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The Journal of Neuroscience, December 1, 2000, 20(23):8943-8953
Projection Neurons with Shared Cotransmitters Elicit Different
Motor Patterns from the Same Neural Circuit
Debra E.
Wood,
Wolfgang
Stein, and
Michael P.
Nusbaum
Department of Neuroscience, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104-6074
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ABSTRACT |
Specificity in the actions of different modulatory neurons is often
attributed to their having distinct cotransmitter complements. We are
assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this
nervous system, the stomatogastric ganglion (STG) contains a
multifunctional network that generates the gastric mill and pyloric
rhythms. Two identified projection neurons [modulatory proctolin
neuron (MPN) and modulatory commissural neuron 1 (MCN1)]
that innervate the STG and modulate these rhythms contain GABA and the
pentapeptide proctolin, but only MCN1 contains Cancer
borealis tachykinin-related peptide (CabTRP Ia). Selective
activation of each projection neuron elicits different rhythms from the
STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct
pyloric rhythm as well as a gastric mill rhythm. We tested the degree
to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this
end, we used the tachykinin receptor antagonist Spantide I to eliminate
the actions of CabTRP Ia. With Spantide I present, MCN1 no longer
elicited the gastric mill rhythm and the resulting pyloric rhythm was
changed. Although this rhythm was more similar to the MPN-elicited
pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia
partially confers the differences in rhythm generation resulting from
MPN versus MCN1 activation. This result suggests that different
projection neurons may use the same cotransmitters differently to
elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including
using distinct cotransmitter complements, to elicit different outputs
from the same neuronal network.
Key words:
neuropeptides; tachykinin; proctolin; stomatogastric
nervous system; crustacea; neuromodulation; motor pattern
generation
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INTRODUCTION |
The prevalence of colocalized
neurotransmitters suggests that the combination of transmitters
released by a neuron attributes specificity to its actions (Hokfelt,
1991
; Agnati et al., 1995; Brezina and Weiss, 1997; Marder et al.,
1997). However, the extent to which this hypothesis explains the
distinct actions of different input neurons influencing a single
neuronal network has not been examined. This is because there are few
systems containing multiple identified neurons that influence the same
network and have identified cotransmitters (Kulhman et al., 1985a,b;
Thorogood and Brodfuehrer, 1995; McCrohan and Croll, 1997; Blitz et
al., 1999). Peptidergic neurons in particular may require distinct
cotransmitters to specify their actions, because neurally released
peptides commonly have a broad sphere of influence, potentially giving
them access to all relevant receptors. This is a result of their
release from nonsynaptic sites and their ability to diffuse relatively
long distances to reach their target receptors (Jan and Jan, 1982; Mayeri et al., 1985; Agnati et al., 1995; Zupanc, 1996).
We aim to understand how different peptidergic projection neurons
elicit distinct motor patterns from a multifunctional network. The
stomatogastric nervous system (STNS) of the crab Cancer
borealis is well suited for this study because it contains well
characterized pattern generating circuits in the stomatogastric
ganglion (STG) (Harris-Warrick et al., 1992). In the C. borealis STG, the gastric mill and pyloric rhythms are
generated by distinct circuits composed of overlapping sets of neurons
(Weimann et al., 1991; Weimann and Marder, 1994). Different versions of
these rhythms are elicited by selective stimulation of different
modulatory projection neurons that innervate the STG (Norris et al.,
1994, 1996; Blitz et al., 1999). For example, modulatory commissural
neuron 1 (MCN1) elicits both gastric mill and pyloric rhythms (Coleman
and Nusbaum, 1994; Bartos and Nusbaum, 1997; Bartos et al., 1999),
whereas the modulatory proctolin neuron (MPN) elicits a pyloric rhythm
but no gastric mill rhythm (Blitz and Nusbaum, 1997; Blitz et al.,
1999). Both neurons contain GABA and the peptide proctolin (Nusbaum and
Marder, 1989a; Blitz et al., 1999). MCN1 also contains Cancer
borealis tachykinin-related peptide Ia (CabTRP Ia) (Blitz et al.,
1999).
Bath-applied proctolin and MPN stimulation produce the same pyloric
rhythm (Nusbaum and Marder, 1989b). Because neurally released peptides
have a relatively broad sphere of influence, proctolin may have the
same actions on the pyloric circuit regardless of the projection neuron
from which it is released. If so, then the distinct actions of MCN1 may
result from its peptide cotransmitter, CabTRP Ia. We tested this
possibility by comparing the MCN1- and MPN-elicited rhythms in the
presence of Spantide I, a tachykinin receptor antagonist (Folkers et
al., 1984; Christie et al., 1997a; Nässel, 1999). Under this
condition, the actions of MPN were not changed, but MCN1 elicited an
altered pyloric rhythm and no longer elicited the gastric mill rhythm.
The altered pyloric rhythm was more similar to, but still distinct from
the MPN-elicited pyloric rhythm. Thus, the peptide cotransmitter CabTRP
Ia contributes to, but does not completely account for the distinct
rhythms elicited by these two projection neurons.
Parts of this work appeared previously in abstract form (Wood and
Nusbaum, 1998).
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MATERIALS AND METHODS |
Animals. Adult male crabs (C. borealis)
were purchased from the Marine Biological Laboratory (Woods Hole, MA)
and from commercial sources (Boston, MA). Crabs were maintained in
filtered, aerated artificial seawater (10-12°C). Animals were
anesthetized by packing them in ice for 20-40 min before dissection.
The dissection of the STNS was done in physiological saline at ~4°C
as described previously (Blitz and Nusbaum, 1997). Experiments were
performed on the isolated STNS (Fig.
1A). Data were obtained
from 91 animals.
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Figure 1.
Schematic of the stomatogastric nervous system and
the response of the gastric mill and pyloric circuits to individual
stimulation of two different modulatory projection neurons.
