Department of Biology, Bowdoin College, Brunswick, Maine 04011
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dickinson, Patsy S., Jane Hauptman, John Hetling, and Anand Mahadevan. RPCH Modulation of a Multi-Oscillator Network: Effects on the Pyloric Network of the Spiny Lobster. J. Neurophysiol. 85: 1424-1435, 2001. The neuropeptide red pigment concentrating hormone (RPCH), which we have previously shown to activate the cardiac sac motor pattern and lead to a conjoint gastric mill-cardiac sac pattern in the spiny lobster Panulirus, also activates and modulates the pyloric pattern. Like the activity of gastric mill neurons in RPCH, the pattern of activity in the pyloric neurons is considerably more complex than that seen in control saline. This reflects the influence of the cardiac sac motor pattern, and particularly the upstream inferior ventricular (IV) neurons, on many of the pyloric neurons. RPCH intensifies this interaction by increasing the strength of the synaptic connections between the IV neurons and their targets in the stomatogastric ganglion. At the same time, RPCH enhances postinhibitory rebound in the lateral pyloric (LP) neuron. Taken together, these factors largely explain the complex pyloric pattern recorded in RPCH in Panulirus.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The often extensive role that
neuromodulators play in altering the output of rhythmic pattern
generators has been demonstrated in a number of systems (e.g.,
Tritonia swimming, Katz and Frost 1995, 1997
;
Katz et al. 1994
; Willows et al. 1987
;
leech swimming, Angstadt and Friesen 1993
; Mangan
et al. 1994a
,b
; Willard 1981
; leech heartbeat,
Thompson and Calabrese 1992
; crustacean stomatogastric system, Blitz et al. 1995
; Cazalets et al.
1990
; Dickinson and Marder 1989
;
Dickinson et al. 1990
; Flamm and Harris-Warrick
1986
; Harris-Warrick et al. 1997
;
Hooper and Marder 1987
; Katz and Harris-Warrick 1990
; Nagy and Dickinson 1983
; Nusbaum
and Marder 1989
; Weimann et al. 1993
). By
altering such parameters as the cycle frequency, the number of neurons
active in the pattern, and spike frequency in neurons of a target
pattern generator, modulators can substantially change the motor output
of the pattern generator. One such modulator, the peptide red pigment
concentrating hormone (RPCH), has been shown to activate the cardiac
sac motor pattern in the spiny lobster, Panulirus
interruptus (Dickinson and Marder 1989
;
Dickinson et al. 1993
), and to activate the pyloric
rhythm in the crab, Cancer borealis (Nusbaum and
Marder 1988
). Immunocytochemical studies have shown that an
RPCH-like peptide is present in all the ganglia of the stomatogastric
system of Panulirus (Dickinson and Marder 1989
). However, the effects of RPCH on the pyloric rhythm in
Panulirus have not yet been described.
In recent years, it has become clear that the pattern
generators that underlie rhythmic behaviors in a variety of both
vertebrate and invertebrate systems are not independent but instead
interact in a variety of ways (Chrachri and Neil 1993;
Chrachri et al. 1994
; McFarland and Lund
1993
; Meyrand et al. 1994
; Weimann and Marder 1994
). These interactions have been most extensively
examined in the four motor pattern generators of the crustacean
stomatogastric nervous system (for reviews, see Dickinson
1995
; Dickinson and Moulins 1992
). The
functional boundaries of these pattern generators are fluid, and
neurons can be shared between two networks, providing a pool from which
appropriate combinations of neurons are selected to form the pattern
generators needed at any given time, as is seen in the pyloric and
gastric networks of the crab, C. borealis (Weimann
and Marder 1994
; Weimann et al. 1990
, 1991
). In
other cases, single neurons can switch from one pattern generator to another, as occurs in Palinurus vulgaris (Hooper and
Moulins 1989
, 1990
) and P. interruptus
(Dickinson and Marder 1989
), where neurons that commonly
fire in pyloric or gastric time switch to fire in cardiac sac time. In
more extreme cases, the gastric mill and cardiac sac patterns can fuse
to form a single, conjoint pattern (Dickinson et al.
1990
), or all of the stomatogastric pattern generators can be
broken down and the neurons used to construct a novel pattern generator
that is thought to control swallowing movements (Meyrand et al.
1991
, 1994
). Similar, though much less studied, interactions
have been shown to occur in many other systems as well, including, for
example, the respiratory and locomotor patterns in vertebrates
(Viala 1986
; for review, see Dickinson 1995
).
