Volen Center and Department of Biology, Brandeis University,
Waltham, Massachusetts 02454-9110
 |
INTRODUCTION |
The pyloric rhythm of the stomatogastric ganglion
(STG) of adult lobsters and crabs is a highly regular motor
pattern that moves the muscles of the stomach. Casasnovas and
Meyrand (1995)
showed that the STG is present and active by
mid-embryonic development, before the stomach is functional and
connected to the hindgut. Early spontaneous and irregular neural
activity without obvious functional or behavioral relevance has been
seen in many systems and may be important for tuning developing
networks (Bradley and Bekoff 1992
; O'Donovan
1999
; Sillar 1994
; Wong 1999
).
Understanding the role of embryonic and larval rhythms in the
maturation of adult circuits requires their quantitative
characterization. Therefore we studied the developmental changes in
frequency, regularity, and periodicity of the motor discharge of one of
the neurons of the pyloric network of the lobster, Homarus
americanus, throughout embryonic and larval life.
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METHODS |
The eggs and larvae of H. americanus were obtained
from the lobster-rearing facility located at the New England Aquarium. Embryos and larvae were staged as in Helluy and Beltz
(1991)
. The larvae were either fed live or frozen brine shrimp,
Artemia salina. Adult animals of both sexes were
purchased from local fishermen.
Adult stomatogastric nervous systems were dissected from the stomach,
pinned in a dish, and superfused with chilled (9-13°C) saline.
Saline composition was as follows (in mM): 479.12 NaCl, 12.74 KCl, 10 MgSO4, 3.91 Na2SO4, 13.67 CaCl2, and 5 HEPES, pH 7.45. Extracellular pin electrodes
were used to record from the motor nerves (Fig.
1A). The entire stomachs
of embryonic and larval animals were dissected and pinned in the dish.
Intracellular electrodes were used to record from the p1
muscle, innervated by the LP neuron (Fig. 1A). The
p1 excitatory junctional potentials (EJPs) were used as
an assay for rhythmic motor pattern generation in the STG
(Casasnovas and Meyrand 1995
). The intracellular
electrodes were filled with 0.6 M K2SO4 and had
a resistance of 50-80 M
.

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Fig. 1.
Measuring the frequency of the pyloric rhythm with the power spectrum.
A: schematic diagram of the adult stomatogastric nervous
system showing the position of the recordings in this paper. The
stomatogastric ganglion (STG), was left attached to inputs from the
esophageal ganglion (OG) and commissural ganglia (CoGs) via the
stomatogastric nerve (stn). The motor nerves indicated
include the llvn (low lateral ventricular nerve), the
pyn (pyloric nerve), and pdn (pyloric
dilator nerve). The position of the p1 muscle,
innervated by the lateral pyloric (LP) neuron is indicated.
B: simultaneous extracellular recordings made in an
adult preparation from the motor nerves at the positions indicated in
A. Activity of the LP, pyloric (PY), and pyloric dilator
(PD) neurons are indicated. C and D:
power spectra calculated from pdn and
llvn recordings, respectively, from an adult
preparation. F is the frequency of the peak. PR indicates the fraction
of the power in the peak region defined by the horizontal bar.
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Data were recorded continuously for 3-20 min. Action potential and EJP
times of occurrence were extracted off-line from digitized electrode
recordings, using in-house and commercial software (MiniAnalysis, Jaejin). Due to the irregularity of the LP motor activity in most of
the embryonic preparations, extraction and analysis of burst onsets and
durations was not generally possible. Instead, cycle frequency of the
pyloric rhythm was determined from the principal peak of the power
spectrum of the action potential or EJP times (Miller and
Sigvardt 1998
; Rosenberg et al. 1989
). For each
trial, the corresponding list of event times was divided into three
equal-time sections, and the final power spectrum was obtained as an
average of the spectra calculated for each section. Each spectrum was normalized to the power at zero frequency to allow comparison of
spectra. The full spectra show peaks at high-frequency (20-40 Hz) that
correspond to the interspike intervals that occur when EJPs are tightly
grouped, as in bursts, and peaks at lower frequency that roughly
correspond to the frequency of bursts. In this paper we were concerned
with the burst frequency but not with the frequency of firing within
the burst, and therefore restricted our analyses to the lower frequency
range of the spectra. An estimate of the variability in the activity
was computed as the ratio of the area under the principal peak region
of the power spectrum to the area under the spectrum from 0 to 3 Hz
[including the peak region, and with the spectral value at frequency 0 (DC offset) set to 0]. The resulting "power ratio" can range
between 0 and 1, is high (>0.5) for highly periodic activity (i.e.,
most of the power in the signal is found at the cycle frequency of
bursting), and decreases with increasing variability in the motor
rhythm (i.e., power is found at frequencies other than the cycle
frequency). When bursting is highly regular so that the recorded signal
approximates a square wave, "harmonic" peaks are seen in the power
spectrum at multiples of the peak frequency. These harmonics are much
less evident in the spectra of irregular activity. Although it is
relatively straightforward to remove the harmonics from highly regular
spectra, identification and removal of the harmonics from the cases of irregular activity is not possible. Therefore all of the power ratio
calculations include the harmonics, and the relative power in the main
spectral peak is underestimated because of the harmonic peaks.