A, The stomatogastric nervous system includes four
ganglia plus their connecting and peripheral nerves. The ganglia are
the stomatogastric ganglion (STG), oesophageal ganglion
(OG), and the paired commissural ganglia
(CoGs). The STG contains the gastric mill and pyloric
circuits, whereas modulatory projection neurons that innervate the STG
are located in the OG and CoGs. Modulatory commissural neuron 1 (MCN1) occurs as a single copy in each CoG, whereas the
modulatory proctolin neuron (MPN) occurs as an
equivalent pair of somata in the OG. The axonal projection patterns of
only one MCN1 and MPN are shown. The inferior (ion) and
superior oesophageal nerves (son) are severed (as
indicated by the broken lines) to eliminate the
modulatory influence from CoG projection neurons that innervate the
STG. Anterior is to the left. B,
Schematic of the cotransmitter complements of MCN1 and MPN, plus their
actions on the STG circuits. Also illustrated are the two synaptic
actions that occur onto the STG terminals of MCN1 that are pivotal
for enabling this neuron to elicit the gastric mill rhythm (Coleman et
al., 1995; Blitz et al., 1999). T-bars represent
transmitter-mediated excitation, filled circle
represents synaptic inhibition, and resistor symbol
represents electrical coupling. C, Tonic stimulation of
MCN1 and MPN elicits distinct STG motor patterns. MCN1 stimulation
enhances and modifies the pyloric rhythm (lvn, mvn) and
activates the gastric mill rhythm (dgn,
LG). MPN stimulation enhances and modifies the pyloric
rhythm but does not activate a gastric mill rhythm. Note that the
pyloric rhythm during stimulation of these two projection neurons is
distinct (e.g., mvn). Most hyperpolarized LG
Vm: saline superfusion
(left),  66 mV; MCN1 stimulation
(middle), 72 mV; MPN stimulation
(right), 69 mV. Nerves: dpon, dorsal
posterior oesophageal nerve; dgn, dorsal gastric nerve;
ion, inferior oesophageal nerve; lgn,
lateral gastric nerve; lvn, lateral ventricular nerve;
mvn, medial ventricular nerve; pdn,
pyloric dilator nerve; pyn, pyloric constrictor nerve;
son, superior oesophageal nerve; stn,
stomatogastric nerve. STG neurons: DG, dorsal gastric
neuron; IC, inferior cardiac neuron; LG,
lateral gastric neuron; LP, lateral pyloric neuron;
PD, pyloric dilator neuron; PY, pyloric
neuron; VD, ventricular dilator neuron.
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Solutions. C. borealis physiological saline had
the following composition (in mM): NaCl, 440;
MgCl2, 26; CaCl2, 13; KCl,
11; Trisma base, 10; maleic acid, 5; pH 7.4-7.6. Spantide I was
obtained from Peninsula Laboratories (Belmont, CA), Sigma (St. Louis,
MO), or was synthesized at the Cancer Research Center, University of Pennsylvania School of Medicine (Philadelphia, PA). Spantide solutions were made by dissolving Spantide I into C. borealis
physiological saline. Each application of Spantide I was continued for
at least 15-20 min before stimulation of MCN1 or MPN. Preparations
were washed by superfusing saline for at least 40 min before
postapplication controls were performed.
Neuropeptide applications. CabTRP Ia
(10
4
M in pipette) and proctolin
(104
M in pipette) were separately pressure-ejected (2-4 sec
duration, 6 PSI) onto the desheathed STG neuropil using a Picospritzer
II (General Valve, Fairfield, NJ). Pressure pipettes had resistances of
1-2 M
.
Electrophysiology. Electrophysiology was performed using
standard methods as described previously (Bartos and Nusbaum, 1997; Blitz and Nusbaum, 1997). The STNS was pinned down in a silicone elastomer-lined (SYLGARD 184; KR Anderson, Santa Clara, CA) Petri dish
and superfused continuously (7-12 ml/min) with chilled physiological saline (10-13°C). To facilitate intracellular recordings and access for applied solutions, the STG and the oesophageal ganglion (OG) were
desheathed and visualized with white light transmitted through a
dark-field condenser (Nikon, Tokyo, Japan). Microelectrodes (15-30
M) were filled with 4 M potassium acetate plus 20 mM KCl. Intracellular current injections were accomplished
using Axoclamp 2 (Axon instruments, Foster City, CA) amplifiers in
single-electrode discontinuous current-clamp (DCC) mode. Sample rates
in DCC mode ranged from 2 to 3 kHz.
Identification of STG neurons was done by documenting their activity
patterns, synaptic interactions, and axonal projection paths as
described previously (Weimann et al., 1991; Bartos and Nusbaum, 1997;
Blitz and Nusbaum, 1997). Identification of the projection neurons MPN
and MCN1 was made on the basis of their soma (MPN, Nusbaum and Marder,
1989a,b) or axon location (MCN1, Coleman and Nusbaum, 1994; Bartos and
Nusbaum, 1997) and their interactions in the STG (Blitz et al., 1999).
In most experiments, modulatory influence from projection neurons in
the commissural ganglia (CoGs) onto the STG was eliminated by
transecting both inferior (ions) and superior oesophageal
nerves (sons) (Fig. 1A). Under
these conditions the pyloric rhythm either stops or slows (0.2-0.8 Hz)
from cycle frequencies that are typically faster than 1 Hz. If it was
active before transection, the gastric mill rhythm always terminated
after transection. Selective activation of MCN1 was achieved by
tonically stimulating the ion or ions (10-20 Hz)
(Coleman et al., 1995; Bartos and Nusbaum, 1997). Stimulation of the
sensory pathway contained in the dorsal oesophageal posterior nerve
(dpon) was used to elicit a gastric mill rhythm that is distinct from the MCN1-elicited gastric mill rhythm (Blitz et al.,
1998; Beenhakker et al., 2000). The dpons were stimulated in
a 0.06 Hz train (15 Hz intraburst, 6 sec duration) for 2.5-3 min. In
the dpon stimulation experiments, the ions were
transected, but the sons were left intact so that the
influence of the activated sensory pathway, as well as CoG projection
neurons other than MCN1, still reached the STG.
The pyloric cycle period was defined as the interval between the onset
of an impulse burst in the pyloric dilator neuron (PD) and the onset of
the subsequent PD neuron burst. Pyloric cycle frequency was determined
as the reciprocal of the pyloric cycle period. Extracellular recordings
were used to determine the mean number of action potentials per
burst and the intraburst firing frequency for several pyloric
neurons including the lateral pyloric (LP), inferior cardiac (IC), and
ventricular dilator (VD) neurons. LP neuron activity was assessed from
the lateral ventricular nerve (lvn), whereas IC and VD
neuron activity was measured from the medial ventricular nerve
(mvn) (Fig. 1C). For phase analysis, the activity
of the PD and lateral posterior gastric (LPG) neurons was also
determined. PD neuron activity was monitored from the pyloric dilator
nerve (pdn), whereas LPG neuron activity was determined from
intracellular recordings (Fig. 1A). Mean values for
all pyloric-related parameters were determined from measurements of 20 consecutive cycles of pyloric activity. In all experiments, MCN1
activity was controlled directly by the stimulation protocol, as each
extracellular stimulation (duration, 1 msec) elicited a single action
potential from MCN1 (Coleman et al., 1995; Bartos and Nusbaum, 1997).
Data analysis during MPN stimulation came from stretches during which MPN maintained a consistent firing frequency (15-20 Hz) over the sample interval.
The gastric mill rhythm was monitored by activity in the lateral
gastric (LG) and dorsal gastric (DG) neurons. The LG neuron was
recorded either extracellularly from the lateral gastric nerve (lgn) or intracellularly, and the DG neuron was recorded
extracellularly from the dorsal gastric nerve (dgn) (Fig.
