that
recorded in other decapod species (Fig.
3). The core pyloric pattern consisted of bursts in the two
pyloric dilator (PD) neurons, followed by a brief silent period before bursts
in the lateral pyloric (LP) neuron, then pyloric (PY) neurons. Bursts in the
inferior cardiac (IC) neuron began soon after the end of the PD burst, and
overlapped the LP burst, whereas those in the ventricular dilator (VD) neuron
began at the end of each PY burst, and overlapped the next PD burst. However,
the burst period in this species was considerably longer than has been
reported for other decapods: average burst period in P. producta was
2.9±0.32 s (N=15), whereas it is approximately 1 s in the
spiny lobster Panulirus interruptus
(Selverston et al., 1976) and
in the crab Cancer borealis
(Hooper et al., 1986), and
about 1.5 s in the lobsters Homarus americanus and Homarus
gammarus (Meyrand et al.,
1991; Richards et al.,
1999; Mizrahi et al.,
2001). In addition, we noted that the pattern in P.
producta was highly dependent on input from the anterior ganglia.
Blocking the stn with isotonic sucrose eliminated all pyloric
bursting within 15 min in all but two preparations (N=22). With the
exception of the PY neurons, which fired tonically in sucrose block, the
pyloric neurons were largely silent, although there were occasional spikes in
many of the neurons (e.g. in the PD neurons in the preparation shown in
Fig. 3). This effect was fully
reversible, and the complete pyloric pattern was restored within 2–3 min
after the sucrose was replaced with saline
(Fig. 3). We considered the
possibility that this level of dependence on modulatory input was an artifact
of the saline used in the experiments, and so tried a number of saline
formulations, based on other decapod salines as well as on the measured
concentrations of ions in P. producta hemolymph
(Cornell, 1979). A
physiological saline formulation based on one routinely used for C.
borealis (Hooper et al.,
1986), another brachyuran crab, was used in all physiological
experiments as it gave the fastest recovery from sucrose block.
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Because the modulator complement of the P. producta STG and neuroendocrine organs appeared to be similar to those of other decapod species that have been studied, we tested the physiological effects of a number of modulators on the pyloric pattern. The goal of these experiments was to determine whether or not they functioned as neuromodulators in this system. To eliminate the confounding effects of modulatory substances spontaneously released from axons projecting from the somata present in anterior ganglia, and to preclude false negatives due to a ceiling effect (if a preparation was already maximally active), we tested each putative modulator on both intact and stn-blocked preparations. However, as we were primarily interested in whether or not the modulators were able to modulate the pyloric pattern, we did not fully characterize the effects of each modulatory transmitter, although we did determine its effects on cycle period, phase of the pyloric neurons, burst durations, and spike frequencies in each group of neurons. In order to avoid damaging neurons with microelectrodes, all neuronal activity was recorded extracellularly. Thus, the measurements of spike frequency represent a composite of all the neurons firing, and as such are useful only as a point of comparison between conditions in this species.
Oxotremorine and proctolin are strong and consistent modulators of the pyloric rhythm
When modulatory inputs from the anterior ganglia (CoGs and OG) were
eliminated by the application of a sucrose block to the stn, only
three of the modulators assayed in these experiments, oxotremorine, proctolin
and dopamine, modulated the pyloric pattern in most preparations tested. Of
these, only oxotremorine and proctolin consistently activated a complete
triphasic pyloric pattern.
Oxotremorine
At a concentration of 10–6 mol l–1, the
muscarinic agonist oxotremorine routinely activated the pyloric pattern in
blocked preparations (seven of seven), in which there was no previously
ongoing pyloric activity (Fig.
4), and enhanced it in unblocked preparations (seven of eight)
with already active patterns (Fig.
5). In the blocked preparations, only the core pyloric pattern was
activated, with firing in the PD, LP and PY neurons, but not in the IC or VD
neurons. Moreover, the cycle period in the presence of oxotremorine, with the
stn blocked, was 4.5 s, somewhat slower than the control frequency of
less than 3 s in unblocked preparations (P=0.06, unpaired
t-test, N=15 control, 4 in oxotremorine). In unblocked
preparations, as can be seen in Fig.