 |
RESULTS |
Figure 1 shows a schematic of the recording configurations used in
this work. Recordings of the adult motor patterns were made
extracellularly from the motor nerves. Figure 1B shows the pyloric rhythm from an adult H. americanus. The traces
labeled pdn and llvn show the alternate patterns
of lateral pyloric (LP), pyloric (PY), and pyloric dilator (PD) neuron
activity. To measure the period and the regularity of the pyloric
rhythm we recorded data and performed a Fourier analysis of the spike
times extracted from the raw data (see METHODS). Figure 1,
C and D, shows the power spectra of the
recordings shown in Fig. 1B calculated from the activity of
the PD (pdn recording) and the LP (largest spike on
the llvn) neurons, respectively. Both spectra show peaks at the same frequency: 0.46 Hz. The activity of this preparation was
highly regular (power ratio of 0.82 for calculated for the PD neuron
activity and 0.71 for LP neuron activity). The average cycle frequency
in six adult preparations was 0.64 ± 0.05 (SE) Hz as
measured with both the LP and PD traces. The power ratio was 0.72 ± 0.064 measured from the PD activity and 0.61 ± 0.031 as
measured from the LP neuron activity. These values are not statistically different (P > 0.07), although in
all cases the regularity was slightly lower when measured with LP
neuron activity than with PD neuron activity, possibly because there
are two PD neurons contributing to the PD spectra, thus increasing the
signal-to-noise ratio of the power spectrum.
Figure 2 shows raw data and power
spectra from individual representative preparations at embryonic and at
each larval stage, as monitored by an intracellular recording from the
p1 muscle. Unlike the data shown in Fig. 1, in all of
these recordings there were episodes in which the LP neuron fired in
relatively regular bursts, and other stretches in which it fired quite
irregularly. The E75% recording shows an example of a preparation that
had several single EJPs, making burst characterization difficult by conventional means. In the recordings shown from the larval times, bursts of EJPs were common, but the bursts were of considerably different durations and had largely variable interburst intervals. The
recordings shown for the LIV preparation are considerably more regular
than those seen at earlier times. Despite the irregularity of the early
rhythms, all of the spectra showed one peak with more power than all
the others (preferred peak frequency; Fig. 2). Table
1 presents pooled data for peak frequency
and percent power in the peak for embryos, LI, LII, LIII, LIV, and
adult animals. Both the frequency and regularity of the rhythm are low
during embryonic and the early larval stages and increase as
development proceeds. The mean frequency increased by ~2.5-fold
between embryonic and adult times, whereas the power ratio more than
doubled. The actual regularity of adult was most likely greater because
the power ratio calculation is preferentially underestimated in the more regular preparations (METHODS).

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Fig. 2.
LP neuron evoked excitatory junction potentials (EJPs) recorded in the
p1 muscle during embryonic and larval times. The
left side of each panel shows an intracellular recording
from a muscle fiber in the p1 muscle from an animal of
the stage indicated. The right side of each panel shows
the power spectrum of the event times of the experiment represented to
the left. F and PR are as in Fig. 1. Vertical bars, 10 mV. Horizontal
bars, 5 s. The resting membrane potentials were 66, 56, 70,
66, and 69 mV, respectively.
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DISCUSSION |
Spontaneous rhythmic activity may function to tune both sensory
and motor circuits during development (O'Donovan 1989
;
Wong et al. 1993
, 1995
). In particular,
irregular activity that precedes functional behavior could be important
in the formation of synaptic connections, and in modifying the
strengths of synapses and the intrinsic properties of the neurons
within a circuit (O'Donovan 1999
). Casasnovas
and Meyrand (1995)
showed that the embryonic STG is
rhythmically active, well before the stomach is processing food. Here
we demonstrate that this activity is quite irregular, only slightly
periodic, and is slower than the adult pyloric rhythm. It is possible
that these early and irregular activity patterns may provide
maturational signals that allow the STG to coordinately tune its
synaptic and intrinsic properties.
