Neuroscience Letters 328 (2002) 85–88
www.elsevier.com/locate/neulet
Correlation between activity in neuron B52 and two features of
fictive feeding in Aplysia
Romuald Nargeot*, Douglas A. Baxter, John H. Byrne
Department of Neurobiology and Anatomy, W.M. Keck Center for the Neurobiology of Learning and Memory, The University of TexasHouston Medical School, P.O. Box 20708, Houston, TX 77225, USA
Received 21 March 2002
Abstract
The present study examined the correlation between the level of activity neuron B52 and the transition from protraction to retraction phases of buccal motor patterns (BMPs) and the termination of the BMPs. The level of activity in B52
during the protraction phase was positively correlated with the duration of that phase. A second burst of activity in B52
was associated with the termination of the retraction phase. An apparent monosynaptic inhibitory connection from B52
to B64, may mediate the effects of B52. The first burst of activity in B52 delays the onset of activity in B64, thereby
prolonging the protraction phase, and the second burst inhibits activity in B64, thereby terminating the retraction phase.
These results suggest that activity in B52 may contribute to switching between ingestion-like and rejection-like BMPs by
regulating both phase transition and termination of BMPs. q 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Buccal motor patterns; Pattern selection; Central pattern generator; Neural network; Buccal ganglia; Aplysia; B52
Dynamical assemblies of neurons mediate adaptive behaviors by switch between different functional states in
response to external or internal stimuli. Feeding behavior
in Aplysia is a useful model system that has provided
insights into the neuronal mechanisms that underlie network
dynamics and motor pattern switching. The buccal ganglia
contain a central pattern generator network (CPG) that
mediates the rhythmic movements (protraction, retraction
and closure) of the odontophore/radula (a tongue-like
organ used to manipulate food) during feeding [7]. This
CPG is a multifunctional network that is dynamically reconfigured to generate several different types of buccal motor
patterns (BMPs), which in turn, mediate different aspects of
feeding (i.e. ingestion and rejection) [1,6,8,9].
Data in reduced preparations indicate that ingestion and
rejection differ partly by the relative duration of the protraction and retraction of the odontophore/radula [1,3]. During
rejection, the protraction, which ejects food from the buccal
cavity, is more prolonged than the subsequent retraction.
During ingestion, the duration of the protraction is briefer
* Corresponding author. Present address: Université Bordeaux
1 – CNRS, UMR 5816, Laboratoire de Neurobiologie des
Réseaux, Bâtiment Biologie Animale-B2, Avenue des Facultés,
33405 Talence Cedex, France. Tel.: 133-5-5796-2560; fax: 1335-5796-2561.
E-mail address: [email protected] (R. Nargeot).
than the subsequent retraction, which draws food into the
buccal cavity. Ingestion and rejection also differ by overlap
of the closure of the radula that grasps food, relative to the
protraction/retraction cycle [8]. In in vitro buccal ganglia,
ingestion- and rejection-like BMPs, which represent fictive
ingestion and rejection, can be distinguished using the duration of retraction phase [11] and the overlap of closure
motor activity relative to phases of the protraction/retraction
motor activities [5,8,11]. Several CPG neurons that mediate
aspects of the closure motor activity and the duration of the
retraction phase have been identified [4,9,11]. The goal of
the present study was to identify neurons whose activity is
associated with changes in the protraction duration and with
termination of BMPs.
The methods for recording BMPs from preparations of
isolated buccal ganglia have been described previously [10].
Briefly, the buccal ganglia from animals anesthetized with
isotonic MgCl2 were removed and placed in a recording
chamber that contained artificial seawater, which was maintained at 158C. Extracellular recordings were made from
three peripheral nerves: I2 n., n.2,1. and R n.1. to monitor
activity in motor neurons that mediate protraction, retraction and closure, respectively (see ref. [10]). Simultaneous
intracellular recordings were made from identified neurons.
In addition, tonic, low frequency (4 Hz) extracellular stimulation of the afferent nerve n.2,3 was used to induce rhyth-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S03 04 - 394 0( 0 2) 00 46 8- 8
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R. Nargeot et al. / Neuroscience Letters 328 (2002) 85–88
mic activity in the CPG. The stimulation of n.2,3 was maintained for 20 min. Data were collected beginning 10 min
after the onset of the stimulation. The present study
summarized data from 186 BMPs (ingestion-like pattern:
54.3%; rejection-like pattern: 19.9%; intermediate pattern:
25.8%) that were recorded in ten preparations.
Fig. 1A illustrates ingestion-like and rejection-like BMPs
that are generated by the isolated buccal ganglia and that are
similar to BMPs recorded in vivo during feeding [8]. These
BMPs were distinguished by overlap of closure activity (i.e.
large-unit activity in R n.1 with a frequency $0.25 Hz)
relative to the retraction phase (i.e. large-unit activity in
n.2,1), which immediately followed the protraction phase
(i.e. large-unit activity in I2 n.) [11]. In ingestion-like
BMPs (Fig. 1A1), $50% of closure activity occurred during
a long (12.4 ^ 1.7 s; mean ^ SEM; n ¼ 9) retraction phase.
