1Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York 10032; and 2Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029
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ABSTRACT |
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Xin, Yuanpei, Klaudiusz R. Weiss, and Irving Kupfermann. Multifunctional Neuron CC6 in Aplysia Exerts Actions Opposite to Those of Multifunctional Neuron CC5. J. Neurophysiol. 83: 2473-2481, 2000. The controls of somatic and autonomic functions often appear to be organized into antagonistic systems. This issue was explored in the bilaterally paired C cluster neuron, CC6, which was found to have properties that suggested that it might function antagonistically to the previously identified multiaction neuron, CC5. Similar to CC5, CC6 is an interganglionic neuron that sends its sole axon to the ipsilateral and contralateral pedal and pleural ganglia. Synaptic inputs to CC6 were opposite to those of CC5. For example, CC6 receives inhibitory inputs from mechanical touch to the lips and tentacles and is excited by firing of C-PR, a neuron involved in the control of a head extension response. Also during rhythmic buccal mass movements CC6 receives synaptic inputs that are out of phase with those received by CC5. CC6 is inhibited during a fictive locomotor program, whereas CC5 is excited, but unlike CC5, the inputs to CC6 are not rhythmic. CC6 has extensive mono- and polysynaptic outputs to many identified and unidentified neurons located in various central ganglia. Firing of CC6 evoked ipsilateral contraction of the transverse muscles of the neck, whereas CC5 contracts longitudinal neck muscles. CC6 monosynaptically inhibits the pedal artery shortener neuron, whereas CC5 monosynaptically excites the pedal artery shortener neuron. Specific motor neurons in the pedal ganglion receive synaptic inputs of opposite sign from CC5 and CC6. Although the inputs and most of the effects of CC6 were opposite to those of CC5, both cells were found to produce polysynaptic excitation of the abdominal ganglion neuron RBhe, a cell whose activity excites the heart. CC5 and CC6 appear to be multifunctional neurons that form an antagonist pair.
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INTRODUCTION |
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Research in a number of invertebrate species as
well is in some verterbrates has shown that behavior and the activity
of pattern-generating circuits can be initiated or modulated by the
firing of single or relatively few higher order interneurons
(Arshavsky et al. 1988, 1989
;
Bartos and Nusbaum 1997
; Delaney and Gelperin
1990
; Didomenico et al. 1988
; Frost and
Katz 1996
; Kupfermann and Weiss 1978
;
Rosen et al. 1991
; Wiersma and Ikeda
1964
). Some of these higher order neurons exert relatively
large effects and have been called various names, including command
neurons (Kupfermann and Weiss 1978
; Wiersma and
Ikeda 1964
), commandlike neurons (Deodhar et al.
1994
), or influential neurons (Arshavsky et al.
1988
). In Aplysia a commandlike neuron (CC5) has
been recently described, which is of special interest because it is
active in many different behaviors (Xin et al. 1996a
,b
).
All of the diverse behaviors in which CC5 participates involve neck
shortening, and CC5 evokes both somatic and visceral concomitants of
this response. The precise functional contribution of CC5, however, is
different for the various behaviors in which it participates. Because
the nervous system may be organized into groups of neurons that exert
opposite actions (Marder and Calabrese 1996
;
Schneirla 1959
; Sharp et al. 1996
), we
have attempted to find other neurons with complex outputs similar to
that of CC5, but serving opposite functions. In this paper we describe
one such bilaterally paired neuron, which we term CC6.
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METHODS |
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Animals
Experiments were performed on 200- to 300-g wild-type Aplysia californica (Marinus, Long Beach, CA). A total of ~200 animals were used. The Ns for most experiments were 3-5 and are indicated in the figure legends. The animals were maintained at 14-16°C in holding tanks containing aerated, filtered artificial seawater (ASW; Instant Ocean, Aquarium Systems; composition in mM: 543 Cl, 467 Na, 9.9 Ca, and 54 Mg) and held for 3-6 days before being used for experiments.