1A). Ten consecutive gastric mill cycles (10 cycles
of LG neuron bursting) were used for computing both the mean number of
action potentials per LG neuron burst and the mean value for the
gastric mill cycle period.
Data analysis. Data were recorded onto videotape (Vetter
Instruments, Rebersburg, PA) and chart recorder (Astromed MT-95000; Astro-Med/Grass Inc., West Warwick, RI). Figures were prepared by
scanning sequences of recordings using a Hewlett Packard Scanjet IIc
with Deskscan II (version 2.0) software. Final figures were prepared
with CorelDraw (version 3.0 for Windows). Graphics and statistics were
generated using Sigma Plot 4.0 and Sigma Stat 2.03 (SPSS, Chicago, IL).
Statistical tests used to analyze data were one-way ANOVA, repeated
measures ANOVA, or Friedman repeated measures ANOVA on ranks (with
Tukey's test for multiple comparisons). Data are presented as
means ± SD.
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RESULTS |
Comparison of the MCN1- and MPN-elicited STG rhythms
Selective stimulation of MCN1 and MPN elicits different motor
patterns from the STG network, whether or not the CoGs remain connected
with the STG (Nusbaum and Marder, 1989b; Coleman and Nusbaum, 1994;
Bartos and Nusbaum, 1997; Blitz et al., 1999). Our aim was to determine
the extent to which these different motor patterns resulted from the
actions of CabTRP Ia released by MCN1 within the STG. Therefore, to
avoid the complication resulting from the synaptic actions of MPN on
MCN1 in the CoGs (Blitz and Nusbaum, 1997), we compared these rhythms
in preparations in which the CoGs were removed by transection of the
ions and sons (Fig. 1A).
MCN1 stimulation elicits specific gastric mill and pyloric rhythms
(Coleman et al., 1995; Bartos and Nusbaum, 1997; Blitz et al., 1999),
whereas MPN elicits only a pyloric rhythm, which is distinct from the
MCN1-elicited pyloric rhythm (Nusbaum and Marder, 1989b; Blitz and
Nusbaum, 1997; Blitz et al., 1999) (Fig. 1B,C).
Furthermore, some STG network neurons are activated only during MCN1
stimulation. This includes the LG, DG, and VD neurons (Fig.
1C). The LG and DG neurons are generally active only with the gastric mill rhythm (Weimann et al., 1991). The VD neuron is
generally active with both the pyloric and gastric mill rhythms, yet is
not activated by MPN stimulation with the CoGs disconnected (Nusbaum
and Marder, 1989b; Weimann et al., 1991).
The differences in the pyloric rhythms elicited by these two projection
neurons include distinct phase relationships among the pyloric circuit
neurons (Blitz et al., 1999). We found that there were also differences
in the pyloric cycle frequency and the activity levels of individual
pyloric neurons (Fig.
2A,B). For example, the
pyloric cycle frequency is increased significantly, relative to the
prestimulation level, by both MPN and MCN1 activation (p < 0.05; MPN, n = 17; MCN1,
n = 44) (Fig. 2B). However, during comparable stimulation rates (20 Hz), MCN1 elicits a faster pyloric rhythm than MPN (p < 0.05) (Fig.
2B). Additionally, whereas both MPN and MCN1 enhance
the activity of the LP and IC neurons, these neurons fire significantly
more spikes per burst during the MPN-elicited pyloric rhythm
(p < 0.05) (Fig. 2B). In
contrast, the VD neuron is significantly more active during the
MCN1-elicited pyloric rhythm (p < 0.05) (Fig.
2B). Note also that, during MCN1 stimulation, there
are additional rhythmic changes in the activity levels of the IC and VD
neurons that occur regularly across several pyloric cycles. These
rhythmic changes occur because these two STG neurons also participate
in the gastric mill rhythm that is elicited by MCN1 stimulation (Figs.
1C, 2A).
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Figure 2.
The modulatory neurons MCN1 and MPN elicit
distinct pyloric rhythms. A, Extracellular recordings of
the pyloric rhythm response to comparable stimulation of MCN1 and MPN.
B, The pyloric cycle frequency and the activity level of
several pyloric neurons are significantly enhanced by stimulation of
either MCN1 or MPN, relative to ongoing pyloric rhythms during saline
superfusion in the absence of stimulation. Additionally, there are
significant differences in each of the analyzed parameters during MCN1
versus MPN stimulation. Each bar represents the mean ± SD for the
indicated parameter. MCN1 stimulations: n = 44; MPN
stimulations: n = 17. Comparison between MCN1 and
saline: * p  0.05. MPN is significantly different
from both saline alone and MCN1 stimulation:  p 0.05.
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Spantide I blocks the effects of neurally released CabTRP Ia
MPN and MCN1 share two transmitters, GABA and the peptide
proctolin, whereas MCN1 also contains the peptide cotransmitter CabTRP
Ia (Fig. 1B; Blitz et al., 1999). We therefore aimed
to determine whether the different STG circuit responses to MPN and MCN1 stimulation resulted from the actions of CabTRP Ia. To this end,
we studied the STG response to MCN1 stimulation in the presence of the
broad spectrum tachykinin receptor antagonist Spantide I (Folkers et
al., 1984; Nässel, 1999). We showed previously that Spantide I
blocks the actions of bath applied CabTRP Ia, but not proctolin, on the
STG (Christie et al., 1997a).
Spantide I (2 × 105
M) effectively and reversibly diminished the actions of
MCN1 stimulation on the gastric mill and pyloric rhythms (Fig.
3). In the absence of MCN1 stimulation,
the gastric mill rhythm is never spontaneously active with the CoGs
disconnected. Under these conditions, MCN1 stimulation routinely
activates this rhythm (Coleman and Nusbaum, 1994). During MCN1
stimulation in the presence of Spantide I, however, the gastric mill
rhythm was no longer activated (n = 41 of 41) (Fig. 3).
This inability to elicit the gastric mill rhythm appeared to result
primarily from the reduction or elimination of the CabTRP Ia-mediated
actions of MCN1 on the LG neuron. The LG neuron receives both
modulatory and electrical excitation from MCN1 (Coleman et al., 1995).
This excitation is necessary for generation of this gastric mill rhythm (Coleman et al., 1995; Nadim et al., 1998; Bartos et al., 1999). In
many of these preparations (30 of 41, 73%), Spantide I superfusion completely eliminated LG neuron impulse activity in response to MCN1
stimulation. All that remained of the LG neuron response to MCN1
stimulation in these preparations were the EPSPs resulting from their
electrical coupling (Fig. 3). In the remaining preparations (11 of 41, 27%), there was weak and intermittent spiking in the LG neuron when
MCN1 was stimulated.
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Figure 3.