5, oxotremorine led to a significant decrease in pyloric cycle
period (Fig. 5D, paired
t-test, P<0.05), which was largely due to a decrease in
the PY burst duration (Fig. 5F,
paired t-test, P<0.05).
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Dopamine regularly modulates the pyloric pattern, but is inconsistent in its effects
Although most preparations were modulated by dopamine (seven of seven
stn-blocked preparations and five of seven stn-intact
preparations), the preparation-to-preparation variability in the qualitative
effects seen in both conditions was high, as can be seen in the two examples
shown in Figs 8 and
9. This variability effectively
precluded pooled quantification of the patterns. In all cases, however,
dopamine enhanced at least one aspect of the pyloric pattern.
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The pyloric rhythm is not activated by either CabTRP I or CCAP
Two peptides that are present in the P. producta stomatogastric
nervous system and/or neuroendocrine organs, and that consistently activate
the pyloric pattern in other decapod species, CabTRP I and CCAP, had no effect
on the pyloric pattern in either stn-blocked (N=4 for both
peptides) or stn-intact (N=5 for both peptides)
preparations. As can be seen in Fig.
12 (CabTRP I, N=5) and
Fig. 13 (CCAP, N=5),
there were no changes in cycle period, burst duration or spike frequency of
any of the pyloric neurons, nor were there changes in the phase relationships
of the pyloric pattern in actively cycling preparations. Neither peptide
induced any rhythmicity in any preparation when the stn was blocked
(N=4 for each peptide).
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Because kelp is available only seasonally in the northern portion of the range of P. producta (including the Puget Sound region where the animals used here were collected), we considered the possibility that these peptides might exert their effects only in the winter, when P. producta is reported by some to eat a more varied diet. We therefore tested the effects of both CabTRP I and CCAP on crabs collected in late December and fed on mussels. As in P. producta collected during the summer and fed kelp, there was no effect of either peptide in either stn-intact or stn-blocked preparations (CabTRP I, N=4; CCAP, N=3; data not shown).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Harris-Warrick et al., 1992;
Nusbaum and Beenhakker, 2002;
Marder and Bucher, 2007). Work
on a number of species has shown that these neural circuits are extensively
influenced both by locally released and circulating substances, which results
in the expression of a large number of distinct motor patterns. Given that the
species commonly used for study have all been highly opportunistic feeders,
this extensive modulation has been hypothesized to be an evolutionary response
to the need to process multiple food types. In this report, we have
investigated the neuromodulatory control of the P. producta STNS,
which, unlike previously studied species, is a dietary specialist, consuming a
relatively uniform diet consisting primarily of kelp and other brown algae
(Hines, 1982).
In terms of general organization, we found that the overall structure of
the P. producta STNS is essentially identical to that of
opportunistically feeding brachyurans (i.e. it consists of the same four
ganglia, which are interconnected by a complement of nerves that are similar
in location and innervation patterns to those of the other species thus far
investigated). Likewise, we found that within the system, the location and
organization of putative synaptic regions are conserved between P.
producta and the other species. Our immunohistochemical survey of
putative transmitters in the STG and neuroendocrine organs of P.
producta suggested that a number of bioactive compounds are readily
available to function as locally and/or hormonally delivered modulators in its
STG. In fact, the distribution of each of the investigated substances is
essentially identical to that seen in the highly modulated and
opportunistically feeding crabs Cancer borealis and/or Cancer
productus (Marder et al.,
1986; Goldberg et al.,
1988; Blitz et al.,
1995; Christie et al.,
1995; Christie et al.,
1997a; Christie et al.,
1997b; Fu et al.,
2005). Thus, in terms of gross structure and the availability of
modulators, there appeared to be few, if any, large-scale differences between
the STNS of the dietary specialist P. producta and those of more
opportunistically feeding crabs.