Given that such interactions can occur, and in many cases are
essential, to generate appropriate behavioral output, it seems likely
that the different pattern generators might in some instances be
modulated in concert rather than individually. Indeed, several neuromodulators or modulatory neurons have been shown to affect more
than one network in the stomatogastric system. For example, the peptide
proctolin co-activates the pyloric (Hooper and Marder 1987; Marder et al. 1986
), gastric
(Heinzel 1988
; Heinzel and Selverston
1988
), and cardiac sac (Dickinson and Marder
1989
) pattern generators in the spiny lobster. The anterior
pyloric modulator neuron modulates both the pyloric (Dickinson
and Nagy 1983
; Nagy and Dickinson 1983
) and
gastric (Dickinson et al. 1988
; Nagy et al.
1988
) pattern generators in the spiny lobster. Likewise, several of the modulatory commissural neurons influence both the pyloric and gastric mill patterns in the crab (Bartos and
Nusbaum 1997
; Coleman and Nusbaum 1994
;
Coleman et al. 1995
; Norris et al. 1994
,
1996
; Nusbaum et al. 1992
). RPCH, like
proctolin, can modulate more than one pattern, as seen by the fact
that, in addition to activating the cardiac sac pattern, it can promote
either the switching of neurons from gastric to cardiac sac time
(Dickinson and Marder 1989
) or the complete fusion of
the cardiac sac and gastric mill patterns (Dickinson et al.
1990
). Interestingly, the modulator dopamine, which activates
the pyloric network, also alters the interactions between the cardiac
sac and pyloric networks, essentially canceling the effects the cardiac
sac normally exerts on the pyloric pattern and restoring a normal
pyloric pattern even during intense cardiac sac bursting (Ayali
and Harris-Warrick 1998
).
The system we have chosen to study here presents an interesting
opportunity to examine the actions of modulators on related interacting
neural networks. In this system, there are several known interactions
between two patterns, the cardiac sac pattern and the pyloric pattern,
in spiny lobsters (Hooper and Moulins 1989, 1990
;
Moulins and Vedel 1977
; Sigvardt and Mulloney
1982b
). Additionally, both of these patterns are modulated by
RPCH in at least some species. We therefore examined the effects of
RPCH on the pyloric pattern in spiny lobsters to complement previous work documenting the peptide's influence on the cardiac and gastric networks of this species, and thus to give a more global view of the
peptide's effects on this group of interacting neural networks.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments were conducted on a total of 82 male and
female California spiny lobsters, P. interruptus, weighing
150-600 g. Animals were purchased from Marinus (Longbeach, CA) or Don
Tomlinson Commercial Fishing (San Diego, CA) and were kept in
recirculating seawater at 12-15°C for 6 wk before use.
Stomachs were removed from lobsters, and the complete stomatogastric
system was dissected from the stomach wall (Selverston et al.
1976) and placed into cold Panulirus saline
[composition (in mM/l): 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10 MgSO4, 11 Trizma base, and 4.8 maleic acid; pH
7.5-7.6] in a silicone elastomer (Sylgard)-lined petri dish.
Preparations included the four ganglia of the system (stomatogastric,
STG; 2 commissural, CG; esophageal, OG), the connecting nerves, and the
motor nerves, as shown in Fig. 1. The
preparation was superfused with saline at 10-12 ml/min throughout the
experiment. Temperature was maintained at 16-18°C using a Peltier
cooling device.
|
The STG was desheathed to allow access to the cell bodies for intracellular recordings. Additionally, in some experiments, the stomatogastric nerve (stn) or the superior and inferior esophageal nerves (sons, ions) were desheathed so that conduction could be blocked with isotonic (750 mM) sucrose; in other experiments, these nerves were cut to block conduction irreversibly. A petroleum jelly wall across the dish allowed the STG to be superfused separately from the other ganglia.
Standard electrophysiological techniques were used in all experiments.
Nerves were recorded extracellularly using A-M Systems AC amplifiers
and stainless steel pin electrodes isolated from the bath with
petroleum jelly. The same electrodes were used for nerve stimulation
via a switch box. For intracellular recordings, we used glass
microelectrodes filled with 2.5 M K Acetate or 3 M KCl (tip
resistances: 10-30 M), and WPI M707 microprobe systems, A-M Systems
DC amplifiers, or an Axoclamp 2B. Data were recorded on a Gould TA4000
recorder and on videotape with a Vetter VCR adapter. In some
experiments, data were taken directly onto a PC or Macintosh computer
using PowerLab (AD Instruments) software for analysis. Current for
controlling intracellular membrane potentials as well as for
extracellular stimulation was from a Grass S88 stimulator via stimulus
isolation units.