Preparations
Before dissection, animals were injected with isotonic magnesium chloride at 25% of their body weight. Three types of preparations were utilized: 1) isolated head ganglia, 2) semi-intact preparations, and 3) reduced preparations. The isolated head preparation consisted of all of the structures in the head and included the cerebral, buccal, and pedal-pleural ganglia. The ganglia were pinned to a silicone elastomer (Sylgard) floor of a recording chamber containing instant ocean ASW. The cerebral and pedal-pleural ganglia were pinned dorsal side up.
The semi-intact preparations consisted of head ganglia and portions of
the head including the mouth, lips, anterior tentacles, and cephalic
artery that supplies the head region. The preparations were set in a
clear Lucite recording chamber consisting of two compartments
containing ASW. The head ganglia were pinned in one compartment in the
same orientation described above for the isolated ganglia preparation.
The mouth, lips, anterior tentacles, and the cephalic artery were set
in the second compartment. The second compartment was deeper than the
first chamber, so that the tissue could be completely immersed in the
ASW. The partition between the two compartments contained fine grooves
that allowed the peripheral nerves to pass through. The grooves were
filled with petroleum jelly (Vaseline) to maintain a watertight seal
between two compartments. The cephalic artery was cannulated, and fresh
ASW was pumped into the vascular system at a rate of ~0.5 ml/min to
perfuse the tissue and to simulate the hydroskeleton of the animal. A
suction tube for the outflow was set in the compartment to control the
fluid level. The preparation could be presented with mechanical or
chemical stimuli. Mechanical stimuli were provided by the tip of a
heat-sealed glass Pasteur pipette. Combined chemo-mechano stimuli
consisted of pieces of moistened dried seaweed (Laver, Vega Trading,
New York, NY), which were applied to the lips or tentacles with
a blunt forceps. A pure chemical stimulus consisted of a seaweed extract solution that was applied by a 1-ml syringe and slowly injected
into the ASW 1 cm near one side of the lip and tentacle region
(Susswein et al. 1978).
Reduced preparations included the head ganglia and neck muscles with attached pedal nerves that connect the muscles to the pedal ganglion.
Electrophysiology
All in vitro experiments were carried out at room temperature
(19-21°). For the intracellular recording and stimulation, neurons were impaled with double-barreled microelectrodes that were made of
thin-walled glass (World Precision Instruments) filled with 2 M
potassium acetate. The electrodes were flow beveled so that their
impedances ranged from 10 to 15 M. To identify neurons and examine
their morphology, the potassium acetate in one barrel was replaced by a
solution of 3% 5(6)-carboxyfluorescein dye (Kodak) in 0.1 M potassium
citrate, titrated to pH 8.0 with KOH (Rao et al. 1986
).
These electrodes were beveled so that the impedance of the electrodes
containing the dye was 15-20 M
and the impedance of the potassium
acetate electrode was 10-15 M
. To test for monosynapticity of
connections between cells, the threshold for action potential generation was raised by bathing the ganglia in a high divalent cation
solution (3 times Ca2+, 30 mM, and 3 times
Mg2+, 450 mM; or 2 times
Mg2+, 300 mM, and 5 times
Ca2+, 50 mM).
For extracellular recording or stimulation of various nerves, the cut
ends of nerves were drawn into small-diameter polyethylene suction
electrodes. Nerve recordings were made with AC amplifiers (A-M
Systems), and electrical stimulation of the nerves was provided by a
Grass 88 stimulator. An isotonic transducer (Harvard Bioscience) was
used to record muscle movement (Xin et al. 1996a).
Morphology
Because many identified cells have distinctive morphologies, to
aid the identification of cells, neurons were routinely filled with 3%
5(6)-carboxyfluorescein dye. Successful intracellular labeling was
achieved by iontophoretic injection of the dye for 15-30 min, followed
by a 48-h incubation at 4°C to allow the dye to fully fill the
processes. To reduce active transport of the dye from the cells during
incubation, the bathing ASW solution included 10 mM probenecid (Sigma)
final concentration (Rosen et al. 1991; Steinberg
et al. 1987
). The living ganglia were cleared in 50% glycerol
in ASW, and the fluorescence was visualized with a Nikon fluorescence
microscope, and the labeled cell body with its processes was
photographed, or drawn with the aid of a camera lucida.
