ACKNOWEDGEMENTS
I would like to thank my advisor, Stuart Thompson, for his help
and encouragement. I would also like to thank William Gilly for his
patience, answers, and the use of much of his equipment. Thank you
also to George Mackey for the methylene blue. Mark Denny deserves
many thanks for the use of his tools and the Fisher bathroom key.
Bruce Hopkins was very helpful in setting up the Aquatron that made
the whole-animal preparation possible. My labmates, John, Shannon,
and Simone should be canonized for putting up with me through many
frustrating hours. Thanks to the friendly people at Hopkins in
general, who were always willing to take time out and lend a hand.
A sigh of thanks to my roommate and best friend, Terri, who bore the
brunt of my weary, edgy demeanor. And finally, last but far from
least, hearty thanks to my TAs, Sam Wang and Jennifer Levitt, each
of whom rescued me one way or another throughout the quarter.
ABSTRACT
Some of the behaviors of the marine nudibranch Melibe leonina
have been described, such as feeding, "galloping", the crumple
response, and its fascinating swimming behavior (Hurst, 1968:
Thompson). However, a new behavior, the shrug response, has been
noticed and is described here. It was noted via visual observations
that this behavior habituated quickly and was inhibited during
swimming and locomotion. The purpose of this study was to observe
and describe the properties of this behavior, trace it
neuroanatomically, and find its relation to other motor behaviors.
Results of behavioral manipulations and electrophysiological
recordings showed that the shrug response is part of a behavioral
hierarchy and, because it is mediated through one, central pathway,
would be good for studies of habituation.
INTRODUCTION
Melibe leonina is a nudibranch mollusc belonging to the family
Tethyidae. It is found along the west coast of the United States,
from Alaska to Mexico (McDonald, et al, 1980). Melibe leonina is so
named (leonina="lion-like") because of its large oral hood complete
with two rows of oral tentacles. On the dorsal surface of the oral
hood are two rhinophores, which are chemoreceptive organs (see
Figures 1, 2).
Melibe were collected by snorkelers and SCUBA divers off Del
Monte Beach on Monterey Bay and were kept at Hopkins Marine
Station in tanks of flowing seawater filled with kelp. An additional
tank was kept at the Monterey Bay Aquarium in order to ensure that
many of the Melibe would stay alive in case problems arose with the
tanks at Hopkins.
Melibe leonina is an ideal organism for behavioral and
neurophysiological study for many reasons. For one, unlike other
nudibranchs, Melibe has a wide variety of active motor behaviors.
including feeding, copulation, locomotion, defense responses, and
swimming. In addition, it has a nearly translucent body wall, which
facilitates dissection. Melibe has a "brain" consisting of three
paired ganglia that are centrally located, and its large, round
neurons are amenable to electrophysiological recordings.
Swimming behavior is controlled by a Central Pattern
Generator (Thompson). The swimming consists of rhythmic side-to¬
side contractions (see Figure 3) that occur when the foot becomes
dislodged from the substratum and ceases when the foot comes in
contact with the substratum again.
This paper describes a new response, referred to as the shrug
response (described in detail below), which cannot be elicited during
swimming. This inhibition during swimming raises some interest
questions. Is there active inhibition of the response during
swimming? Is this centrally mediated? Or is the shrug a local
retlex? Is the shrug inhibited during swimming because there are
muscles common to the two behaviors, or for some other reason? If
some other reason, what could it be?
The present studies addressed these and other questions about
the nature of the shrug response in Melibe leonina,
MATERIALS AND METHODS
Behavioral Methods
A precondition for accurate neuroethological studies is
knowledge of the experimental animal's behavioral repertoire in its
natural context (Ewert, 1980). Thus an aquarium was set up for
behavioral observations. Three animals were placed in the tank
simultaneously (animals were rotated) and their behaviors observed
and manipulated. Feeding, copulation, locomotion, and swimming
were observed naturally. The aquarium was supplied with flowing
seawater (11-15 C), and the tank was guarded from harsh sunlight.
In addition to observing behaviors in natural context, behaviors were
also manipulated. A glass rod was used to test the crumple response
(Thompson), a defensive response to tactile stimulation.
Tracing the Neuroanatomy
In an attempt to isolate the nerve responsible for mediating
the shrug response, the nerve roots from the brain innervating the
oral hood were mapped. This was accomplished by careful
dissection and methylene blue staining. Since the nerves in Melibe
appear appreciably more grainy than the muscles under the
dissecting scope, it was possible to trace the nerve roots out into
the hood via careful dissection.
In order to reinforce the dissections, specimens were stained
with methylene blue (dilution ratio = 3 drops/20mL dH2O) for
approximately 50 hours. This helped make the nerve roots stand out
clearly and was especially helpful in getting a depth perspective on
the roots as they dove out of the brain region and into the
surrounding tissue. Once the particular nerve responsible for
mediating the shrug response was isolated by electrophysiological
methods (see below), it was traced thoroughly into the hood (see
Figure 4).
Electrophysiology
In order to correlate behavioral activity with neural activity, a
whole-animal preparation was used, similar to that used by Willows