RPCH, purchased from Peninsula, was dissolved in 5% dimethyl sulfoxide
(DMSO). Deionized water was added to make a
103 M solution, which was
kept frozen as a stock solution. It was diluted to the final
concentration of 10
6 M in
Panulirus saline just before use. At this RPCH
concentration, DMSO was at a final concentration of 0.005%, which had
no effects in control experiments.
Data were analyzed using two-tailed paired Student's t-tests with GBStat (Dynamic Microsystems, Silver Spring, MD) or a binomial test, as indicated in the text. All n values reported refer to the number of preparations analyzed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since neurons originating in the commissural ganglia (CGs) release
a number of neuromodulators into the STG that act in concert on the
pyloric network, most of our experiments were conducted with input from
the CGs removed, as shown in Fig. 1. This was accomplished either by
cutting the ions and sons or by blocking conduction in those nerves. Under these conditions, the cardiac sac
pattern is never spontaneously active, and the pyloric pattern is less
active than in the unblocked condition; thus excitatory effects,
particularly those that are state dependent (Dickinson and Nagy
1983; Nusbaum and Marder 1989
), are clearer and
more easily studied.
The pyloric pattern in RPCH
(106 M) displayed
different patterns of activity in two time domains after the start of
superfusion. Initially, the pyloric pattern alone was enhanced (Fig.
2A), while later, once the
cardiac sac pattern was activated (see Fig. 5), the pyloric pattern
assumed a more complex form because of the influence of the cardiac sac
network on the pyloric network. In a first step to assess the influence
of RPCH on the pyloric network itself, we examined the changes that
occurred in the pyloric pattern before the cardiac sac pattern was
initiated.
|
While the effects of RPCH may not have been maximal in the relatively
short time (11 min after the onset of RPCH application) before the
cardiac sac pattern was activated, it was nonetheless clear that the
peptide directly activates the pyloric network (Fig. 2). In the
presence of RPCH, preparations that did not express spontaneous pyloric
activity began to display a robust pyloric pattern (Fig.
2A), and pyloric activity increased in those preparations that were already cycling (Fig. 2B). This was seen, for
example, as a significant increase in cycle frequency [from 0.53 ± 0.05 to 0.58 ± 0.04 (SE) Hz, n = 38, paired
t-test; P = 0.02]. However, this increase
was state dependent and varied as a function of control frequency
(i.e., frequency before RPCH was applied; Fig. 2C). Thus, in
preparations that were cycling slowly, cycle frequency increased
substantially (by 46%, from 0.24 ± 0.02 to 0.35 ± 0.05 Hz
in the slowest 25% of preparations, P = 0.017, paired
t-test, n = 10), while it increased little
or not at all in those preparations that were already cycling at a
relatively high frequency.
In the presence of RPCH, but with no cardiac sac pattern, activity in both the pyloric dilator (PD) and lateral pyloric (LP) motor neurons increased significantly as measured by both spike frequency within pyloric bursts and by the number of spikes per burst (Fig. 3A; LP spike frequency, P = 0.003; PD spike frequency, P = 0.032; Fig. 3B, LP spikes/burst, P = 0.039; PD spikes/burst, P = 0.002; paired t-test, n = 17). Additionally, burst duration increased in both the LP and PD neurons (Fig. 3C; LP, P = 0.027; PD, P = 0.012; paired t-test, n = 17). It should be noted that in nearly half of the preparations (30 of 74), the LP neuron was silent in the control. In 17% of these 30 preparations, the LP was activated by RPCH. Neither burst duration nor spike frequency within bursts increased significantly in the ventricular dilator (VD) neuron, although the number of spikes per burst increased significantly, as it did in the other neuronal types (Fig. 3; P = 0.020; paired t-test, n = 14). Activity in the inferior cardiac (IC) neuron also increased in some preparations (n = 2/11), though both IC and PY were usually silent in both control (IC, n = 10/11; PY, n = 30/34) and RPCH (IC, n = 9/11; PY, n = 28/34).