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RESULTS |
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Morphology
CC6 is located bilaterally on the dorsal surface of cerebral
ganglion, within a distinct cluster of neurons that comprise the
cerebral C cluster (Jahan-Parwar and Fredman 1976). CC6
was generally found anterior and lateral to the previously described neuron, CC5 (Xin et al. 1996a
,b
). The size of its cell
body is ~80-100 µm. CC6 sends some processes into the region deep
to the somata in the C cluster. Its main axon enters the ipsilateral pedal ganglion via the cerebral-pedal connective, and then continues to
the contralateral pedal ganglion via the pedal-pedal commissure. It
sends out small processes in both pedal ganglia, and the processes extend to both pleural ganglia (Fig. 1).
No axons of CC6 were observed to enter any peripheral nerves. Dye
backfills (data not shown), indicate that in addition to CC6, there is
another cell in the cerebral C cluster that sends its axon to the
ipsilateral cerebral-pedal connective. This other cell is located
anterior-medial to CC6, and its cell body is much smaller than that of
CC6. Firing of this cell could evoke rhythmic activity in pedal nerves
(data not shown), suggesting that it may be one of the previously
described locomotor commandlike neurons (Fredman and
Jahan-Parwar 1983
). CC6 was usually identified on the basis of
the characteristic inhibition it produces in the pedal artery shortener
neuron (PAS, see CC6 inhibits the pedal
artery shortener neuron).
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Inputs to CC6
CC6 receives inputs from different sources. Application of seaweed
to the anterior tentacle or lip evoked inhibition in CC6 (Fig.
2A). The preparations used in
these studies did not typically exhibit feeding responses, and seaweed
stimuli may have elicited defensive withdrawal responses rather than
appetitive feeding responses. We found that pure mechanical stimulation
of the lip or tentacles, provided by a polished glass pipette, also
evoked inhibition in CC6, and the response was indistinguishable from that evoked by seaweed. Previously we reported that tactile stimuli applied to the tentacles of the animal evoked excitatory responses in
CC5 (Xin et al. 1996b).
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In preparations consisting of the head ganglia and buccal mass,
mechanical stimuli applied to the buccal mass produced several cycles
of buccal mass forward and backward movements, and during the
movements, CC6 received cyclic inhibitory inputs that were in phase
with each backward movement of the buccal mass (Fig. 2B).
The phase of this input is opposite to that previously reported for CC5
(Xin et al. 1996b).
We next examined the connections between CC6 and C-PR, a neuron
involved in the head lifting component of appetitive feeding responses
(Nagahama et al. 1993; Teyke et al.
1990
). Firing of C-PR produced excitation of CC6 (Fig.
3A). In preparations in which
intracellular records were obtained simultaneously from CC5 and CC6,
firing of C-PR resulted in excitation of CC6 and inhibition of CC5
(Fig. 3B).
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To determine whether CC6 receives synaptic input in phase with a
locomotor program, we electrically stimulated a P9 nerve (Jahan-Parwar and Fredman 1998). Simultaneous
extracellular recordings from the P9 and P10 nerves were obtained to
monitor fictive locomotor activity. It was found that stimulation of P9
evoked a long-duration hyperpolarization in CC6 (Fig.
4A) and inhibited firing of
the cell. The hyperpolarization persisted during the time that a
fictive (presumably escape) motor program was present, as indicated by bursts of firing of units in the P9 and P10 nerves (Fig.
4B). Previously, we showed that during a fictive locomotor
program CC5 received excitatory input (Xin et al.
1996b
), but unlike the tonic hyperpolarization seen in CC6, the
excitation was phasic. Direct firing of CC6 during an evoked locomotor
program could excite activity of one or more units in P9 and P10 and
could temporarily suppress rhythmic output recorded from the nerves
(Fig. 5). Although the activity evoked in
the nerves appeared to be tonic, we cannot rule out the possibility
that rhythmic activity of small units might be obscured by the larger
tonic units. Rhythmic activity of the large unit returned when the
depolarizing current injected into CC6 was terminated and CC6 no longer
fired (Fig. 5).