Application of the tachykinin receptor antagonist
Spantide I effectively weakens the gastric mill and pyloric circuit
responses to stimulation of MCN1. During saline superfusion, MCN1
stimulation elicited the gastric mill rhythm (dgn,
LG) and enhanced the pyloric rhythm (lvn,
mvn). When MCN1 was stimulated during Spantide I (2 × 105 M)
superfusion, the gastric mill rhythm was not activated. Note that,
during Spantide I superfusion, the LG neuron response to MCN1 was
subthreshold and consisted of only unitary electrical EPSPs, whereas
the DG neuron fired tonically instead of rhythmically. Also, the
pyloric rhythm response was reduced relative to MCN1 stimulation in
saline. The effects of Spantide I were reversible with saline wash. The
middle amplitude unit in the dgn represents activity of
the anterior gastric receptor (AGR) sensory neuron, whereas the
smallest units are stimulus artifacts resulting from tonic stimulation
of the ions to activate MCN1. Most hyperpolarized LG
Vm: saline, 71 mV; MCN1 during saline,
73 mV; MCN1 during Spantide, 68 mV; saline after Spantide, 73
mV.
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Another consistent consequence of MCN1 stimulation during Spantide I
application was that DG neuron activity switched from rhythmic impulse
bursts to tonic firing (83% of preparations; n = 24)
(Fig. 3). Although the timing of DG neuron bursts during the gastric
mill rhythm is regulated by LG neuron activity, DG rhythmicity during
MCN1 stimulation normally persists when the LG neuron is
hyperpolarized, and the gastric mill rhythm is thereby suppressed
(Coleman and Nusbaum, 1994).
The pyloric system response to MCN1 stimulation was also consistently
weakened by the application of Spantide I (Fig. 3). This included a
significantly slower pyloric cycle frequency and weaker activity in at
least several pyloric circuit neurons (Fig. 4). The pyloric cycle frequency was
reduced from 0.93 ± 0.12 Hz during MCN1 stimulation in normal
saline to 0.78 ± 0.13 Hz (p < 0.001;
n = 29) when the stimulation occurred during Spantide I
superfusion. The cycle frequency occurring during MCN1 stimulation with
Spantide I present did, however, remain significantly faster than that
of the ongoing pyloric rhythm without MCN1 stimulation (0.47 ± 0.26 Hz; p < 0.001; n = 29).
Similarly, the LP, IC, and VD neurons all showed significant decreases
in their activity when MCN1 was stimulated during Spantide I
superfusion (p < 0.001; n = 29)
(Figs. 3, 4). All of these neurons, however, remained more active than
without any MCN1 stimulation (Fig. 4). Without MCN1 stimulation,
Spantide I did not alter either ongoing pyloric rhythm cycle frequency
or the activity level of individual pyloric neurons (t test,
n = 29, p > 0.05).
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Figure 4.
Quantitative comparison of pyloric rhythm
parameters during tonic stimulation of MCN1 before and during Spantide
I application (2-5 × 105
M). The pyloric cycle frequency was significantly increased
when MCN1 was stimulated, whether or not Spantide I was present.
However, MCN1 stimulation elicited a significantly faster cycle
frequency during saline superfusion than when Spantide I was applied.
The same results pertained to the activity level of at least three
pyloric neurons, including the lateral pyloric (LP),
inferior cardiac (IC), and ventricular dilator
(VD) neurons. Significant difference from saline control
indicated by * (p 0.001;
n = 29). Significant difference from saline control
and MCN1 stimulation in saline indicated by (p 0.001; n = 29).
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In the experiments described above, it was difficult to determine the
immediate cause of the changes in these rhythms during Spantide I
application because the MCN1-elicited gastric mill and pyloric rhythms
also influence one another via synaptic pathways within the STG (Bartos
and Nusbaum, 1997; Bartos et al., 1999). Additionally, several STG
neurons participate in both of these rhythms (e.g., IC and VD neurons;
Fig. 1C). The LG neuron, whose activation by MCN1 is
suppressed by Spantide I, is the gastric mill neuron that regulates the
actions of MCN1 on the pyloric circuit, via its presynaptic inhibition
of MCN1 (Coleman and Nusbaum, 1994; Bartos and Nusbaum, 1997).
Therefore, we aimed to determine whether the reduced response of the
pyloric circuit to MCN1 stimulation during Spantide I superfusion was
actually a secondary consequence of the elimination of the gastric mill
rhythm under these conditions. To this end, we compared the pyloric
system response to MCN1 stimulation with and without Spantide I present
during times when LG neuron activity was suppressed by hyperpolarizing
current injection.
As reported previously (Bartos and Nusbaum, 1997), the pyloric rhythm
was still excited by MCN1 stimulation when the LG neuron was
inactivated by hyperpolarizing current injection. This excitation was
stronger in normal saline than during Spantide I superfusion (Fig.
5). With Spantide I present and LG neuron
activity suppressed, MCN1 stimulation elicited a pyloric rhythm that
was significantly slower than the comparable stimulation in normal
saline (p < 0.02; n = 22), but
still significantly faster than the spontaneously active pyloric rhythm
without MCN1 stimulation (p < 0.001;
n = 22) (Fig. 5B). This result was
consistent with the effects of Spantide I seen when LG neuron activity
was not suppressed (Fig. 4). The same results were obtained with
respect to changes in pyloric neuron activity levels. With LG neuron
activity eliminated, MCN1 stimulation in normal saline increased the
activity of the LP, IC, and VD neurons (Fig. 5B). When
Spantide I was added to the bath, MCN1 stimulation still significantly
increased the activity of these three neurons relative to their
prestimulation activity levels, but to a lesser degree than in normal
saline (p < 0.02; n = 22) (Fig.
5B). Thus, the reduced level of excitation provided by MCN1
to the pyloric system during pharmacological block of the actions of
CabTRP Ia are independent of the MCN1 actions on the gastric mill
circuit.
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Figure 5.
The MCN1-elicited pyloric rhythm is still altered
when CabTRP Ia actions are blocked with Spantide I during suppression
of the gastric mill rhythm. A, When the gastric mill
rhythm was suppressed by maintained hyperpolarization of the LG neuron
(note the lack of gastric mill-timed activity in the IC and VD
neurons), the pyloric rhythm (lvn, mvn) was still
enhanced by MCN1 stimulation during superfusion of saline either alone
or with Spantide I (2 × 105 M). However,
the MCN1-elicited pyloric rhythm is slower and weaker during the latter
condition. B, Quantitative comparison of several pyloric
rhythm parameters under these conditions shows that the pyloric cycle
frequency and activity level of the LP, IC, and VD neurons are all
enhanced by MCN1 stimulation. However, all of these parameters are
significantly reduced during Spantide I application. Significant
difference from saline control indicated by *
(p 0.05; n = 22).
Significant difference from saline control and MCN1 activation with LG
hyperpolarized in normal saline is indicated by (p 0.05; n = 22).
