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
Patsy S. Dickinson1,2,*,
Elizabeth A. Stemmler3 and
Andrew E. Christie4,5
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
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Fig. 1. Schematic representation of the stomatogastric nervous system (STNS) of
Pugettia producta, including the locations (blue stippling) of
putative synaptic neuropils, as indicated by the presence of synapsin-like
immunoreactivity, within the STNS. coc, circumesophageal connective,
CoG, commissural ganglion; dpon, dorso-posterior esophageal nerve;
dvn, dorsal ventricular nerve; ion, inferior esophageal
nerve; lvn, lateral ventricular nerve; mvn, medial
ventricular nerve; OG, esophageal ganglion; on, esophageal nerve;
pdn, pyloric dilator nerve; son, superior esophageal nerve;
STG, stomatogastric ganglion; stn, stomatogastric nerve;
vlvn, ventro-lateral ventricular nerve.
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Fig. 2. MALDI-FTMS identification of native Pugettia producta
neuropeptides. (A–C) Representative direct tissue spectra of the (A)
sinus gland (SG), (B) stomatogastric ganglion (STG) and (C) pericardial organ
(PO). In the SG, a peak corresponding to the [M+Na]+ ion of
pELNFSPGWamide (red pigment concentrating hormone; RPCH) was routinely
detected. In the STG, the [M+H]+ ion corresponding to
APSGFLGMRamide (authentic Cancer borealis tachykinin-related peptide
I; CabTRP I) was present in most spectra. In PO samples, the [M+H]+
ion of PFCNAFTGCamide (crustacean cardioactive peptide; CCAP) was commonly
seen. In addition, peaks corresponding to other peptides were also detected in
these tissues, some of which are labeled in the spectra. m/z,
mass/charge; Gly1-SIFa, GYRKPPFNGSIFamide.
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Fig. 3. The pyloric motor pattern of Pugettia producta is similar to that
of other decapod species, though its expression is more highly dependent on
the presence of modulatory inputs from anteriorly located sources. (A) In P.
producta, the pyloric motor pattern strongly resembled that recorded in other
decapod species, and consisted of alternating bursts of action potentials in
the three core pyloric neuronal types: the pyloric dilator (PD), lateral
pyloric (LP) and pyloric (PY) neurons, recorded on the lateral ventricular
nerve (lvn). The ventricular dilator (VD) and inferior cardiac (IC)
neurons, recorded on the medial ventricular nerve (mvn), fired weaker
bursts that were more or less in phase with the bursts in the LP and PY
neurons, respectively. (B) Blocking the stomatogastric nerve (stn),
which provides the only neuronal input to the STG, with isotonic sucrose
eliminated all pyloric bursting within 15 min in most preparations, as shown
here. (C) When the sucrose was replaced with saline, so that normal conduction
was restored in the stn, the complete pyloric pattern recovered
within 2–3 min. Nerves: mvn, medial ventricular nerve
(recording action potentials of the VD and IC neurons); pdn, pyloric
dilator nerve (recording action potentials of the PD neurons); lvn,
lateral ventricular nerve (recording action potentials of the PD, LP and PY
neurons).
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Fig. 4. The muscarinic acetylcholine agonist oxotremorine routinely activated the
pyloric pattern in preparations in which the stn was blocked with
isotonic sucrose, and there thus was no ongoing pyloric pattern. (A)
Representative recordings of pyloric activity in a preparation with conduction
in the stn blocked with isotonic sucrose and superfused with normal
saline, then superfused with oxotremorine (10–7 mol
l–1), followed by a wash in normal saline. A complete core
pyloric pattern, with intense firing in all three neuronal types (PD, LP, and
PY) was induced by the oxotremorine (seen in pdn, PD, and
lvn, PD, LP and PY, recordings); in contrast, regular bursting was
not initiated in the VD and IC neurons (mvn). (B) Phase plot, showing
two cycles of the pyloric pattern recorded in oxotremorine, taken from four
preparations, showing that oxotremorine activated the core pyloric pattern,
but not the VD and IC neurons. (C) Graph of average cycle period: in control
saline there was no activity, while cycle period was approximately 4 s in the
presence of oxotremorine (Oxo); this is somewhat longer than cycle period in
control saline when the stn is not blocked (approximately 3.3 s in
Fig. 3, for example.) (D,E)
Graphs of the spike frequency during bursts and burst duration in each
neuronal type during oxotremorine superfusion with the stn blocked.