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Effect of CC6 on neck muscles
To explore the effect of CC6 on neck muscles, extracellular
records were obtained from pedal nerves (P3, P4, P5) that innervate the
neck (Bablanian et al. 1987; Jahan-Parwar and
Fredman 1978
). Firing of CC6 strongly excited unit activity in
neck nerves as well as other pedal nerves including P7, P9, and P10
(Fig. 6, A-C). We therefore
examined the effect of firing of CC6 on neck muscles. By adjusting the
plane of movement of the movement transducer, we could distinguish
transverse from longitudinal contractions. We observed that firing of
CC6 produced distinct transverse contractions of neck muscles (Fig.
7). Such transverse contractions should result in extension of the neck, and indeed CC6 firing evoked a
distinct lengthening of the neck that was of sufficient magnitude that
it could be observed visually without a microscope. This effect of CC6
contrasts with that of CC5, whose firing results in neck shortening
(Xin et al. 1996b
).
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Intracellular recordings from unidentified pedal neurons that send axons into pedal nerves revealed that many of them were either excited (Fig. 8), or inhibited (Fig. 9) when CC6 was fired. Typically the excitation or inhibition of these presumptive pedal motor neurons was not associated with synaptic potentials that were one-for-one with the CC6 spikes, suggesting that the effects were polysynaptic. In some instances, however, discrete excitatory postsynaptic potentials (EPSPs) that were time-locked to CC6 were obtained from the presumptive pedal motor neurons (Fig. 8B). In three experiments in which both CC5 and CC6 were recorded simultaneously, we found presumptive pedal motor neurons that received a synaptic input of one sign (either excitatory or inhibitory) when CC5 was fired but received input of the opposite sign (either inhibitory or excitatory) when CC6 was fired (Fig. 10, A and B). In experiments in which CC5 and CC6 were recorded simultaneously, it was observed that firing of CC5 or CC6 could evoke a weak slow membrane potential shift in the cell that was not fired, but mutual effects were not always observed (e.g., Fig. 10B), and we have not specifically investigated the sources of possible interaction between the cells.
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CC6 inhibits the pedal artery shortener neuron
We previously reported (Xin et al. 1996a) that
neuron CC5 controls the pedal artery shortener neuron
(PAS), which contracts the pedal artery
(Skelton and Koester 1992
) during various behaviors that
involve shortening of the neck. Because our data indicated that CC6
extends or lengthens the neck muscle, we hypothesized that CC6 would be
involved in lengthening of the pedal artery and might therefore inhibit
the pedal artery shortener neuron (PAS).
Initially we obtained extracellular recordings from the pedal artery
nerve (PAn) to monitor the activity of PAS, which is the only cell to send a large-diameter axon into the nerve (Skelton and Koester 1992
). In these experiments we
recorded simultaneously from CC5 and CC6. Firing of CC6 inhibited the
firing of the PAS unit recorded in the
ipsilateral pedal artery nerve. Confirming previous results, firing of
CC5 was found to excite the PAS unit recorded in
the ipsilateral pedal artery nerve (Fig. 10, A and B).
Simultaneous recordings from the left and right pedal artery nerves and a single CC6 cell showed that, in addition to strongly inhibiting the ipsilateral PAS unit in the nerve, CC6 had a weak inhibitory action on the contralateral unit (Fig. 11). Conversely, simultaneous recordings from a CC5 cell on the opposite side of the CC6 cell showed that CC5 had no effect or a very weak excitatory effect on the contralateral PAS unit, but strongly excited the ipsilateral unit.
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We next directly examined the synaptic connectivity of CC6 to PAS by recording directly from the PAS neuron cell body. Firing of CC6 produced strong inhibition in the PAS neuron (Fig. 12A), and at a fast sweep speed, small inhibitory postsynaptic potentials (IPSPs) one-for-one with CC6 spikes could be observed (Fig. 12B). The IPSPs persisted in a high divalent cation solution (Fig. 12C), suggesting that they are monosynaptic.