In this study we used the activity in the muscle innervated by the LP
neuron as an assay of the rhythmic activity in the pyloric region of
the stomach. In our adult recordings, the frequency of the rhythm
obtained with the LP and PD neurons was always identical, and the
regularity of the rhythm seen with the LP neuron activity was
statistically indistinguishable from that seen with the PD neurons.
Figure 3 outlines a number of
possibilities that could account for the lower frequency and more
variable rhythms seen in the early developmental stages.

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Fig. 3.
Possible causes for irregular motor patterns seen early in development.
Top panel: left shows the circuit responsible for
regular motor patterns in the adult. The commissural pyloric oscillator
(CPO) in each commissural ganglion (CoG) drives the pacemaker ensemble
( ), which inhibits the LP neuron ( ). The LP
therefore fires regularly in alternation with the PD neurons.
Bottom panel: shows 3 cases, each of which can produce
irregular LP neuron activity (see text). AB, anterior
burster.
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Figure 3, top panel, illustrates the circuit that is
responsible for the adult pyloric rhythm in H. gammarus and
almost certainly as well in H. americanus (Cardi and
Nagy 1994
; Nagy and Cardi 1994
; Nagy et
al. 1994
; Robertson and Moulins 1981a
,b
). The
commissural pyloric oscillator (CPO) network is found in each
commissural ganglion (CoG; Fig. 1) and projects to the STG where it
rhythmically drives and entrains the pyloric network (Cardi and
Nagy 1994
; Nagy and Cardi 1994
; Nagy et
al. 1994
; Robertson and Moulins 1981a
,b
). The
rhythmically active PD and anterior burster (AB) neurons
inhibit the LP neuron, which is thus entrained to fire in tight
pyloric-timed bursts (Fig. 3, adult). Therefore in the adult, the
frequency (measured either from the LP or the PD neurons) will be a
consequence of the frequency of the entraining CPO.
The three cases shown in Fig. 3 illustrate that the lower frequencies
and lower regularity seen early in development can result from
variability occurring at a number of different sites in the circuit. In
Case 1, the CPO and the pacemaker ensemble may or may
not be regular, but the LP neuron fails to follow its rhythmic drive,
either because of its intrinsic properties or because the synapses from
the PD and AB neurons to the LP neuron are not properly tuned. In this
case, the frequency extracted from the LP neuron activity could be
different from that of the CPO and the PD neurons. In Case
2, the CPO may be strongly rhythmic, but unable to drive reliably the PD/AB ensemble, either because the projection to the STG
is not present, the synapses are not properly tuned or because the
AB/PD neurons do not have the appropriate intrinsic properties. In this
case, the PD neurons (and therefore the LP neuron) may have a lower
frequency than that of the CPO and may appear irregular. In Case
3, the CPO itself may not be regular or may be absent. The slow
and irregular patterns produced by the LP neuron would therefore be
indicative of lack of strong rhythmicity in the entire circuit.
In addition to the CPO, there are a large number of other modulatory
inputs to the STG, which may modify both the intrinsic properties of
neurons and the synaptic connections among them (Harris-Warrick
and Marder 1991
; Marder and Calabrese 1996
).
Although some of the neuromodulatory inputs are already present by
mid-embryonic development, others are not present until late in larval
life (Fénelon et al. 1998
, 1999
;
Kilman et al. 1999
). Therefore the irregularity and low
frequency of the early rhythms may be a consequence of the modulatory
environment seen early in development. Although the stomach starts to
process food at LI, its full mechanical and structural properties
develop slowly between LI and LIV (Factor 1995
), during
which time the remaining modulatory inputs to the STG first become
apparent (Fénelon et al. 1999
; Kilman et
al. 1999
). The increase in frequency and regularity in late
larval and postlarval animals may occur because this is the time at
which the activating modulatory inputs are fully present and active. Alternatively, the major changes in both frequency and regularity reported here could reflect developmental processes that tune both the
intrinsic properties of circuit neurons and the synaptic connections
among them.
We thank C. Soto-Treviño for help early on in this project
and L. F. Abbott, X. J. Wang, M. Goldman, and K. Kemptes for
useful discussions on data analysis and statistics.
This research was supported by National Institute of Neurological
Disorders and Stroke Grant NS-17813 to E. Marder, Individual National
Research Service Award NS-10770 to K. S. Richards, and the W. M. Keck Foundation.
Address for reprint requests: E. Marder, Volen Center, MS 013, Brandeis
University, 415 South St., Waltham, MA 02454-9110.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.