with Tritonia (Willows, 1967). With the brain suspended from a
platform, the animal was able to behave in the tank while recordings
trom its nerve cells were obtained (see Figure 5).
Using focal extracellular electrodes, recordings were made
from the anterior nerve roots in an attempt to isolate the nerve
responsible for mediating the shrug response. In order to
accomplish this, a portion of the nerve was sucked up into the tip of
the electrode, spontaneous activity was noted, and any changes in
firing when the shrug was elicited were noted.
Is the shrug centrally commanded? The fact that a behavior
can be commanded centrally does not rule out the possibility that
there is a peripheral pathway sufficient to maintain the response in
the absence of central excitation. This is the case with the Aplysja
gill withdrawal (Peretz, et al, 1976). In order to address this, the
C2 nerve (see Figures 4, 6) was cut both unilaterally and bilaterally
and the effects on behavior noted. A control, in which each of the
other anterior nerve roots was cut, tested for effects of trauma.
As mentioned, the shrug appears to be inhibited during
swimming. Therefore the activity of C2 was recorded while the
animal was swimming versus when its foot was attached to the
kelp.
It the motor commands for the shrug response are being sent
to the periphery via C2, stimulation of C2 should elicit (at least
some component of) the behavior. Electrical stimulation was
applied to C2, first intact and then to the cut end. Stimulation
frequency was 5.5 Hz, with a delay of 2msec, and a duration of 120
msec. Voltage was the parameter varied.
Since C2 attaches to the dorsal surface of the cerebral
ganglion, the search for the shrug motoneurons began there. The
method employed was the extracellular hunting technique.
Recordings were made from various areas in the paired cerebral
ganglia, the origin/termination site of C2 nerve root. It was
difficult to reliably identify neurons, since sizes, shapes, and
coloration varied from brain to brain. However, the cerebral
ganglion has a few (3-6) readily identifiable giant cells, which are
thought to be important hood motoneurons (Hurst, 1968), and
recordings were made from these cells and the cells in this area
(see Figure 6). Recordings included neural firing during swimming,
while the animal was on the kelp, and while the shrug was elicited.
RESULTS
Behavioral Results
While mapping the receptive fields for the crumple response,
the shrug response was first noticed. When the animal was touched
on the dorsal surface of the oral hood between the rhinophores, the
hood (and the hood ALONE) was retracted toward the body and
flattened (see Figure 7). The fact that the shrug is localized to the
hood distinguishes it from the crumple, which is a full-body
response. The shrug habituates rapidly. It habituated more rapidly
with a smaller interstimulus interval (see Figure 8), and a 30¬
second interval yielded full recovery of the response. There are two
components to this habituation: (1) amount of
contraction/withdrawal and (2) time to re-extend the hood. The
latter was reduced more rapidly. The shrug response was not tested
for the other remaining eight characteristics necessary for true
habituation (Shabb, 1984; Peeke and Herz, 1973).
Other features of this response include the fact that a crumple
response can be elicited if the novel stimulus is severe, and that
this response is inhibited during swimming and during locomotion.
Previous authors have noted that the hood is in a "neutral", semi¬
contracted state during swimming (Hurst, 1968; Thompson). Perhaps
this posture is not only characteristic but very important to
swimming, such that the shrug is actively inhibited during
swimming
Neuroanatomical Results
Four anterior nerve roots were clearly identifiable from
specimen to specimen (see Figure 6). These have been labelled as
nerve roots C1-C4 (Hurst, 1968), although 1 found that C1 actually
attaches to the tentacular lobe, and C4 appears to come from the
ventrolateral surface of the pleural ganglion.
In tracing the nerves from the cerebral ganglion out into the
oral hood, it was found that the C2 nerve branched twice, which is in
agreement with Hurst's results (Hurst, 1967). The terminal points
of the first branch were found to be located in the region between
the rhinophores, while other terminal points were in the longitudinal
muscles of the hood (see Figure 4).
Electrophysiological Results
From recordings made from the anterior nerve roots, C2 was
the only one that showed bursts when the shrug was elicited. It
appeared to be somehow involved in mediating the shrug response.
while the other nerve roots did not.
C2 was identified as the nerve which mediates the shrug
response by ablation experiments. Unilateral ablation of C2 resulted
in a loss of the shrug. It was clear that this loss was not simply due
to sensory information being cut off, because: 1) the rhinophores
still withdrew when a stimulus was applied, and 2) the crumple was
clearly elicited when the strength of the stimulus was increased.
The control experiment showed that the loss of the shrug was not
due to trauma. When each of the other nerve roots was cut, the
shrug remained. It was only when C2 was cut that the shrug was
abolished. Bilateral ablation of C2 resulted in loss of the crumple in
response to mechanical stimulation between the rhinophores, while
it still occurred in response to mechanical stimulation of the body
wall. It appeared, then, that while the sensory information carried
in C2 unilaterally was enough to elicit the crumple, C2 was needed
bilaterally in order for the shrug to occur.
Recordings from intact C2 showed clear bursts (with a mean
duration of 890 msec and 10.3 spikes; see Figure 9) when the shrug