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Effects of CC6 on neurons in the abdominal ganglion
CC5 and a number of cells located near CC5 and CC6 in the C
cluster have been found to produce varied effects on neurons in the
abdominal ganglion (unpublished observations). For example, it
was previously reported that firing of CC5 evokes polysynaptic excitation of RBhe, a neuron whose activity
excites the heart (Mayeri et al. 1974) and is involved
in a general arousal response of the animal (Koch et al.
1984
). Whereas CC5 and CC6 typically have opposite actions, CC6
had the same action as CC5 on RBhe, that is, it excited the cell (Fig.
13A). The excitation of RBhe appeared to be polysynaptic, as would be expected because CC6 does not
send an axon to the abdominal ganglion. Additional polysynaptic effects
of CC6 on abdominal ganglion neurons included excitation of left upper
quadrant (LUQ) cells (Fig. 13B), neurons involved in
kidney function (Koester and Alevizos 1989
), and
excitation of L9 (Fig. 13C), a motor neuron that contracts
the gill (Kupfermann and Kandel 1969
).
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Effects of CC6 on neurons in the cerebral ganglion
Firing of CC6 evoked polysynaptic input to a number of neurons
whose cell bodies are located in the cerebral ganglion. Firing of CC6
evoked fast, polysynaptic potentials in the metacerebral cell (MCC;
Fig. 14, A and
B), a serotonergic neuron that modulates feeding behavior
(Rosen et al. 1989). The occurrence of the individual synaptic potentials outlasted the firing of CC6, and they were not
one-for-one with the CC6 spikes. Although the evoked synaptic potentials appeared to be depolarizing, they were not associated with
an increase of the rate of firing of the MCC (Fig. 14A), but rather with a small but distinct decrease (note the increased interspike interval of the MCC immediately following the firing of
CC6). It is possible that the evoked synaptic potential represents an
example of the conjoint EPSP-IPSP previously described to occur in the
MCC (Weiss and Kupfermann 1976
), but when the MCC was
prevented from spiking by means of a low level of hyperpolarizing
current, the synaptic potentials did not exhibit any obvious multiple
components.
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Because the connections of CC6 suggested that it may be involved in
head extension, we determined whether it might inhibit cerebral buccal
interneuron 1 (CBI-1), which appears to be involved in buccal mass
withdrawal, and presumably head withdrawal (Rosen et al.
1991). CBI-1 was identified by the presence of the
characteristic bilateral axons that it sends into the cerebral-buccal
connectives, as established by dye fills. We found that firing of CC6
strongly inhibited CBI-1 (Fig. 15).
Similarly, Bn cells, which have been shown to be involved in head
withdrawal (Teyke et al. 1989
), were strongly inhibited
by CC6 (Fig. 16). As was the case for
CBI-1, the inhibition of spiking in Bn cells was associated with a
smooth hyperpolarization (Fig. 16A) or with no clear
membrane potential shift (Fig. 16B). We have not attempted
to determine whether this inhibition might be monosynaptic.
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DISCUSSION |
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It has been suggested that somatic (Schneirla 1959)
as well as autonomic (Patton 1989
) responses are often
controlled by antagonistic functional mechanisms. The present results
suggest that the multifunctional neurons CC5 and CC6 comprise a
functional antagonist pair that contributes importantly to the control
of opposing movements. Previously, it was shown that CC5 is active in a
variety of behaviors, all of which involve shortening of the neck. The
current data support the hypothesis that CC6 is a counterpart of CC5,
but is involved in lengthening of the neck. In some sense the two cells operate like a half-center pair. The notion of half-centers
(Marder and Calabrese 1996
; Sharp et al.
1996
), however, has been traditionally applied to sets of
mutually inhibitory neurons that can form an oscillatory circuit. Our
data do not indicate that CC5 and CC6 are integral parts of an
oscillatory pattern-generating circuit. Furthermore, head shortening
and lengthening often occur as independent components of behaviors,
such as feeding and defensive withdrawal, and presumably in these
behaviors the activity of the two cells will not be directly linked.