|
An additional change seen in RPCH without cardiac sac activity was in the phase relationships of the PD, LP, and VD neurons (Fig. 4, n = 7; measured only in preparations in which all 3 neurons were active in the control). While the phases in which the PD and VD neurons were active did not change significantly, the end of the LP neuron burst was relatively later in the cycle in RPCH than in the control (paired t-test, P = 0.01). Consequently, the LP burst was relatively longer in RPCH than in control saline (paired t-test, P = 0.01). Additionally, the onset of firing in the VD neuron was in phase with that of the LP neuron in RPCH. As a result, the LP and VD bursts were closer to being in phase with one another in RPCH than in control, in which the VD burst began before that of the LP neuron (paired t-test, P = 0.008).
|
Effects of RPCH when the cardiac sac pattern is active
Later during RPCH superfusion, the pyloric pattern became complex
because the peptide eventually also activates the cardiac sac pattern
(Dickinson and Marder 1989) and because the cardiac sac
pattern itself, via synapses from the inferior ventricular (IV) neurons
onto a number of pyloric neurons (Russell and Hartline 1981
; Sigvardt and Mulloney 1982a
), can then
influence the pyloric pattern (Moulins and Vedel 1977
).
Instead of the regular alternation of bursts in the pyloric neurons
seen in the control (Fig. 5A), the pyloric pattern recorded after longer exposure to RPCH included regular interruptions in cycling, coincident with dilator bursts recorded in cardiac sac neurons (CD2 neuron, Fig. 5B).
Although the details of the complex pattern differed considerably
between preparations, RPCH generally enhanced the pyloric pattern,
particularly in preparations that showed weak pyloric activity under
control conditions (i.e., normal saline, with anterior inputs blocked). One reflection of this enhancement was a highly state-dependent increase in average pyloric cycle frequency, measured between cardiac
sac bursts (Fig. 6). Thus in those
preparations in which the initial cycle frequency was low, cycle
frequency increased significantly [from a mean of 0.24 ± 0.02 to
0.42 ± 0.03 Hz (P < 0.0001, paired t-test,
n = 11) in preparations in which initial cycle
frequency was <0.35 Hz, and from a mean of 0.33 ± 0.03 to 0.43 ± 0.02 Hz (P = 0.027, n = 21) in preparations in which cycle frequency was less than the mean of
all initial cycle frequencies in control preparations with anterior
inputs blocked, i.e., <0.53 Hz]. There was, however, no significant
increase in cycle frequency in those preparations that were already
cycling at higher frequencies (>0.53 Hz). It should be noted that
because blocking the input from the commissural ganglia decreases the
level of activity in the pyloric network, 80% of unblocked
preparations (i.e., 16 of 20) fell into the latter category, whereas
only 27% (i.e., 12 of 44) of the blocked preparations burst at these
higher frequencies in control saline.
|
|
During cardiac sac bursts, seen in Fig. 5 as bursts of action potentials in the cardiac dilator 2 motor neuron (CD2), both the PD and VD neurons fired tonically, while the LP neuron was silent. Moreover, as was the case in RPCH before the onset of cardiac sac bursting, the PY and IC neurons (not shown) were completely silent in both control saline and RPCH in most preparations (PY, n = 28/34; IC, n = 9/11). At the end of a cardiac sac burst, the first pyloric neuron to fire after a cardiac sac burst was in most cases (n = 33/38) the LP. This was then followed by alternating bursts in the PD and LP neurons.
Between cardiac sac bursts, pyloric activity was not constant but instead gradually changed with time. Thus, although average pyloric cycle frequency, measured between cardiac sac bursts, was enhanced as described in the preceding text, these average frequencies are in some respects misleading. In fact, cycle frequency remained constant between cardiac sac bursts in only about half of the preparations, while it gradually increased in the others (see Fig. 7). Likewise, levels of activity within most of the pyloric neurons changed gradually between cardiac sac bursts. In most cases, pyloric activity, as measured by spike frequency and burst duration in the LP and PD neurons, was maximal either immediately after each cardiac sac burst or within the first two pyloric cycles after the cardiac sac burst (n = 24/39). Pyloric activity then gradually diminished until it either ceased or was interrupted by the next cardiac sac burst (Fig. 8, A-D), though these changes in burst characteristics over time were much less pronounced in the PD than in the LP neuron. In some cases (n = 15/39), however, as in the example shown in Fig. 5B, peak pyloric activity (i.e., maximum burst duration and amplitude in the LP neuron) was reached only after several pyloric cycles (3 cycles in 8 preparations; 4 cycles in 7 preparations).