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Specificity and effectiveness of Spantide I block of CabTRP
Ia actions
The gastric mill and pyloric neurons that exhibit reduced
responses to MCN1 stimulation with Spantide I present are also among the subset of STG neurons that are excited during CabTRP Ia application (Christie et al., 1997b) (D. E. Wood, A. E. Christie, W. Stein, and M. P. Nusbaum, unpublished observations). Moreover,
Spantide I appears to selectively block only the CabTRP Ia component of the MCN1 actions in the STG. For example, at Spantide I concentrations (104
M) that eliminate the actions of bath-applied CabTRP Ia
(106
M) on the STG, there is no change in the pyloric rhythm
response to proctolin
(106
M) superfusion (Christie et al., 1997a). Proctolin and GABA
are cotransmitters of CabTRP Ia in MCN1 (Blitz et al., 1999).
Additionally, Spantide I (2-5 × 105
M) did not interfere with the actions of proctolin and GABA
on pyloric neurons when these transmitters were released from MPN (Fig.
6). Specifically, MPN stimulation
enhanced the pyloric cycle frequency and the activity of individual
pyloric neurons to the same extent, relative to prestimulation levels,
whether or not Spantide I was present (p < 0.001; n = 8) (Fig. 6B). This
included excitation of those STG neurons (LP, IC, and VD neurons) that showed decreased responsiveness to MCN1 activation with Spantide I
present.
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Figure 6.
The pyloric circuit response to MPN stimulation is
not altered by application of Spantide I. A, The MPN (20 Hz)-elicited pyloric rhythm was not changed when Spantide I (2 × 10 5 M) was added to the
bath. B, Comparison of parameters of the MPN-elicited
pyloric rhythm before and during Spantide I application shows no
significant differences in the pyloric rhythm cycle frequency or the
number of spikes per burst fired by the LP, IC, or VD neurons.
Significant difference from saline control indicated by *
(p 0.05; n = 8).
Prestimulation Vm: MPN, 55 mV.
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We also tested whether there were nonspecific effects of Spantide I on
the gastric mill circuit. This was done by examining the actions of
Spantide I on a gastric mill rhythm that does not involve the
participation of MCN1. We elicited this gastric mill rhythm via
extracellular stimulation of the dpon, which activates a
sensory pathway that projects through this nerve to the CoGs and STG
(Blitz et al., 1998; Beenhakker et al., 2000) (Fig.
1A). Stimulating this pathway for 2-3 min activated
a gastric mill rhythm that persisted for an additional 5-10 min. In
these experiments, we transected both ions to prevent any
influence of MCN1 on the STG. The gastric mill rhythm elicited by
dpon stimulation had similarities and differences to that
elicited by selective stimulation of MCN1. One similarity that is
evident in Figure 7 is that both rhythms
included rhythmic alternating bursting between the LG and DG neurons.
One distinction between these rhythms is that the gastric mill (GM)
neurons generated regular bursting activity only during the
dpon-elicited rhythm (Fig. 7). Another difference, not shown
in Figure 7, was that IC neuron activity was eliminated during each LG
neuron burst only in the dpon-elicited rhythm. Consistent
with our earlier experiments (Fig. 3), we were not able to elicit the
gastric mill rhythm by stimulating MCN1 during Spantide I
(105
M) superfusion (Fig. 7A). In contrast,
in these same preparations during Spantide I superfusion, the gastric
mill rhythm was still readily elicited by dpon stimulation
(Fig. 7B) (n = 5 of 5 preparations). This
included rhythmic bursting in all the same gastric mill neurons, including the LG, DG, and GM neurons (Fig. 7B).
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Figure 7.
The tachykinin receptor blocker Spantide I
selectively blocks gastric mill rhythm activation by MCN1.
A, Tonic stimulation of MCN1 elicited the gastric mill
rhythm during saline superfusion but not during Spantide I application
(105 M). The
gastric mill rhythm is represented by rhythmic alternating impulse
bursts in the LG and DG neurons. The two nonrhythmic units recorded on
the dgn are the AGR sensory neuron and the gastric mill
(GM) motor neurons. B, Stimulation
of the dorsal posterior oesophageal nerve (dpon) during
saline superfusion activated a different version of the gastric mill
rhythm, which then persisted for ~10 min after stimulation. MCN1 did
not contribute to this gastric mill rhythm because the ions
were transected. This gastric mill rhythm was again activated by
dpon stimulation in the presence of Spantide I
(105 M). Note that,
in contrast to the MCN1-elicited rhythm, the GM neurons
(dgn) participated in the dpon-elicited
gastric mill rhythm. A and B were from
the same experiment, and stimulations occurred during the same
applications of Saline (middle) and Spantide I
(right). In each case, dpon stimulation was
performed after the effects of MCN1 stimulation subsided. Most
hyperpolarized LG Vm: Prestimulation, 64
mV; Saline, 67 mV; Spantide, 68 mV.
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We next aimed to verify that Spantide I completely eliminated the
gastric mill and pyloric responses to neurally released CabTRP Ia. To
this end, we attempted to strengthen the actions of neurally released
CabTRP Ia within the STG by increasing both its local concentration and
the duration of its presence, by preventing its enzymatic degradation.
Many tachykinins are cleaved and inactivated in the extracellular space
by neutral endopeptidase-24.11 (NEP), and NEP activity is effectively
inhibited by phosphoramidon in both vertebrates and invertebrates
(Turner et al., 1987; Roques et al., 1993; Saleh et al., 1996; Zappulla
et al., 1999).
We first examined whether phosphoramidon
(105
M) superfusion would selectively enhance the actions of
applied CabTRP Ia in the crab STG. We found that this endopeptidase
inhibitor
(105
M) did indeed reliably enhance the actions of focally
applied CabTRP Ia (pipette concentration,
104
M). For example, we determined the time required for each
measured parameter to return to preapplication levels after the end of a standard duration (2 sec) and intensity (6 PSI) presentation of this
neuropeptide. As shown in Figure 8, the
LG neuron response to applied CabTRP Ia was intensified and prolonged
in the presence of phosphoramidon (p < 0.01;
n = 8). So too was the pyloric cycle frequency, which
was increased by CabTRP Ia application for 64.8 ± 40.4 sec during
saline superfusion and for 136.6 ± 83.8 sec with phosphoramidon
present (p < 0.05; n = 7). We
showed previously that there is also an increased pyloric rhythm
response to both applied and neurally released proctolin after blocking
extracellular aminopeptidase activity in the STG (Coleman et al., 1994;
Nusbaum and Wood, 1999). Because proctolin is a cotransmitter of CabTRP Ia within MCN1, we also examined whether phosphoramidon altered the STG
response to proctolin application. There were no changes in the pyloric
rhythm response to focally applied proctolin (pipette concentration,
104
M) during phosphoramidon superfusion
(105
M) (Fig. 8, p > 0.05;
n = 7). The LG neuron was not directly affected by
proctolin application whether or not phosphoramidon was present.
