Download Read as PDF

THE JOURNAL OF COMPARATIVE NEUROLOGY 395:466–480 (1998)
Serotonin Immunoreactivity
in the Central Nervous System of the
Marine Molluscs Pleurobranchaea
californica and Tritonia diomedea
LELAND C. SUDLOW, JIAN JING, LEONID L. MOROZ, AND RHANOR GILLETTE*
Department of Molecular and Integrative Physiology and the Neuroscience Program,
University of Illinois, Urbana-Champaign, Urbana, Illinois 61801
ABSTRACT
The central nervous systems of the marine molluscs Pleurobranchaea californica
(Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were
examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired
clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In
the cerebropleural ganglion complex, these were the metacerebral giant neurons (both
species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including
identified neurons of the escape swimming network (both species), and a dorsal lateral cluster
in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both
species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia
had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal
commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal
ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both
species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. A few asymmetrical
5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the
left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known
physiologic properties of their identified members are consistent with a loosely organized arousal
system of serotonergic neurons whose components can be generally or differentially active in
expression of diverse behaviors. J. Comp. Neurol. 395:466–480, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: serotonin; immunohistochemistry; Mollusca; Aplysia; Lymnaea
Serotonin (5-HT) plays multiple roles in modulating the
behavioral state in molluscs, arthropods, annelids, and
vertebrates (Jacobs and Fornal, 1993; Weiger, 1997). Its
roles range across regulation of affective state in behavior,
setting arousal levels in neural networks, and fine-tuning
sensory pathways. In molluscs, 5-HT innervation within
the central nervous system (CNS) is prominent and so
influential in network up-regulation that it has been
proposed to act as a general arousal factor (Kupfermann
and Weiss, 1981; Sakharov, 1990). Thus, 5-HT acts in
arousal mechanisms for defensive behavior, as in gill/
siphon withdrawal in Aplysia (Kandel and Schwartz,
1982; Glanzman et al., 1989), and analogous behaviors in
the pteropod Clione (Satterlie and Norekian, 1996) and the
pulmonate snail Helix (Zakharov et al., 1995). Serotonin
acts as a facilitatory modulator in feeding networks of
Aplysia (Kupfermann and Weiss, 1981) and Lymnaea
(Kyriakides and McCrohan, 1989), and in the respiratory
network of Lymnaea (Moroz, 1991) as well. Serotonin is
r 1998 WILEY-LISS, INC.
also an intrinsic neuromodulator of motor network and
muscle activity in the escape swim of Tritonia (Katz et al.,
1994; McClellan et al., 1994; Katz and Frost, 1995a,
1995b) and in parapodial swimming in both Aplysia (McPherson and Blankenship, 1991c) and Clione (Panchin et
al., 1996; Satterlie and Norekian, 1996). In Tritonia and
Lymnaea, 5-HT may regulate ciliary locomotion by means
of serotonergic neurons in the pedal ganglia (Audesirk et
al., 1979; Syed et al., 1988). The bilateral pair of serotonergic giant cerebral ganglion cells, the metacerebral giants
Grant sponsor: National Institutes of Health; Grant numbers: RO1
NS26838 and PHS 5 T32 MN18412.
*Correspondence to: Dr. R. Gillette, Department of Molecular and
Integrative Physiology, University of Illinois, Urbana-Champaign, 524
Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801.
E-mail: [email protected]
Received 2 September 1997; Revised 3 February 1998; Accepted 6
February 1998
SEROTONIN IN MOLLUSCS
(MCGs) conserved in many opisthobranch and pulmonate
species (Weinreich et al., 1973; Senseman and Gelperin,
1974; Sakharov, 1976; Weiss and Kupfermann, 1976;
Gillette and Davis, 1977; Granzow and Rowell, 1981; Croll,
1987), exert varying degrees of influence in feeding behavior (Gillette and Davis, 1977; Weiss et al., 1978; Yeoman et
al., 1996).