|
|
In contrast to the PD and LP neurons, in which bursting activity was strongest within a few cycles after a cardiac sac burst, the VD neuron fired only weakly, if at all, immediately after a cardiac sac burst (n = 44). Instead, the VD neuron was often silent for a prolonged period after a cardiac sac burst, then gradually resumed its pyloric activity (n = 21/44; Fig. 5B; mvn). Thus, in most cases (n = 27/44), both spike frequency and burst duration increased with time during the cardiac sac interburst interval in the VD neuron (Fig. 8, E and F). In 31% of experiments (n = 12/39), however, there was little change in spike frequency with time after a cardiac sac burst. In the remaining five (of 44) preparations, VD remained entirely silent between cardiac sac bursts. It should be noted that, in many cases (n = 21/44), as in the one shown in Fig. 5B, the activity of the VD neuron was only marginally time-locked to the pyloric rhythm between cardiac sac bursts.
Role of the IV neurons
One major question raised by these results is the extent to which
the effects of RPCH on the pyloric pattern are due to the peptide
itself and the extent to which they are indirect, due to the activation
of the cardiac sac pattern by RPCH. Because the IV neurons, which begin
bursting in the presence of RPCH (Dickinson et al.
1993), synapse directly onto a number of pyloric neurons (Sigvardt and Mulloney 1982a
), a number of the effects
of RPCH on the pyloric network are certainly indirect and due to these synaptic potentials.
We addressed this question in two ways. First, in preparations with CG inputs blocked to remove other modulatory inputs, we examined the effects of RPCH on the pyloric pattern before peptide-induced cardiac sac bursting started. As described in the preceding text, there were very clear effects of RPCH on the pyloric pattern as well as on characteristics of the firing of the LP neuron as is discussed later. Thus it is evident that RPCH exerts at least a part of its effects directly on the pyloric network.
The second way we addressed the contribution of the IV neurons to the RPCH activation of the pyloric pattern was by electrically stimulating the ivn in a burst pattern similar to that occurring during RPCH-evoked cardiac sac activity (average spike frequency, 21.4 Hz; average burst duration, 6.5 s). By stimulating the ivn in repeated bursts (5-s duration, 20 Hz, once every 30 s) in control saline, we produced a complex pyloric pattern that was strikingly similar to that seen during superfusion with RPCH (n = 12; Fig. 9). Both the number of spikes per burst and spike frequency increased in the LP neuron immediately after each stimulated ivn burst (P = 0.009 for number of spikes/burst, P = 0.015 for spike frequency, paired t-tests, n = 7), then gradually decreased as they did after the cardiac sac bursts in RPCH. (See Table 1 for a comparison of burst parameters in the 2 bursts before and after each ivn stimulation.) However, LP burst duration did not increase significantly. In the PD neurons, spike frequency, number of spikes per burst, and burst duration all increased after the stimulated ivn bursts, then gradually decreased (P = 0.0004 for spike frequency; P = 0.0005 for number of spikes/burst; P = 0.043 for burst duration, paired t-tests, n = 8; Table 1). In contrast, both burst duration and number of spikes per burst in the VD neurons decreased after stimulated ivn bursts (P = 0.036 for number of spikes/burst; P = 0.048 for burst duration, paired t-tests, n = 6) though average spike frequency did not change (Table 1). In 33% of preparations (4 of 12), the VD neuron completely stopped firing in the pyloric pattern between stimulated ivn bursts, instead firing only during the ivn stimulation.
|
|
These data suggest that the complex pyloric pattern recorded in RPCH was due largely to the effects of the synapses from the IV neurons. However, the pyloric pattern recorded when the ivn was stimulated in control saline was not identical to that recorded when the ivn was stimulated in RPCH (not shown) or when the IV neurons fired in spontaneous bursts in RPCH. In many preparations, one of the most striking differences was the order in which the LP and PD neurons resumed firing after a cardiac sac burst. After a stimulated ivn burst, the PD neuron fired strong bursts and was most often the first pyloric neuron to fire (75%; 9 of 12 preparations). During the PD burst, the LP neuron was inhibited and so only fired at the end of the PD burst (Fig. 9A). In contrast, after a cardiac sac burst in RPCH, the LP neuron was, nearly always the first neuron to fire (87%; n = 33 of 38; Fig. 9B). It fired strongly after each burst, then gradually decreased its firing intensity.