In rejection-like BMPs (Fig. 1A2) the closure activity was
Fig. 1. (A) The protraction phase of ingestion- and rejection-like
BMPs have different durations. BMPs are composed of a protraction (P) phase (i.e. activity in I2 n.), a retraction (R) phase (i.e.
activity in n.2,1) and a closure (C) activity (i.e. large-unit activity
in R n.1). The durations of each phase are indicated under
recordings by the bars labeled P, R, C, respectively. BMPs were
categorized as being either ingestion- (A1), rejection-like (A2) or
intermediate on the basis of the amount of overlap of closure
activity with the retraction phase (see text for details). (B) In each
preparation (n ¼ 10), the mean duration of the protraction phase
for each type of BMP was calculated in a 10 min period (see text
for details). The mean duration of the protraction phase of ingestion-like BMPs was significantly shorter than rejection-like and
intermediate BMPs.
restricted to the protraction phase that preceded a shorter
(7.8 ^ 0.9 s; n ¼ 7) retraction phase. BMPs that did not
meet the criteria for either ingestion- or rejection-like
BMPs were categorized as intermediate (not shown;
n ¼ 7) although their behavioral signification remains
unclear.
BMPs can also be distinguished by the duration of the
protraction phase (Fig. 1B). The average duration of the
protraction phase during an ingestion-like BMP was
5.9 ^ 1.4 s, during a rejection-like BMP was 13.8 ^ 1.5 s
and during an intermediate BMP was 13.3 ^ 3.1 s. A single
factor analysis of variance (one-way ANOVA) indicated a
significant difference among these values (F2;20 ¼ 4:8;
P , 0:02), and post hoc pair-wise comparisons (StudentNewman–Keuls) indicated that the duration of the protraction phase during ingestion-like BMPs was significantly
shorter than the protraction phases of either rejection-like
or intermediate BMPs. Thus, in addition to differences in the
activity of closer motor neurons [9] and the duration of the
retraction phase [11], BMPs that are generated by the
isolated buccal ganglia can be distinguished by the duration
of the protraction phase.
A search was undertaken to identify cells that regulate the
duration of protraction. Previous studies found that B52 fires
a burst of spikes at the end of the retraction phase [2]. This
final burst of activity in B52 may function to terminate the
BMP (see below and refs [2,12]). However, as illustrated in
Fig. 2A, an initial burst of spikes in B52 can also occur
during the protraction phase of some BMPs. The occurrence
of the B52 burst during the protraction phase was significantly more variable (McNemar’s test, x2 ¼ 42:023;
df ¼ 1; P , 0:001) than the occurrence of the B52 burst
that terminates the retraction phase. The B52 burst that
terminates the retraction phase was recorded in all BMPs
(regardless of whether they were rejection- or ingestionlike), whereas the initial B52 burst was absent in 24% of
BMPs. To determine whether there was a relationship
between the level of B52 activity during the initial burst
and the duration of the protraction phase, the number of
spikes that occurred in B52 during the protraction phase
was counted and the duration of the protraction phase was
measured in all of the 186 BMPs. As illustrated in Fig. 2B,
there was a significant (F1;184 ¼ 188:5; P , 0:001), positive
correlation between the number of spikes in B52 and the
duration of the protraction phase. This correlation accounted
for 51% of the variation of the duration of protraction. In
addition, these BMPs were classified as being either ingestion-, rejection-like or intermediate and the level of activity
in B52 that occurred during the protraction phase for each
type of BMP was determined. Less activity occurred during
the protraction phase of ingestion-like BMPs than rejectionlike and intermediate BMPs (Fig. 2C). The mean number of
spikes in B52 that occurred during the protraction phase of
ingestion-like BMPs (2.1 ^ 0.8) was significantly less than
the mean number occurring during rejection-like BMPs
(12.8 ^ 2.6) (F2;20 ¼ 8:1, P , 0:005; pair-wise comparison
R. Nargeot et al. / Neuroscience Letters 328 (2002) 85–88
Fig. 2. B52 activity in different types of BMPs. (A) Simultaneous
extracellular and intracellular recordings were used to monitor
BMPs and activity in neuron B52. Activity in B52 during rejectionlike BMPs (labeled Rej.) differs from that during ingestion-like
BMPs (labeled Ing.) by the occurrence of a large burst of spikes
during the protraction phase. (B) The number of spikes in B52
during the protraction phase and the duration of the protraction
phase of each BMP were positively correlated. The regression
line and the coefficient of determination (r 2) were calculated
from 186 BMPs that were recorded in ten preparations. Similar
results (r 2 ¼ 0:47, P , 0:001) were obtained after excluding the
24% of BMPs in which B52 did not fire during the protraction
phase. (C) The mean number of spikes in B52 during the protraction phase, which was calculated from each type of BMP (ingestion-like, rejection-like and intermediate), indicates that
significantly more spikes occurred during the protraction
phase of rejection-like BMPs as compared to ingestion-like and
intermediate BMPs.