Three lines of evidence support the idea that CC6 produces,
actions that are antagonistic to those of CC5. First, the two cells
have opposite synaptic inputs (either direct or polysnaptically) from
common sources. For example, the higher order neuron C-PR excites CC6
but inhibits CC5. Furthermore, the two cells receive opposite sensory
inputs following tactile stimulation of the head. The observed
inhibition in CC6 following tactile stimuli to the head is consistent
with a role of CC6 in responses that involve lengthening of the neck
muscles, because such tactile stimuli typically produce head withdrawal
and bilateral shortening of the neck (Teyke et al.
1989). A second line of evidence indicating an antagonist
relationship between CC5 and CC6 comes from examination of their
monosynaptic and polysynaptic effects on various follower neurons. A
prominent feature of CC5 is that it produces strong monosynaptic
excitation of the pedal artery shortener neuron, PAS (Skelton and Koester 1992
). In
contrast, CC6 inhibits the PAS, and similar to
CC5 the synaptic effect appears to be monosynaptic. Many other cerebral
and pedal neurons receive polysynaptic input of opposite sign from the
two neurons. The final evidence of antagonist actions of CC5 and CC6 is
that firing of the individual cells indirectly evokes strong muscle
contractions that produce opposing directions of movement of the neck.
Interestingly, although in most cases the responses and outputs of CC6
are diametrically opposite to those of CC5, this is not always the
case. For example, during a locomotor program elicited by a presumably
noxious stimulus, CC5 fires rhythmically (Xin et al.
1996b), whereas CC6 is completely inhibited rather than firing
rhythmically in antiphase to CC5. These observations, however, do not
preclude the possibility that the cells may exhibit antiphasic burst
activity during locomotion that is not elicited by a strong noxious
stimulus. It is also noteworthy that the two cells do not appear to
provide strong mutual inhibition of one another, which is often, but
not always (Lu et al. 1999
), a feature of theoretical
and actual neural systems that mediate opposite or conflicting actions
(Blitz and Nusbaum 1997
; Brooks 1986
;
Dickinson 1995
; Edwards 1991
; Jing
and Gillette 1995
; Krasne and Lee 1988
; Kristan and Shaw 1997
; Maes 1990
;
Redgrave et al. 1999
; Svoboda and Fetcho
1996
).
A second type of nonopposite behavior of CC6 and CC5 is seen in their effects on the heart exciter neuron Rbhe, which is located in the abdominal ganglion. CC6 as well as CC5 produce polysynaptic excitatory input to RBHE (Fig. 16A). Similar to CC5, CC6 does not send an axon to the abdominal ganglion, so that its effect on RBhe presumably involves intermediary interneurons located in head ganglia. The example of similarity of action of CC6 and CC5 may reflect the operation of a common "arousal-like" function, in which various motor activities, even opposing actions, nevertheless engage certain common functions, particularly "autonomic" responses that aid in the execution of motor output.
In addition to effects on RBhe CC6 also has actions on other abdominal ganglion neurons. Similar to CC5, CC6 appears to be involved in the control of both somatic as well as visceral muscles, and thus is involved in generating a highly complex behavioral response rather than a simple component of a response.
Studies of CC5 have shown that it is involved in a large number of
ostensibly different behaviors (Xin et al. 1996a,b
) and that its specific functional role in each of the responses is different. In some behaviors CC5 appears to be necessary and sufficient for a component (arterial shortening) of a complex withdrawal response,
and thus functions as a command neuron for that component. We have now
provided evidence that CC6 may similarly play multiple roles in
different behaviors, but based on its parallel, albeit opposite effects
to those of CC5, it seems likely that it is a member of a growing class
of multifunctional neurons that exert widespread actions. Definitive
elucidation of the behavioral role of CC6 will require lesion and
recording experiments in intact animals. These experiments are
currently unfeasible but may become possible with future technical advances.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Mental Health Grants MH-35564, MH-36730, K05 MH-01591, and K05 MH-01427.
Present address of Y. Xin: Genetic Research Lab, 410 Chipeta Way, Rm. 220, Salt Lake City, UT 84108.
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FOOTNOTES |
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Address for reprint requests: I. Kupfermann, Center for Neurobiology and Behavior, 1051 Riverside Dr., Box 87, New York, NY 10032.
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.
Received 25 October 1999; accepted in final form 11 January 2000.
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REFERENCES |
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