With respect to the motor output produced by the P. producta STG
circuit, we found that its gross pyloric motor pattern is similar to those
described from other brachyurans [i.e. the core pyloric pattern is triphasic
(PD, LP, PY), with bursts in the VD and IC neurons more or less in phase with
those in the PD and LP or PY neurons, respectively
(Nusbaum and Beenhakker,
2002)]. As in other species, the expression of the P.
producta pyloric rhythm appears to be dependent on modulatory influences
provided by descending inputs from the CoGs and OG, as blocking impulse
activity in the stn (the sole route of input to the STG from these
ganglia) always diminished or stopped production of this motor pattern. In
fact, the extent to which the pyloric rhythm was suppressed by stn
blockade suggested that it might have a stronger dependence on input from
these anterior ganglia than do the opportunistic feeders, such as C.
borealis. Given these results, we were quite surprised to find that many
of the modulators we localized to the STG neuropil and/or identified as
putative hormones in P. producta exerted little or no modulatory
action on the pyloric rhythm in this species (i.e. the amine dopamine and the
peptides CabTRP I, CCAP and RPCH), despite their strong modulatory influence
on this motor pattern in all other decapods thus far investigated. In fact,
only the muscarinic acetylcholine receptor agonist oxotremorine and the
peptide proctolin showed strong modulatory effects on the system that were
similar to those seen in other decapod species. Thus, whereas P.
producta possesses a number of neuromodulators known to influence the
output of the stomatogastric circuit in many opportunistically feeding
crustaceans, our results show that it is relatively insensitive to many of
them, perhaps as it needs only a limited repertoire of motor outputs to
process the relatively uniform food types it commonly ingests.
What is responsible for the decreased modulation in Pugettia producta
The two most likely differences between P. producta and other
decapods that might account for the decreased modulation in this species are
changes in the modulatory environment or changes in the receptors to those
modulators. We found that the modulators we examined all appear to be present
in P. producta in locations similar to those in other species.
Moreover, even the amino acid sequences of the native peptide isoforms we
examined were identical to those reported in other crab species, with the
possible exception of proctolin. Ironically, proctolin was the peptide with
effects that most strongly resembled those seen in other species, but was the
only peptide that was not detected in the STNS or in the neuroendocrine organs
we examined using direct tissue MALDI-FTMS. In contrast to the MALDI results,
our immunohistochemistry experiments indicate that either proctolin itself, or
a proctolin-like peptide is present in the STG, the CoGs, the pericardial
organ and the sinus gland. One explanation is that the proctolin
concentrations are below the level of detection by MALDI or that it does not
ionize under the conditions we used. We are, however, consistently able to
detect proctolin in the sinus gland of other brachyuran crabs. Another
intriguing possibility is that the amino acid sequence of the native proctolin
isoform differs from that of other species, as has been suggested to be the
case in the Colorado potato beetle
(Spittaels et al., 1995).
With respect to CabTRP I, one additional possibility is that, as has
recently been reported for several Cancer species
(Stemmler et al., 2007), there
is a second TRP (tachykinin-related peptide) isoform present in P.
producta. This peptide, TPSGFLGMRamide, is present in P.
producta in the highest proportion relative to CabTRP I of any of the
Brachyuran species we have examined (E.A.S., unpublished observations).
Virtually 50% of the TRP in P. producta is TPSGFLGMRamide (CabTRP
II), compared to approximately 15% in C. borealis and C.
productus, and 30% in C. irroratus. In the only species in which
it has been tested, C. borealis, the effects of CabTRP I and CabTRP
II are identical (Stemmler et al.,
2007). This suggested the possibility that the active TRP in
P. producta was CabTRP II rather than CabTRP I. However, in
preliminary experiments, CabTRP II, like CabTRP I, had no effect on the
pyloric pattern in P. producta (P.S.D., unpublished
observations).
Changes in receptors to the modulators could also account for the lack of effect seen with many of the modulators we tested. Although we could not directly test this hypothesis, it is interesting to consider that kelp is available only seasonally in the waters in which P. producta were collected. Thus, in the winter, P. producta from the Puget Sound area may become opportunistic feeders. To test the possibility that P. producta seasonally express receptors for the inactive peptides, thus increasing their modulatory repertoire in the winter when they are eating a more varied diet, we collected animals in late December, after the kelp had been gone for over 2 months and P. producta were feeding opportunistically, and tested the two peptides that had no effect in the summer. Neither CCAP nor CabTRP I caused any effect in these animals, suggesting that the receptors to CCAP and CabTRP I in the neurons of the pyloric circuit may have been evolutionarily lost in this species.