However, under these conditions the LG neuron did exhibit subthreshold
pyloric-timed membrane potential oscillations (Fig.
8B). These were because of proctolin excitation of
the pyloric rhythm. The LG neuron receives pyloric-timed synaptic
inhibition (Bartos et al., 1999). The presence of phosphoramidon itself
did not alter any aspect of STG activity in the absence of either peptide application or projection neuron stimulation
(p > 0.05; n = 23).
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Figure 8.
The endopeptidase inhibitor phosphoramidon
selectively enhances the actions of focally applied CabTRP Ia on the
STG. A, Focal application of CabTRP Ia
(104 M in puffer
pipette, 6 PSI) onto the desheathed STG neuropil excited the LG neuron
and enhanced the pyloric rhythm (lvn). The response of
both the pyloric neurons and the LG neuron to CabTRP Ia was increased
in strength and duration during superfusion of phosphoramidon
(105 M), in a
reversible manner. B, Focal application of proctolin
(104 M in puffer
pipette, 6 PSI) onto the STG neuropil excited the pyloric rhythm
(lvn) but did not excite the LG neuron. Note that there
was no sustained depolarizing response in the LG neuron. The onset of
the pyloric-timed subthreshold oscillations in the LG neuron after
proctolin application was an indirect result of proctolin excitation of
the pyloric circuit. There was no change in the response of either the
pyloric neurons or the LG neuron to proctolin application during
phosphoramidon (105
M) superfusion. Most hyperpolarized LG
Vm: 62 mV. C, Superfusion
of phosphoramidon significantly prolonged the excitatory actions of
focally applied (2 sec, 6 PSI) CabTRP Ia, but not proctolin, on the
pyloric rhythm and the LG neuron. The asterisks indicate
that, relative to pre and postapplication saline controls,
phosphoramidon significantly increased the duration of CabTRP Ia action
on the pyloric cycle frequency (p < 0.05;
n = 7), the number of LP spikes/pyloric-timed burst
(p < 0.01; n = 7), and
the depolarization of the LG neuron (p < 0.01; n = 8). In contrast, phosphoramidon neither
enhanced nor prolonged the pyloric rhythm response to proctolin
application (p > 0.05;
n = 7). Duration of response of all three
parameters was measured from the end of the peptide application to the
return of that parameter to prepeptide application levels.
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Phosphoramidon
(105
M) also enhanced the STG response to MCN1 stimulation. For
example, during the MCN1-elicited gastric mill rhythm, there was an
increased number of spikes per burst in the LG neuron
(p < 0.02; n = 5) and an
increase in the gastric mill cycle period (p < 0.02; n = 5) (Fig. 9). DG
neuron burst duration also appeared to increase during these gastric
mill rhythms with phosphoramidon present (Fig. 9A), but we
did not determine whether this change was a direct consequence of
phosphoramidon application or an indirect result of the change in LG
neuron activity. During MCN1-elicited gastric mill rhythms, the LG
neuron regulates the timing and duration of DG neuron activity (Coleman
and Nusbaum, 1994).
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Figure 9.
Phosphoramidon does not enable neurally
released CabTRP Ia to overcome the blocking actions of Spantide I. A, The presence of the peptidase inhibitor
phosphoramidon (105
M) enhanced the LG neuron response to MCN1 stimulation.
Note the considerable increase in both the number of action potentials
and the burst duration in the LG neuron when MCN1 was stimulated in the
presence of phosphoramidon. As in Figure 3, the larger tonically active
unit is the AGR neuron, whereas the smaller tonic unit represents
stimulus artifacts from ion stimulation. Most hyperpolarized LG
Vm: 68 mV. B,
Phosphoramidon did not rescue the LG neuron response to MCN1
stimulation in the presence of Spantide I. Activation of MCN1 (20 Hz)
readily elicited the gastric mill rhythm during saline superfusion
(Sal.) or in the presence of phosphoramidon
(P; 105
M). However, during phosphoramidon superfusion, both LG
neuron activity (number of LG spikes per burst) and the gastric mill
cycle period were significantly increased relative to MCN1 stimulation
during saline superfusion (both parameters: * represents
p < 0.02; n = 5). In contrast,
there was neither LG neuron activity nor a gastric mill rhythm elicited
when MCN1 was stimulated in the presence of Spantide I
(S; 2-5 × 105 M), with or
without coapplication of phosphoramidon
(105 M)
(n = 5). Neither the LG neuron nor the gastric mill
rhythm were active without MCN1 stimulation during saline superfusion.
C, Pre-MCN1 stimulation controls during saline
superfusion; Sal. 1, saline superfusion before Spantide
I application; S, Spantide I application;
P, phosphoramidon application; P/S,
phosphoramidon and Spantide I coapplication; Sal. 2,
saline superfusion after washout from P/S.
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Despite the ability of phosphoramidon to increase and prolong the
actions of focally applied and neurally released CabTRP Ia, the STG
response to MCN1 stimulation during coapplication of phosphoramidon and
Spantide I was the same as during application of Spantide I alone (Fig.
9B). For example, under both conditions the LG neuron
response to MCN1 stimulation was limited to the presence of electrical
EPSPs, as shown for Spantide I alone in Figure 3. There was no spiking
activity elicited from the LG neuron during these stimulations. Thus,
the gastric mill rhythm was never activated (5 of 5 preparations) (Fig.
9B). Similarly, the DG neuron continued to respond to MCN1
stimulation by firing tonically when phosphoramidon was coapplied with
Spantide (n = 5). There was also no difference in the
pyloric rhythm response to MCN1 stimulation when Spantide I was
superfused with or without phosphoramidon (p > 0.05; n = 5). The reduced effectiveness of MCN1
stimulation under both conditions was readily reversible with
subsequent saline superfusion (Fig. 9B). These results are
therefore consistent with the conclusion that Spantide I is effectively
blocking the actions of CabTRP Ia on the gastric mill and pyloric rhythms.