Because there was no rhythmic activity, and therefore no bursts in any of the
neurons, values during saline superfusion are not shown. N=4 for all
graphs. Bars indicate standard deviations. Nerves: mvn, medial
ventricular nerve (recording action potentials of the VD and IC neurons);
pdn, pyloric dilator nerve (recording action potentials of the PD
neurons); lvn, lateral ventricular nerve (recording action potentials
of the PD, LP and PY neurons).
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Fig. 5. In preparations in which the stn was intact, the muscarinic
acetylcholine agonist oxotremorine enhanced ongoing pyloric activity. (A)
Recordings of pyloric activity in a representative preparation in control
saline, during superfusion of 10–6 mol l–1
oxotremorine, and during the wash with normal saline. Oxotremorine enhanced
the overall pyloric pattern, seen most clearly here as an increase in pyloric
cycle frequency and as increased activity in the PD (pdn and
lvn) and VD (mvn) neurons. (B) Phase diagram, showing two
cycles of the activity of the pyloric pattern in control saline. (C) Phase
diagram of the pyloric pattern when the STG was superfused with
10–6 mol l–1 oxotremorine. (D) Plot of the
cycle period in both control saline and oxotremorine, showing the decreased
cycle period during oxotremorine application. (E,F) Graphs of spike frequency
during bursts and burst duration, respectively, in each of the pyloric
neuronal types in control saline and when the STG was being superfused with
oxotremorine. N=5 for all graphs. Bars indicate standard deviations.
Asterisks indicate a value significantly different from control
(P<0.05). Nerves: mvn, medial ventricular nerve
(recording action potentials of the VD and IC neurons); pdn, pyloric
dilator nerve (recording action potentials of the PD neurons); lvn,
lateral ventricular nerve (recording action potentials of the PD, LP and PY
neurons).
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Fig. 6. (A) When axonal conduction in the stn was blocked with isotonic
sucrose, and there was no ongoing pyloric rhythm, superfusion of the
pentapeptide proctolin (10–6 mol l–1)
induced rhythmic pyloric activity in 55% of preparations, including the one
shown here. (B) Phase plot, showing two cycles of the pattern induced by
proctolin in those preparations (N=6) that were activated. Phases of
firing in the VD and IC neurons are not shown, because they were active in
only three preparations (including the one shown in A). (C) Graph of average
cycle period. In control saline there was no activity, whereas cycle period
was nearly 15 s in the presence of proctolin; this is considerably longer than
cycle period in control saline when the stn is not blocked
(approximately 3.3 s in Fig. 3,
for example.) (D,E) Graphs of spike frequency during bursts and of burst
duration in each of the core pyloric neurons (the PD, LP and PY neurons) that
were regularly activated during proctolin superfusion with the stn
blocked. Because there was no rhythmic activity, and therefore no bursts in
any of the neurons, values during saline superfusion are not shown. Nerves:
mvn, medial ventricular nerve (recording action potentials of the VD
and IC neurons); dlvn, dorso-lateral ventricular nerve (recording
action potentials of the PD neurons); lvn, lateral ventricular nerve
(recording action potentials of the PD, LP and PY neurons).
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Fig. 7. When the anterior inputs were intact and the ongoing pyloric pattern was
active, proctolin did not cause significant changes in cycle period, but it
did cause significant changes in the relative phases of the pyloric pattern
and in the burst duration of some neurons. (A) Recordings of control activity
and activity when 10–6 mol l–1 proctolin was
bath-applied to the STG. (B,C) Phase plots, showing two cycles of the pyloric
pattern in control saline (B) and in the presence of proctolin (C). The LP and
IC neuron bursts were each prolonged, starting at about the same phase, but
continuing longer, in proctolin than in control saline. The burst of PY
neurons started sooner in proctolin than in control saline. (D) Cycle period
was not significantly changed by proctolin when the stn was intact.
(E,F) Graphs of the spike frequency during bursts (E) and of burst duration
(F) in control saline and in proctolin, showing that most parameters were not
altered, but both LP and IC neuron bursts increased in duration. N=5
for all graphs. Bars indicate standard deviations. Nerves: mvn,
medial ventricular nerve (recording action potentials of the VD and IC
neurons); pdn, pyloric dilator nerve (recording action potentials of
the PD neurons); lvn, lateral ventricular nerve (recording action
potentials of the PD, LP and PY neurons).