87
ingestion- versus rejection-like BMPs P , 0:05). Similar
results were observed for the average frequency of B52
spiking during the protraction phase (i.e. total number of
spikes divided by the duration of the protraction phase).
The average spike frequencies were higher for rejectionlike (0.9 ^ 0.16 Hz) than for ingestion-like (0.54 ^ 0.19
Hz) and intermediate BMPs (0.48 ^ 0.12 Hz).
Taken together, these results indicated that high levels of
activity in B52 during the protraction phase were associated
with the prolonged protraction phase of rejection-like
BMPs, whereas low levels or the absence of activity in
B52 were associated with the shorter protraction of ingestion-like BMPs. Thus, B52 in addition to its possible role in
terminating BMPs and thereby in contributing to the duration of the retraction phase, may be one of the CPG neurons
that contributes to the duration of the protraction phase. As
such, levels of activity in B52 may play a role in genesis and
switching the distinguishing features of BMPs.
To investigate how B52 might regulate the duration of the
protraction phase, its synaptic connections to other neurons
of the CPG were examined. B31/32 are a pair of neurons
that function, in part, to sustain the protraction phase [3,13].
B52 does not make a synaptic connection with these cells,
however (data not shown). Thus, if B52 regulates the duration of the protraction phase, it must do so via some pathway
other than a direct connection to the cells that sustain the
protraction phase. B64 is a cell that functions, in part, to
terminate the protraction phase and to mediate the retraction
phase [4]. As illustrated in Fig. 3A, B52 makes an apparent
monosynaptic inhibitory connection to B64. Similar results
were obtained in all preparations (n ¼ 3) in which this
connection was tested. Thus, high levels of activity in B52
during the protraction phase would inhibit B64, which in
turn, would delay the onset of spike activity in B64 and
therefore the transition from protraction to retraction.
Consequently, the protraction phase duration would be
extended.
Simultaneous intracellular recordings from B52 and B64
during ingestion- and rejection-like BMPs support this
hypothesis (Fig. 3B). Although it is not illustrated in the
Fig. 3, BMPs were categorized by the phase relationship
of the closure activity relative to the protraction and retraction phases. During the protraction (P) phase of the ingestion-like BMP, B64 slowly depolarized. This depolarization
eventually reached the threshold for eliciting a plateau-like
potential in B64 and the resulting burst of spikes in B64
contributed to the termination of the protraction phase and
the initiation of the retraction (R) phase [4]. During the
protraction phase of the rejection-like BMP, B64 received
a similar initial slow depolarization, but a burst of activity in
B52 appeared to inhibit B64 and delay the onset of the
plateau-like potential in B64, thereby prolonging the
protraction phase. In addition, the burst of spikes in B52
at the end of the BMP contributed to the termination of
the activity in B64, and thus to termination of the BMP.
Similar results were obtained in the three preparations in
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R. Nargeot et al. / Neuroscience Letters 328 (2002) 85–88
retraction phase and prolong the protraction phase of the
rejection-like BMPs. In addition to these roles in shaping
buccal motor patterns, B52 also functions as a sensory
neuron [2]. Thus, B52 must be considered a multifunctional
cell that plays several important roles in the functional
dynamics of the buccal CPG and hence may contribute to
the switching of feeding motor programs.
This research was supported by National Institute of
Mental Health (NIMH) Grant R01 MH58321.
Fig. 3. B52 inhibits the retraction generator neuron B64. (A)
Spikes in B52 produced one-for-one inhibitory postsynaptic
potentials (IPSPs) in B64. Arrowheads indicate the onset and
offset of depolarizing current injection into B52. (B) Simultaneous extracellular and intracellular recordings monitored
BMPs and activity in cells B52 and B64. Although closure activity
(i.e. extracellular recordings from R n.1) is not illustrated, it was
monitored and used to categorize the ingestion- (Ing.) and rejection-like (Rej.) BMPs. The arrow during the protraction phase of
the rejection-like BMP indicates a burst of activity in B52 that
appeared to inhibit the depolarization of B64. This inhibition
was associated with a delay of the phase transition from protraction to retraction, and thereby with a prolonged duration of the
protraction phase of the rejection-like BMP.
which the synaptic connection from B52 to B64 has been
tested.
B52 was first characterized by Plummer and Kirk [12],
who noted that B52 made inhibitory synaptic connections to
most ventral-cluster motor neurons, and thus, they
suggested that B52 might play a role in terminating bursting
in these cells during BMPs (see also ref. [2]). The results of
the present study are consistent with this hypothesis. B52
produces strong inhibitory input in B64 and in several other
elements of the CPG. Thus, the final burst of activity in B52
would inhibit many cells throughout the feeding circuitry
and thereby contribute to the termination of the BMP. In
addition, the present data suggest that the inhibitory connection from B52 to B64 may explain the way in which B52
helps to shape rejection-like BMPs. Although additional
studies will be necessary to examine the causal role of
B52 in this process, spiking in B52 during the protraction
phase would inhibit B64, and thereby delay the onset of the
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