was elicited. It was clear that these were not just the sensory
neurons from the dorsal surface of the oral hood, because there were
no bursts when stimulation did not produce the shrug (e.g., during
swimming).
Recordings from the cerebral giant area revealed some cells
that appeared to burst when the shrug was elicited. The cells in this
area also fired when hood and tentacular movements were made, in
response to touch or pinch of the oral tentacles, and during feeding¬
type movements. A rather spectacular finding was that all of the
cells surveyed seemed to have decreased firing when the animal was
swimming as compared to when it was on the kelp.
DISCUSSION
The fact that the shrug response is inhibited during swimming
brings up two possibilities for further study. The first is the
mechanism by which the output of a Central Pattern Generator
inhibits other motor behaviors. Because the shrug is inhibited
during swimming , and because all of the cells surveyed in the region
of the giant cells in the cerebral ganglion showed decreased firing
during swim (consistent with results of Thompson), I arrived at the
hypothesis that some neurotransmitter is acting more like a
hormone by being dumped locally into the ganglion instead of being
active at distinct synapses. This type of mechanism has been
worked out for suppression of behaviors in both Aplysia (Kandel,
1976) and Pleurobranchaea (Davis, et al, 1974). This general
inhibition of hood motor activities may be important if the hood
must remain in its "neutral", semi-contracted state (State 2 of
Figure 9) for swimming to be efficient.
Another possibility for further study is the investigation of
the behavioral hierarchy of Melibe. Diagrams detailing the
interrelationships of behaviors have been constructed (and
reconstructed) for many mollusks (Willows, 1985; Davis, et al.
1974; Gillette and Davis, 1974). The present study indicates that
swimming has a fairly central place in the priority sequence of
Melibe (see Figure 10 for preliminary hierarchy). Swimming inhibits
both the shrug response and feeding movements, and it occurs
whenever the animal becomes dislodged from the substratum. A
more detailed study of the behavioral hierarchy of Melibe would
reveal how homologous it is to those of other molluscs and might
give valuable insight into the larger issue of behavioral chojce.
which is common to a wide range of organisms in the animal
kingdom.
Another property common to many organisms is learning.
Studies of training, conditioning, and habituation have served as
models of basic, or simple, learning. Many researchers (Pinsker, et
al, 1969, Kupferman, et al, 1969, and Castellucci, et al, 1969) have
used molluscan preparations for studies of habituation. Howeyer, it
has been shown that such learning studies have involved neural
pathways that are not as straightforward as was once thought. In
the most common preparation, Aplysia californica, three different
sites for habituation of the gill withdrawal reflex have been found.
each of which is sufficient to mediate habituation (Peretz, et al.
1976). The present study confirms that the shrug response in Melibe
leonina is not mediated via a local reflex circuit (i.e., peripheral
pathway, as in Aplysia). Thus, this organism and behavior might
prove useful for the study of simple learning mechanisms, such as
habituation. Behavioral studies revealed that the shrug response
habituates rapidly, while studies of habituation on the cellular level
have yet to be done.
LITERATURE CITED
Davis, W. J., Mpitos, G. J., Siegler, M. V. S., Pinneo, J. M., and Davis, K.
B. (1974). Neuronal Substrates of Behavioral Hierarchies and
Associative Learning in Pleurobranchaea. Ameriçan Zoologist. 14:
1037-1050.
Ewert, Jorg-Peter. Neuroethology. Berlin: Springer-Verlag, 1980.
Gillette, R. and Davis, W. J. (1976). The Role of the Metacerebral
Giant Neuron in the Feeding Behavior of Pleurobranchaea. Journal of
Comparative Physiology. 116: 129-159.
Hurst, Anne. (1968). The Feeding Mechanism and Behaviour of the
Opisthobranch Melibe leonina. Symp. Zool Soc. Lond. 22: 151-166.
Kandel, E. R. Cellular Basis of Behavion. San Francisco: W. H.
Freeman and Company, 1976.
Kandel, E. R. and Schwartz, J. H. Principles of Neural Science. New
York: Elsevier Science Publishing Co., 1985.
Kennedy, D. (1975). Comparative Strategies in the Investigation of
Neural Networks. Journal of Experimental Zoology. 194: 35-50.
Peeke, Harman V. S. and Herz, Michael J., ed. Habituation.. (Two
volumes). New York: Academic Press, 1973.
Peretz, Bertram, Jacklet, Jon W., and Lukowiak, Kenneth. (1976)
Habituation of Reflexes in Aplysia: Contribution of the Peripheral
and Central Nervous Systems. Science. 191: 396-399.
Shabb, Sam Richard. Habituation of the Gill Withdrawal Response
and Mapping of Associated Neurons in the Nudibranch, Doriopsilla
albopunctata. (Master's thesis completed at Hopkins Marine Station).
Thompson, S. H. Personal communication.
Willows, A. O. D. (1967). Behavioral Acts Elicited by Stimulation of
Single Identifiable Nerve Cells. Science, 157: 520-524.
Willows, A.O.D., ed. The Mollusca (vol. 8): Neurobiology and Behavior.
Part 1. Orlando, Florida: Academic Press, Inc., 1985
LIST OF FIGURES
1. Melibe leonina in its natural environment
2. Dorsal view of Melibe with basic anatomy labelled
3. Melibe swimming pattern
4. Sketch of C2 nerve out into the oral hood
5. Whole-animal preparation setup
6. Sketch of central ganglia with anterior nerve roots and cerebral
giant cells labelled
7. Sketch of the shrug response
8. Behavioral habituation, comparing 5- and 15-second
interstimulus intervals (ISl's, aka epochs)
9. Burst of action potentials in C2 nerve during shrug; N = 23, for
means
10. Preliminary behavioral hierarchy ethogram
Fiqure 1
ORAL HOo—
—