Different projection neurons with proctolin and GABA
cotransmitters have distinct actions on the pyloric circuit
One aspect of our working hypothesis, that CabTRP Ia was
responsible for the distinct actions of MCN1 and MPN on the STG
circuits, was confirmed by showing that MCN1 no longer elicited a
gastric mill rhythm in the presence of Spantide I (Fig. 3). We next
examined whether the same pyloric rhythm was elicited by MCN1 and MPN
when Spantide I was present. To ensure a direct comparison, we injected hyperpolarizing current into the LG neuron to eliminate any residual activity occurring during MCN1 stimulation. When these two projection neurons were stimulated at comparable firing rates under these conditions, the pyloric rhythms elicited by each neuron were more similar to one another than in normal saline, but they remained distinct (Fig. 10). Specifically, the
pyloric cycle frequency was now the same during MCN1 stimulation with
the LG neuron hyperpolarized and Spantide I present (0.87 ± 0.11 Hz; n = 29) and during MPN stimulation with Spantide I
present (0.83 ± 0.07 Hz; p > 0.05; n = 17) (Fig. 10C). However, the activity
level of the LP, IC, and VD neurons remained distinct during
stimulation of these two projection neurons (Fig. 10C). The
LP and IC neurons still fired significantly more action potentials per
pyloric cycle during MPN stimulation (p 0.05;
n = 17), whereas VD neuron activity remained stronger
during MCN1 stimulation (p 0.05;
n = 17). There were also differences in the intensity
of firing in some pyloric neurons during MCN1 and MPN stimulation under
these conditions. For example, the intraburst firing frequency of the
PD and IC neurons was significantly higher during MPN than MCN1
stimulation (p 0.05; n = 7)
(Fig. 11), although the firing rates of
these neurons during MCN1 stimulation were higher than during the
spontaneously cycling rhythm (Fig. 11). Although there were more LP
neuron impulses per cycle when MPN was activated than during MCN1
stimulation, in these experiments there was no difference in LP neuron
firing frequency under these two conditions (Fig. 11).
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Figure 10.
The MCN1- and MPN-elicited pyloric rhythms remain
distinct during Spantide I (2 × 105 M) application.
A, An example of the MCN1-elicited pyloric rhythm during
Spantide I application. LG neuron activity was suppressed with
hyperpolarizing current injection. B, An example of the
MPN-elicited pyloric rhythm from the same preparation shown in
A. LG neuron activity was suppressed as in
A. Note the differences between this rhythm and the
MCN1-elicited rhythm in A. For example, the VD neuron
was only active during the MCN1-elicited pyloric rhythm, whereas the IC
neuron was more active during the MPN-elicited rhythm.
Vm: MPN, 55 mV. C,
Quantitative comparison of several parameters from these two pyloric
rhythms reveals there is no difference in the pyloric cycle frequency,
but that differences persist in the activity levels of the LP, IC, and
VD neurons. The IC and LP neurons produced more spikes per burst during
MPN stimulation, whereas the VD neuron was active only during MCN1
stimulation. Significant difference from saline control indicated by *
(p 0.05; n = 20).
Significant difference from saline control and MCN1 stimulation
indicated by (p 0.05;
n = 17).
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Figure 11.
Intraburst firing frequency in some pyloric
neurons is different during the MPN- and MCN1-elicited pyloric rhythms
with Spantide I (2-5 × 105 M) present and
the LG neuron hyperpolarized. The PD, LP, and IC neurons exhibited
increased firing intensity when MCN1 or MPN was stimulated, relative to
their prestimulation values (indicated by *p 0.05; n = 7). PD and IC neuron firing frequency was
significantly higher during MPN than MCN1 stimulation. Significant
difference during MPN stimulation, relative to saline controls and MCN1
activation indicated by (p 0.05;
n = 7).
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Direct comparison of the three-phase pyloric activity patterns produced
by MPN and MCN1, with CabTRP Ia actions blocked and LG neuron activity
suppressed, revealed that these patterns were only equivalent for the
first phase of the pattern (Fig. 12).
During this phase, the coactive PD and LPG motor neurons fired their impulse bursts for the same fraction of the cycle (duty cycle) during
stimulation of either projection neuron. In contrast, the duty cycles
of all of the other pyloric motor neurons were different during these
two pyloric rhythms (p 0.05;
n = 6). Three of these neurons (IC, LP, and PY) had
significantly longer duty cycles during MPN stimulation, whereas the
fourth motor neuron (VD) had a longer duty cycle during MCN1
stimulation. There was no VD neuron activity during MPN stimulation
(Figs. 1C, 2, 6, 9). The difference in the IC, LP, and PY
neuron duty cycles resulted from significant phase delays in their
burst onset during MCN1 stimulation (Fig. 12).
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Figure 12.
Distinct pyloric motor patterns were elicited by
MPN and MCN1 stimulation when CabTRP Ia actions were blocked by
Spantide I (2-5 × 105
M). The phase relationships (the fraction of a normalized
pyloric cycle during which each neuron is active) are shown as a
function of the normalized pyloric cycle period. Pyloric cycle period
is arbitrarily designated as beginning with burst onset in the PD
neuron, and ending with the onset of the next PD burst. Top
panel, Pyloric motor pattern during tonic MPN stimulation (20 Hz). Bottom panel, Activity pattern of the same neurons
during tonic MCN1 stimulation (20 Hz). The phase of burst onset and
offset for the LPG neuron, and offset of the PD neuron, was not
significantly different between the conditions
(p > 0.05; n = 6).
There were also no differences in the phase of burst offset of the IC,
LP, and PY neurons when either projection neuron was stimulated, but
the phase of burst onset for each of these neurons occurred earlier
during MPN stimulation (: p 0.05;
n = 6). Burst onset and offsets phases for the VD
neuron were not compared because VD was not active during MPN
activation (n = 6 of 6).
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| |
DISCUSSION |
In this study we found that cotransmitter complement is one means
by which projection neurons elicit distinct motor patterns from the
same multifunctional network. Our results show that the presence of the
peptide cotransmitter CabTRP Ia in the modulatory projection neuron
MCN1 contributes to the distinct motor patterns elicited from the STG
network by MCN1 and MPN. MCN1 and MPN share the cotransmitters GABA and
proctolin, but only MCN1 contains CabTRP Ia (Fig. 1B)
(Blitz et al., 1999). With CabTRP Ia actions on the STG blocked by
Spantide I, the STG response to MCN1 activation more closely resembled
its response to MPN activity, but differences remained.
The STG response to MCN1 and MPN became more similar in several ways
with the actions of CabTRP Ia blocked. Most dramatically, the presence
of Spantide I eliminated the activation of the gastric mill rhythm by
MCN1, so that in this condition MCN1 only modified the pyloric rhythm.
MPN stimulation never activates the gastric mill rhythm (Blitz and
Nusbaum, 1997). The MPN- and MCN1-elicited pyloric rhythms also became
more similar with Spantide I present as the pyloric cycle frequency
became equivalent during stimulation of either neuron. However, the
activity levels of many pyloric neurons and the pyloric neuron phase
relationships that help define the motor pattern remained distinct.
Different modulatory neurons with a common peptide transmitter,
myomodulin, also have distinct actions on the feeding motor pattern
in Aplysia (Hurwitz et al., 1999; Xin et
al., 1999). However, it is not known whether the different actions
of these neurons result from different actions of myomodulin or as yet
unidentified cotransmitters.