RPCH enhances postinhibitory rebound in the LP neuron
Many of the effects of RPCH and ivn bursts on the
pyloric pattern can be explained by the synaptic input the pyloric
neurons receive from the IV neurons, but the LP neuron is not one of
the neurons that has been shown to receive a direct postsynaptic
potential (PSP) from the IV neurons (Sigvardt and Mulloney
1982a). In spite of this, it was strongly affected by the
cardiac sac pattern in RPCH. The strong firing in the LP neuron after
cardiac sac bursts might be explained by the enhanced inhibition it
would receive from the PD neuron during such a burst, for the IV
neurons powerfully and directly excite the PD neurons. However, this
would not explain the fact that the LP neuron fired before the PD
neuron in RPCH but after it in response to stimulated ivn
bursts. Because postinhibitory rebound makes a major contribution to
the firing of the LP neuron (Miller 1987
), we postulated
that RPCH might alter the extent or characteristics of postinhibitory
rebound in the LP neuron. To determine whether this was the case, we
held the LP neuron at a constant membrane potential using a second
microelectrode, then injected pulses of hyperpolarizing current into
the neuron in both control saline and RPCH. The resultant
postinhibitory rebound was stronger in RPCH, as evidenced by a higher
spike frequency and a larger depolarization after the hyperpolarizing
pulses in RPCH compared with control (Fig.
10; spike frequency, P = 0.006; depolarization, P = 0.003; n = 13; binomial test).
|
RPCH potentiates synaptic potentials from the IV neurons to its pyloric targets
In addition to its direct effects on the LP neuron, RPCH enhanced
the effects of the IV neurons on the pyloric pattern by potentiating
the excitatory synaptic potentials from the IV neuron to the PD and VD
neurons. To demonstrate this, we recorded the PSPs generated in
response to ivn stimulation with an intracellular electrode
in either the PD or VD neuron, while controlling the membrane potential
of the neuron with a second electrode. The amplitude of the PSPs was
substantially greater in RPCH than in control saline (Fig.
11), though this difference was
significant (paired t-tests, P < 0.05 and
P < 0.01) only at membrane potentials that were
relatively hyperpolarized (more hyperpolarized than 60 mV for the PD
neuron and
70 mV for the VD neuron) and thus further from the
predicted reversal potential. Surprisingly, however, the amplitude of
the PSPs in control saline appeared to remain constant in amplitude
rather than increasing with hyperpolarization as we would predict. One
factor that may partially explain this phenomenon is the increased
amplitude of the hyperpolarization-activated inward current,
Ih, and the resulting decrease in
membrane input resistance at hyperpolarized membrane potentials.
However, this current was clearly present in RPCH as well as in control
saline, so this cannot be the only explanation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For a number of years, the stomatogastric system has served as a
model in elucidating many fundamental principles regarding the
functioning of rhythmic motor pattern-generating networks (Marder and Calabrese 1996; Pearson and Ramirez
1992
). In the present study, we used this system to continue
our investigations into the ways that neuromodulatory substances can
alter, in concert, a number of different, but functionally related,
motor patterns. The role of neuromodulators in determining behavioral
output is extensive; they can specify a particular physiological
configuration from an anatomically defined but physiologically flexible
neuronal network (Getting 1989
; Marder
1991
), thus playing an important role in the selection of motor
patterns that are appropriate for a given set of conditions. By
simultaneously affecting more than one motor program, neuromodulators
also have the potential to alter suites of related behaviors in
concert. Moreover, motor pattern generators interact in a variety of
systems (e.g., vertebrate respiration and swallowing or locomotion,
McFarland and Lund 1995
, 1996; Viala
1986
; Aplysia feeding, Perrins and Weiss
1996
; goldfish escape and swimming, Svoboda and Fetcho
1996
; for review, see Dickinson 1995
), and, as
we have shown here, and has been seen previously (e.g., Ayali
and Harris-Warrick 1998
; Dickinson et al. 1990
;
Hooper and Moulins 1989
, 1990
), neuromodulators can alter the strength of such interactions. Consequently modulators may
also play key roles in coordinating related behaviors or in preventing
incompatible behaviors from occurring simultaneously.