RHINOPHRE —


ORAL TENTACLE
CERATA
DORSAL VIEN
or Helibe

S

CENTRAL GANGUA
-

Fiqure 2
FiG. 3. Three successive stages in swimming, taken from a moving film of elibe.
(reprinted from Hurst, 168)
Fiqure 3
ANTERIOR

2
.......

POSTERIOR
Fiqure 4
D
minophore
oral hood
S
platfern
recordi

eleetrede
90

OTRW INFL
CEICTERED SEAMATEE AT 10-158c)
Figure 5
Eve-
PTATOCVST
c3
Ci
C2
ANTERIOR
Cerebral gont cells
CEResent gaNGUDN

e

Pedt
GANGUDN

Prekle
GAUGUON
POSTÉRIOR
Fiqure 6
C4
+--


I
30



—
1.
fiqure 7
101
9+
8+
6
5+
41
3+
2
1+
BEHAVIORAL HABITUATION
= 15-second epochs
- - = 5-second epochs
0 -0-0-0-0—

—
1 2 3 4 5 6 7 8 9 10
Trials
Fqure 8
+ +
ktatatatatattt








Shrug Response
C2nerveroot
X duaton - 810 2120 wsec
spikes - 10.3 * 1. 3
Fiqure 7
CRUMPL
E
SWIMMING
SHRUG
Figure 10
E
—