The fact that the MPN- and MCN1-elicited pyloric rhythms remained
different when the actions of CabTRP Ia were blocked suggest that the
actions of proctolin and/or GABA are different when released into the
STG by MPN and MCN1. With respect to proctolin, these two projection
neurons may differ in the amount that they release, the local
concentration of released proctolin at the proctolin receptors, and/or
in the access of released proctolin to all proctolin receptors. There
is precedence in the Aplysia neuromuscular system for
different identified neurons releasing different amounts of the same
neuropeptide at the same firing frequency (Vilim et al., 2000). Any
differences in levels of released proctolin could produce different
pyloric rhythms, insofar as concentration-dependent modulation of the
pyloric rhythm occurs during bath application of several
neuromodulators (Flamm and Harris-Warrick, 1986; Weimann et al., 1997;
Ayali and Harris-Warrick, 1999). Any differences in local proctolin
levels are likely to result in part from the proctolinergic actions of
MPN and MCN1 being sculpted differently by extracellular peptidase
activity (Nusbaum and Wood, 1999). Extracellular aminopeptidase
activity in the STG degrades and inactivates proctolin (Coleman et al.,
1994). Peptidase activity limits the actions of neurally released
peptides in many systems (Squire et al., 1991; Rose et al., 1996; Saleh
et al., 1996; Zappulla et al., 1999).
These two projection neurons may also make GABAergic synapses on
different target neurons or have qualitatively distinct actions on the
same (or different) target neurons. Focally applied GABA has both
excitatory and inhibitory actions in the crab STG, including having
both actions on many STG neurons (Swensen et al., 2000). It also
remains possible that the persisting distinct actions of these
projection neurons when the CabTRP Ia receptor is blocked results from
one or both of them containing another cotransmitter. It seems unlikely
that an additional transmitter participates in the MPN modulation of
the pyloric rhythm because these actions are mimicked by proctolin
superfusion (Nusbaum and Marder, 1989b). Thus far, each neuron is known
not to contain a number of transmitters that have been localized to
other STG inputs (Blitz et al., 1999).
There are no available pharmacological tools for determining the extent
to which the different pyloric rhythms elicited by MCN1 and MPN in the
presence of Spantide I result from differences in proctolin and/or GABA
actions. There are no effective proctolin receptor blockers available
(M. P. Nusbaum, unpublished observations), and the available GABA
antagonists do not effectively block all GABA actions in the STG
(Swensen et al., 2000).
Aside from the possibility of additional chemical cotransmitters, there
is another form of synaptic transmission that is used differently by
MPN and MCN1. Specifically, electrical transmission plays an important
role in enabling MCN1 to elicit the gastric mill rhythm (Coleman et
al., 1995; Nadim et al., 1998), and MCN1 is also electrically coupled
to several pyloric neurons (Coleman, 1995; Norris et al., 1995). There
is no evidence for electrical transmission involving MPN, and it is
clear that at least some of these electrical synapses are unique to
MCN1 because MPN has no actions whatsoever on at least some of the
gastric mill neurons to which MCN1 is electrically coupled (Blitz and
Nusbaum, 1997).
MCN1 and MPN appear to share many neuronal targets within the STG.
However, two targets that are influenced only by MCN1 are the LG and DG
neurons of the gastric mill system (Coleman and Nusbaum, 1994; Coleman
et al., 1995; Blitz and Nusbaum, 1997). Neither of these neurons are
responsive to proctolin application, but both show reduced responses to
MCN1 stimulation in the presence of Spantide I. In Spantide I, all but
the electrical EPSPs are eliminated in the LG neuron response to MCN1
stimulation, suggesting that MCN1 only uses CabTRP Ia to influence this
neuron. In contrast, the DG neuron response to MCN1 with Spantide I
present is altered from rhythmic oscillatory activity to tonic firing,
suggesting that MCN1 uses both CabTRP Ia and GABA to influence this neuron.
There is also a functional compartmentalization of MPN cotransmitter
actions. Although there is no evidence within the STG regarding the use
of the MPN cotransmitters on individual targets, MPN uses only GABA to
influence some projection neurons in the CoGs (Blitz and Nusbaum,
1999). Similarly, in the neuroendocrine bag cell system of
Aplysia, the coreleased 
bag cell peptide and egg-laying
hormone do not influence all of the same target neurons (Mayeri et al.,
1985; Sigvardt et al., 1986).
Another possible explanation for the continued differences in the STG
response to MCN1 and MPN stimulation is that Spantide I did not
completely block the actions of neurally released CabTRP Ia. This
possibility, however, seems unlikely because increasing the
effectiveness of CabTRP Ia, by using a peptidase inhibitor that
enhanced the actions of this peptide, did not rescue any of the MCN1
actions that were suppressed by Spantide I.
If the STG response to MCN1 and MPN during Spantide I application does
indeed remain different because of their distinct use of proctolin
and/or GABA, this would provide further support for the hypothesis that
the use of distinct cotransmitters is only one of several strategies
used by different modulatory projection neurons to elicit distinct
motor patterns from the same network. In this particular case, one
additional strategy involves the use of electrical transmission only by
MCN1 to influence the STG circuits. There is also a major role played
by the pattern of transmitter release. There is a rhythmic release of
the MCN1 cotransmitters, which results from rhythmic presynaptic
inhibition of the STG terminals of MCN1 by a gastric mill neuron (LG
neuron; Fig. 1B) (Coleman and Nusbaum, 1994; Coleman
et al., 1995; Bartos and Nusbaum, 1997). In contrast, the modulatory
actions of MPN appear to result from its tonic release of
neurotransmitters. There is no indication of rhythmic presynaptic
control of the STG terminals of MPN. This is consistent with the fact
that bath applied proctolin and MPN stimulation elicit the same pyloric
rhythm (Nusbaum and Marder, 1989b). Interactions between projection
neurons can also contribute to motor pattern selection. For example,
MPN activity prevents the activation of the gastric mill rhythm in the
STG via its inhibitory actions on other projection neurons within the
CoGs (Blitz and Nusbaum, 1997). In conclusion, distinct cotransmitters
clearly provide one means by which different modulatory projection
neurons elicit distinct activity patterns from the same neuronal
network. However, motor pattern selection from a multifunctional
network is a more complex process that also involves a constellation of additional strategies.
| |
FOOTNOTES |
Received May 12, 2000; revised Aug. 29, 2000; accepted Sept. 21, 2000.
This work was supported by National Science Foundation Grant
IBN-9808356 (M.P.N.) and National Institute of Neurological Disorders and Strokes Grant NS29436 (M.P.N.). We thank Dr. Dawn M. Blitz for
helpful discussions and for comments on earlier versions of this paper.
Correspondence should be addressed to Dr. Michael P. Nusbaum,
Department of Neuroscience, University of Pennsylvania School of
Medicine, 215 Stemmler Hall, Philadelphia, PA 19104-6074. E-mail: nusbaum{at}mail.med.upenn.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