Why maintain superfluous neuromodulators?
The data presented in our study raises the question `Is the expression of
many well-known neuromodulators in the P. producta STG truly
superfluous?'. Clearly, there are many possible answers to this question, the
most likely of which is that they are not, in fact, superfluous. We have
examined the effects of these neuropeptides on only one target, the neurons of
the pyloric central pattern generator, and they undoubtedly have other
targets. Although we did not examine the distribution of the modulators in
other parts of the nervous system, CabTRP I, proctolin, RPCH and dopamine are
widely distributed in the brain and thoracic ganglia in other species, and the
similarity of neuromodulator distributions within the tissues we examined
suggests that they are likewise present in other parts of the nervous system
in P. producta, where they could still be exerting their effects. It
is also possible that these modulators do alter the expression of the pyloric
pattern, but do so only under certain conditions, which we may not have
tested. The effects of many neuromodulators and modulatory neurons on the
stomatogastric system are known to be state dependent
(Nagy and Dickinson, 1983;
Nusbaum and Marder, 1989a;
Nusbaum and Marder, 1989b).
Moreover, other modulators require the presence or recent presence of another
modulator in order to exert a given effect, as is seen with the activation of
the cardiac sac pattern by the peptide proctolin; proctolin activates the
cardiac sac pattern in an isolated STG only if superfused with or shortly
after superfusion with RPCH (Dickinson et
al., 1997). We did not test combinations of modulators in this
study, but the possibility remains that the inactive peptides could modulate
the pyloric pattern when applied in appropriate combinations.
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Blitz, D. M., Christie, A. E., Marder, E. and Nusbaum, M. P. (1995). Distribution and effects of tachykinin-like peptides in the stomatogastric nervous system of the crab, Cancer borealis. J. Comp. Neurol. 354,282 -294.[CrossRef][Medline]
Christie, A. E., Skiebe, P. and Marder, E. (1995). Matrix of neuromodulators in neurosecretory structures of the crab Cancer borealis. J. Exp. Biol. 198,2431 -2439.[Medline]
Christie, A. E., Baldwin, D. H., Marder, E. and Graubard, K. (1997a). Organization of the stomatogastric neuropil of the crab, Cancer borealis, as revealed by modulator immunocytochemistry. Cell Tissue Res. 288,135 -148.[CrossRef][Medline]
Christie, A. E., Lundquist, C. T., Nässel, D. R. and Nusbaum, M. P. (1997b). Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis. J. Exp. Biol. 200,2279 -2294.[Abstract]
Christie, A. E., Stemmler, E. A., Peguero, B., Messinger, D. I., Provencher, H. L., Scheerlinck, P., Hsu, Y. W., Guiney, M. E., de la Iglesia, H. O. and Dickinson, P. S. (2006). Identification, physiological actions, and distribution of VYRKPPFNGSIFamide (Val1-SIFamide) in the stomatogastric nervous system of the American lobster Homarus americanus. J. Comp. Neurol. 496,406 -421.[CrossRef][Medline]
Christie, A. E., Kutz-Naber, K. K., Stemmler, E. A., Klein, A.,
Messinger, D. I., Goiney, C. C., Conterato, A. J., Bruns, E. A., Hsu, Y. W.,
Li, L. et al. (2007). Midgut epithelial endocrine cells are a
rich source of the neuropeptides APSGFLGMRamide (Cancer borealis
tachykinin-related peptide Ia) and GYRKPPFNGSIFamide
(Gly1-SIFamide) in the crabs Cancer borealis, Cancer
magister and Cancer productus. J. Exp. Biol.
210,699
-714.
Cornell, J. C. (1979). Salt and water balance
in two marine spider crabs, Libinia emarginata and Pugettia
producta. I. Urine production and magnesium regulation. Biol.
Bull. 157,221
-233.