Thus, in the present case, RPCH not only activates the cardiac sac
motor pattern (Dickinson and Marder 1989) and causes the gastric mill and cardiac sac patterns to fuse, thus creating a novel
conjoint pattern (Dickinson et al. 1990
), but it also
activates the pyloric pattern (as it does in crabs; Nusbaum and
Marder 1988
) and enhances the effects of the cardiac sac
pattern on the pyloric pattern. Consequently, the motor behavior of an
entire anatomical part (the foregut) of the animal is altered. The fact
that similar interactions between motor patterns have been seen in
other systems under a variety of conditions suggests that the existence
of such interactions
and perhaps more importantly, the possibility of changes in such interactions, controlled by either hormonal or neuronally released modulators
is widespread among nervous systems.
Interestingly, we found that the time course of the modulation of the
cardiac sac and pyloric patterns by RPCH differed. Early during a bath
application of RPCH, the pyloric pattern alone was modulated. The
cardiac sac pattern was activated later and so did not begin to
influence the pyloric pattern until later in the perfusion. This
difference in timing could be due to different thresholds, with that
for the pyloric pattern being lower, so that it was enhanced before the
concentration reached its final value of
106 M in these
experiments. It might also be attributed to different mechanisms in the
two systems. The cardiac sac pattern might, for example, involve a
longer and/or slower second messenger pathway. Regardless of the
mechanism, this difference in the time of onset of these different
effects of RPCH has important functional consequences: it allows a
single modulator to produce a series of sequential changes in the
overall motor output.
Once the cardiac sac pattern was activated, the pyloric pattern became
more complex, with regular interruptions of pyloric bursting followed
by the return of strong pyloric bursting, which then gradually
diminished. In some respects, the overall pattern resembled a pyloric
rhythm with a cardiac sac pattern superimposed on it. This contrasts
with the conjoint pattern, in which two patterns have completely
merged, that is seen when the gastric and cardiac sac patterns are
recorded in RPCH (Dickinson et al. 1990). It should be
noted that both the gastric mill and cardiac sac patterns were silent
before RPCH application in the previous study, whereas in the
experiments reported here, the pyloric pattern was generally ongoing
while the cardiac sac pattern was silent. Moreover, even in those cases
in which the pyloric network was initially silent, it was activated
before the cardiac sac pattern, whereas gastric mill and cardiac sac
activity began simultaneously in RPCH, with the two patterns fused from
the onset of rhythmic activity.
As is the case with the gastric-cardiac sac interactions in RPCH, a
major mechanism responsible for the increased influence of the cardiac
sac pattern on the pyloric pattern was the increased amplitude of the
PSPs from the IV neurons that drive the cardiac sac pattern onto
several pyloric neurons. These synapses, previously described by
Sigvardt and Mulloney (1982a,b
), Russell and
Hartline (1981)
and Claiborne (1983
, 1984
), are
shown diagrammatically in Fig. 12. In
particular, the IV neurons synapse onto the PD and VD neurons. In both
cases, the synapses are primarily excitatory and are potentiated by
RPCH. In addition, however, the PD neurons are subject to a delayed
inhibition during high-frequency firing (Sigvardt and Mulloney
1982a
,b
) and display enhanced bursting after the end of a train
of IV PSPs (Russell and Hartline 1981
). The pattern of
activity in the PD neurons can be largely explained by this combination
of effects as follows. During the cardiac sac burst, the PD neurons
were usually driven one for one by the larger amplitude excitatory PSPs
(EPSPs) they received from the IV neurons. After the cardiac sac burst,
bursting in the PD neurons was enhanced, as always occurs after a burst
in the IV neurons. There is at present no clear evidence that the burst
enhancement in RPCH differs from that in control saline. As this burst
enhancement decreased with time, so did the bursting in the PD neurons
and the entire pyloric pattern, until it was once again interrupted by
the next cardiac sac burst.
|
The pattern of activity in the VD neurons, which fired vigorously
during each cardiac sac burst, was likewise due at least in part to the
enhanced PSPs from the IV neurons. Again, the large IV PSPs were able
to drive the VD neurons to fire a higher frequency burst, generally one
for one with the IV neurons. After the burst, the VD neurons were
silent for some time before gradually resuming their activity.
Additionally, it is possible that the plateau properties in the VD
neurons of Panulirus, like those of Palinurus, were suppressed by synaptic input from the cardiac sac pattern generator (Hooper and Moulins 1989, 1990
). In contrast
to the case in both the LP and PD neurons, in which neuronal activity is enhanced by RPCH both in the presence and absence of a cardiac sac
pattern, pyloric activity in the VD neuron decreased in RPCH during
cardiac sac activity, but increased slightly (as evidenced by an
increased number of spikes per burst) in RPCH when the cardiac sac
pattern was not activated. This suggests that the cardiac sac pattern
(presumably via the IV neurons) more strongly influences the activity
of the VD neuron than does RPCH itself.