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Fig. 8. In preparations with relatively weak baseline firing, dopamine was capable
of activating a typical pyloric pattern, as was the case in the preparation
shown here. Recordings of the pyloric pattern in (A) control, (B) when
dopamine (10–4 mol l–1) was superfused over
the STG, and (C) in wash. Nerves: mvn, medial ventricular nerve
(recording action potentials of the VD and IC neurons); pdn, pyloric
dilator nerve (recording action potentials of the PD neurons); lvn,
lateral ventricular nerve (recording action potentials of the PD, LP and PY
neurons).
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Fig. 9. In preparations with stronger ongoing pyloric activity, dopamine did not
alter cycle frequency, but did cause changes within the pattern, notably a
strong increase in activity in the VD neuron (mvn) and a decrease in
activity in the LP neuron (large spike on lvn). Recordings of the
pyloric pattern in (A) control saline, (B) in the presence of dopamine
(10–4 mol l–1), and (C) in wash. Nerves:
mvn, medial ventricular nerve (recording action potentials of the VD
and IC neurons); dlvn, dorso-lateral ventricular nerve (recording
action potentials of the PD neurons); lvn, lateral ventricular nerve
(recording action potentials of the PD, LP and PY neurons).
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Fig. 10. The peptide red pigment concentrating hormone (RPCH) did not affect the
pyloric pattern when the stn was intact and an ongoing pyloric
pattern was active. (A,B) Phase plots, showing two cycles of the pyloric
pattern in control saline (A) and in the presence of 10–6 mol
l–1 RPCH (B), showing that the pattern itself remained
virtually unchanged by RPCH. (C–E) Cycle frequency (C), spike
frequencies during bursts (D) and burst durations (E) were all unchanged.
N=4 preparations. Error bars represent standard deviations.
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Fig. 11. Red pigment concentrating hormone (RPCH) is able to affect the pyloric
pattern, shown by the fact that it did so in two of nine preparations with the
stn blocked, one of which is shown here. Recordings of the pyloric
pattern in (A) control saline, (B) during RPCH (10–6 mol
l–1) bath application and (C) when washed with control
saline. Note that, in contrast to the vast majority of preparations, the
pyloric pattern continued even when the stn was blocked in this
preparation. To ensure that this was not due to an incomplete block of
condition in the stn, the stn was later cut, which did not
alter the pattern of activity recorded. RPCH strongly activated the complete
core pyloric pattern [bursting in the PD, LP, PY neurons seen on the
lvn and the dlvn (PD), as well as weak bursting in the IC
neuron]. Nerves: mvn, medial ventricular nerve (recording action
potentials of the VD and IC neurons); dlvn, dorso-lateral ventricular
nerve (recording action potentials of the PD neurons); lvn, lateral
ventricular nerve (recording action potentials of the PD, LP and PY
neurons).
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Fig. 12. In the presence of Cancer borealis tachykinin-related peptide I
(CabTRP I), there were no changes in the ongoing pyloric activity in
preparations with the anterior inputs intact (A–E), nor was there any
activation of the pattern in preparations with the stn blocked (not
shown). (A,B) Phase plots, showing two cycles of the pyloric pattern in
control saline (A) and when CabTRP I (10–6 mol
l–1) was bath-applied to the STG (B). No differences are
apparent. (C–E) There were no changes in cycle period (C), spike
frequency within bursts (D) or burst duration (E) in any of the pyloric
neurons when CabTRP I was bath applied to the STG. N=3. Error bars
indicate standard deviations.
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Fig. 13. The neuropeptide crustacean cardioactive peptide (CCAP) did not affect the
pyloric pattern in preparations in which the stn was blocked (not
shown) or in preparations with the stn intact. (A,B) Phase plots,
showing two cycles of pyloric pattern in control saline (A) and during bath
application of CCAP (10–6 mol l–1) to the
STG (B). (C–E) There were no changes in cycle period (C), spike
frequency within bursts (D) or burst duration (E) in any of the pyloric
neurons when CCAP was bath applied to the STG. N=4. Error bars
indicate standard deviations.
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