Cuello, A. C., Galfre, G. and Milstein, C.
(1979). Detection of substance P in the central nervous system by
a monoclonal antibody. Proc. Natl. Acad. Sci. USA
76,3532
-3536.
Dickinson, P. S., Fairfield, W. P., Hetling, J. R. and Hauptman,
J. (1997). Neurotransmitter interactions in the
stomatogastric system of the spiny lobster: one peptide alters the response of
a central pattern generator to a second peptide. J.
Neurophysiol. 77,599
-610.
Dickinson, P. S., Hsu, Y. A., Labenia, J., Latham, R., Lin, M., Messinger, D. I., Ngo, C. T., Graubard, K. and Christie, A. E. (2004). The pyloric rhythm of the kelp crab contains but is insensitive to peptides that modulate this rhythm in other crustaceans. Program No. 657.11. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Online.
Dircksen, H. and Keller, R. (1988). Immunocytochemical localization of CCAP, a novel crustacean cardioactive peptide, in the nervous system of the shore crab, Carcinus maenas L. Cell Tissue Res. 254,347 -360.
Fu, Q., Kutz, K. K., Schmidt, J. J., Hsu, Y. W., Messinger, D. I., Cain, S. D., de la Iglesia, H. O., Christie, A. E. and Li, L. (2005). Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation. J. Comp. Neurol. 493,607 -626.[CrossRef][Medline]
Goldberg, D., Nusbaum, M. P. and Marder, E. (1988). Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus. Cell Tissue Res. 252,515 -522.[CrossRef][Medline]
Harris-Warrick, R. M., Nagy, F. and Nusbaum, M. P. (1992). Neuromodulation of stomatogastric networks by identified neurons and transmitters. In Dynamic Biological Networks: The Stomatogastric Nervous System (ed. R. M. Harris-Warrick, E. Marder, A. I. Selverston and M. Moulins), pp. 87-138. Cambridge, MA: MIT Press.
Hines, A. H. (1982). Coexistence in a kelp forest: size, population dynamics, and resource partitioning in a guild of spider crabs (Brachyura, Majidae). Ecol. Monogr. 52,179 -198.[CrossRef]
Hooper, S. L., O'Neil, M. B., Wagner, R., Ewer, J., Golowasch, J. and Marder, E. (1986). The innervation of the pyloric region of the crab, Cancer borealis: homologous muscles in decapod species are differently innervated. J. Comp. Physiol. A 159,227 -240.[CrossRef][Medline]
Johnson, E. C., Garczynski, S. F., Park, D., Crim, J. W.,
Nässel, D. R. and Taghert, P. H. (2003). Identification
and characterization of a G protein-coupled receptor for the neuropeptide
proctolin in Drosophila melanogaster. Proc. Natl. Acad. Sci.
USA 100,6198
-6203.
Klagges, B. R., Heimbeck, G., Godenschwege, T. A., Hofbauer, A.,
Pflugfelder, G. O., Reifegerste, R., Reisch, D., Schaupp, M., Buchner, S. and
Buchner, E. (1996). Invertebrate synapsins: a single gene
codes for several isoforms in Drosophila. J. Neurosci.
16,3154
-3165.
Madsen, A. J., Herman, W. S. and Elde, R. (1985). Differential distribution of two homologous neuropeptides (RPCH & AKH) in the crayfish nervous system. Abstr. Soc. Neurosci. 11,941 .
Marder, E. and Bucher, D. (2007). Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs. Annu. Rev. Physiol. 69,291 -316.[CrossRef][Medline]
Marder, E., Hooper, S. L. and Siwicki, K. K. (1986). Modulatory action and distribution of the neuropeptide proctolin in the crustacean stomatogastric nervous system. J. Comp. Neurol. 243,454 -467.[CrossRef][Medline]
Marder, E., Christie, A. E. and Kilman, V. L. (1995). Functional organization of cotransmission systems: lessons from small nervous systems. Invert. Neurosci. 1, 105-112.[CrossRef][Medline]
Messinger, D. I., Kutz, K. K., Le, T., Verley, D. R., Hsu, Y.