Both the PY and the IC neurons were generally silent both in the
control, with the stn blocked, and during bath application of RPCH. Neither neuron receives a PSP from the IV neurons
(Sigvardt and Mulloney 1982a), and these data suggest
that RPCH does not directly affect either neuron.
The pyloric neuron whose activity in RPCH is perhaps most
intriguing is the LP neuron. The LP neuron does not receive a direct PSP from the IV neurons, so its firing cannot be attributed to enhancement of that synaptic activity. In addition, we noted that the
activity of the LP neuron was enhanced even before the onset of cardiac
sac activity, as well as in preparations in which RPCH concentration
was subthreshold for the cardiac sac pattern, but still enhanced
pyloric activity. At least three separate factors contribute to the LP
neuron's enhanced firing. First, postinhibitory rebound, which occurs
when the anterior burster (AB) and PD neurons stop firing and
thus release the LP neuron from inhibition, plays a major role in the
onset of bursts in the LP neuron (Miller 1987). The
strong bursts generated by the PD neurons when they were driven by
cardiac sac bursts in the IV neurons produced strong inhibition in LP,
leading in turn to strong LP neuron rebound. Second, Manor et
al. (1997)
have shown that the synapses from the LP to
the PD neurons are depressed during ongoing pyloric activity. During the long inhibition of the LP neuron that occurs during the IV and PD
neuron bursts in RPCH, that depression would be removed. Thus, when the
LP neuron was released from the PD inhibition at the end of the cardiac
sac burst, it was able to inhibit the PD neuron more strongly than in
previous cycles. This in turn would lead to a greater inhibition of the
LP neuron and consequently a stronger postinhibitory rebound on the
next cycle. As the synaptic depression set in once again and the
enhanced rebound decreased, the intensity of both inhibition and of
postinhibitory rebound would gradually decrease, resulting in the
observed decrease in pyloric intensity. Third, we showed here that RPCH
also enhanced postinhibitory rebound itself in the LP neuron. This
would at least partially account for the enhanced LP neuron activity in RPCH before the cardiac sac pattern was initiated as well as the enhanced activity between cardiac sac bursts after that pattern had
been activated. In combination with the enhanced inhibition due to
recovery from depression, enhanced postinhibitory rebound would be
expected to result in stronger and longer bursts in the LP neuron, as
were observed. While we have not yet examined the mechanisms that might
be responsible for the increased postinhibitory rebound in the LP
neuron, the sag voltage clearly increased in 9 of the 13 preparations
in which we measured postinhibitory rebound (unpublished data; see also
Fig. 10A), suggesting the possibility that a
hyperpolarization-activated inward current
(Ih) might contribute to the enhanced rebound.
Thus, these results clearly illustrate the ways in which multiple mechanisms can contribute to the modulation and altered coordination of multiple motor networks. Using several mechanisms and acting on several parts of the stomatogastric networks, including both synaptic sites and membrane properties, a single neuropeptide (RPCH) is able to alter several neural circuits in concert. Consequently, the activity of three networks, namely the pyloric, gastric mill, and cardiac sac networks, as well as the interactions between them, are altered, resulting in a motor output that is quite different from that observed in the absence of this neuromodulator. Additionally, we have shown that because it can activate different patterns with different time courses, a single modulator, even if released hormonally and thus synchronously onto more than one neural network, can lead to progressive changes in both a single rhythmic motor pattern and a group of interacting motor patterns. Finally, these data demonstrate that not only can neurons (e.g., the LP neuron) within a network be influenced, via network interactions, by other neurons (e.g., the command-like IV neurons) with which they have no direct connections, but also that these indirect effects can be enhanced by changes in the membrane properties of the network neurons themselves as well as by changes in other parts of the network.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. John Simmers and G. Anderson for helpful comments on the manuscript.
This work was supported by National Science Foundation Grant IBN 9723885 and the Human Frontier Science Project.
Present address of J. Hetling: Bioengineering Dept. (M/C 063), 851 S. Morgan St., Rm. 232, Chicago, IL 60607-7052.
![]() |
FOOTNOTES |
---|
Address for reprint requests: P. S. Dickinson, Biology Dept., 6500 College Station, Bowdoin College, Brunswick, ME 04011 (E-mail: pdickins{at}bowdoin.edu).
Received 4 August 1999; accepted in final form 30 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|