W., Ngo, C. T., Cain, S. D., Birmingham, J. T., Li, L. and Christie, A. E.
(2005). Identification and characterization of a
tachykinin-containing neuroendocrine organ in the commissural ganglion of the
crab Cancer productus. J. Exp. Biol.
208,3303
-3319.
Meyrand, P., Simmers, J. and Moulins, M. (1991). Construction of a pattern-generating circuit with neurons of different networks. Nature 351, 60-63.[CrossRef][Medline]
Mizrahi, A., Dickinson, P. S., Kloppenburg, P., Fenelon, V.,
Baro, D. J., Harris-Warrick, R. M., Meyrand, P. and Simmers, J.
(2001). Long-term maintenance of channel distribution in a
central pattern generator neuron by neuromodulatory inputs revealed by
decentralization in organ culture. J. Neurosci.
21,7331
-7339.
Nagy, F. and Dickinson, P. S. (1983). Control
of a central pattern generator by an identified modulatory interneurone in
crustacea. I. Modulation of the pyloric motor output. J. Exp.
Biol. 105,33
-35.
Nusbaum, M. P. and Beenhakker, M. P. (2002). A small-systems approach to motor pattern generation. Nature 417,343 -350.[CrossRef][Medline]
Nusbaum, M. P. and Marder, E. (1989a). A modulatory proctolin-containing neuron (MPN). I. Identification and characterization. J. Neurosci. 9,1591 -1599.[Abstract]
Nusbaum, M. P. and Marder, E. (1989b). A modulatory proctolin-containing neuron (MPN). II. State-dependent modulation of rhythmic motor activity. J. Neurosci. 9,1600 -1607.[Abstract]
Richards, K. S., Miller, W. L. and Marder, E.
(1999). Maturation of lobster stomatogastric ganglion rhythmic
activity. J. Neurophysiol.
82,2006
-2009.
Selverston, A. I. and Moulins, M. (1987). The Crustacean Stomatogastric System: A Model for The Study of Central Nervous Systems. Berlin: Springer-Verlag.
Selverston, A. I., Russell, D. F. and Miller, J. P. (1976). The stomatogastric nervous system: structure and function of a small neural network. Prog. Neurobiol. 7, 215-290.[CrossRef][Medline]
Skiebe, P. (2001). Neuropeptides are ubiquitous chemical mediators: using the stomatogastric nervous system as a model system. J. Exp. Biol. 204,2035 -2048.[Medline]
Skiebe, P. and Ganeshina, O. (2000). Synaptic neuropil in nerves of the crustacean stomatogastric nervous system: an immunocytochemical and electron microscopical study. J. Comp. Neurol. 420,373 -397.[CrossRef][Medline]
Skiebe, P. and Wollenschlager, T. (2002). Putative neurohemal release zones in the stomatogastric nervous system of decapod crustaceans. J. Comp. Neurol. 453,280 -291.[CrossRef][Medline]
Spittaels, K., Vankeerberghen, A., Torrekens, S., Devreese, B., Grauwels, L., Van Leuven, F., Hunt, D., Shabanowitz, J., Schoofs, L., Van Beeumen, J. et al. (1995). Isolation of Ala1-proctolin, the first natural analogue of proctolin, from the brain of the Colorado potato beetle. Mol. Cell. Endocrinol. 110,119 -124.[CrossRef][Medline]
Stangier, J., Hilbich, C., Dircksen, H. and Keller, R. (1988). Distribution of a novel cardioactive neuropeptide (CCAP) in the nervous system of the shore crab Carcinus maenas.Peptides 9,795 -800.[CrossRef][Medline]
Stemmler, E. A., Gardner, N. P., Guiney, M. E., Bruns, E. A. and Dickinson, P. S. (2006). The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations. J. Mass Spectrom. 41,295 -311.[CrossRef][Medline]
Stemmler, E. A., Peguero, B., Bruns, E. A., Dickinson, P. S. and Christie, A. E. (2007). Identification, physiological actions, and distribution of TPSGFLGMRamide: a novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs.J. Neurochem. 101,1351 -1366.[CrossRef][Medline]
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