In the predatory opisthobranch Pleurobranchaea, 5-HT
may play roles in locomotion and in the escape swimming
network similar to those of Tritonia, because the animals
share apparently homologous networks for these behaviors (Jing and Gillette, 1995; Jing et al., 1997; Jing and
Gillette, 1998). In this animal, 5-HT stimulates multiple
aspects of behavioral activity, including feeding behavior
(Palovick et al., 1982; Gillette et al., 1997). Thus, in Pleurobranchaea as in other molluscs, 5-HT may play an important role as a general arousal factor. Previously, we described the serotonergic innervation of the periphery in both
Pleurobranchaea and Tritonia (Moroz et al., 1997). We
found it to be extensive, and entirely central in origin. In
particular, greatest densities of 5-HT-immunoreactive (5HT-IR) elements were found in the foot and reproductive
system, and in multiple feeding structures. Dense 5-HT-IR
innervation of chemosensory epithelia suggested neuromodulatory regulation of chemosensory pathways by central elements.
In the present work, we describe 5-HT immunoreactivity in the CNS of Pleurobranchaea and Tritonia. We undertook this study to identify putative serotonergic neurons in
Pleurobranchaea’s neural networks for feeding, locomotion, and escape swimming, and those cells that innervate
chemosensory areas and the reproductive system. We
included Tritonia for comparison, because it represents a
line of the nudibranch evolutionary radiation derived from
pleurobranchomorphs that conserves some primitive traits
of its pleurobranchomorph ancestors and differs in others
central to its ecologic specialization. It is also used as a
model system of considerable power for which no published immunohistochemical map for 5-HT exists.
We found around 170 5-HT-IR cells in the CNS of
Pleurobranchaea. Identified neurons that were 5-HT-IR
were the MCGs and cells of the dorsal A-cluster group of
the cerebropleural ganglia, and the G neurons of the pedal
ganglia. The buccal ganglion is densely 5-HT-IR innervated, but lacks 5-HT-IR somata, and the sole source of
5-HT is probably the MCGs. Tritonia, with about 220
5-HT-IR cells, shares many apparently homologous 5-HT-IR
cells with Pleurobranchaea, based on their relative sizes
and positions, including the MCGs, G cells, and the
A-cluster cells. Consideration of the anatomical and physi-
Abbreviations
5-HT
5-HT-IR
ABC
BSA
CNS
DAB
DSI
INa,cAMP
MCG
PAP
PBS
PBS-GT
PBST
serotonin
serotonin-immunoreactive
avidin-biotin complex
bovine serum albumin
central nervous system
3,38-diaminobenzidine tetrahydrochloride
dorsal swim interneuron
cAMP gated sodium current
metacerebral giant
peroxidase-antiperoxidase
phosphate buffered saline
phosphate buffered saline with goat serum and Triton
X-100
phosphate buffered saline with Triton X-100
467
ologic attributes of the 5-HT-IR neurons points to their
possible role as a loosely organized arousal network capable of modulating the expression of diverse behaviors.
MATERIALS AND METHODS
Specimens of Pleurobranchaea californica and Tritonia
diomedea were obtained from Mr. Mike Morris (Sea Life
Supply, Sand City, CA) or Dr. Rimmon Fay (Pacific BioMarine, Venice, CA) and maintained in 14°C artificial sea
water. Twenty Pleurobranchaea, ranging from 10 to 300 g,
and four Tritonia, ranging from 20 to 100 g, supplied
ganglia for whole-mounts. Ganglia from an additional
eight Pleurobranchaea were used in control experiments.
Five Pleurobranchaea, ranging from 15 to 300 g, were
studied in cryotome sections. Animals were anesthetized
by chilling; ganglia were quickly dissected free and transferred to artificial Pleurobranchaea saline (in mM: 420
NaCl, 10 KCl, 25 MgCl2, 25 MgSO4, 10 CaCl2, 10 MOPS
buffered with NaOH, pH 7.5).
For whole-mount immunocytochemistry, ganglia were
pinned to Sylgard and fixed for 1 hour at 0oC, then 5 hours
at 4oC in 4% paraformaldehyde in 450 mM NaCl with 20
mM phosphate buffer, pH 7.5. Tissues were washed 24
hours (4 3 6 hours) in equiosmotic phosphate buffered
saline (PBS; 450 mM NaCl, 20 mM phosphate, 0.5%
thimerosal, pH 7.5) and dehydrated through an ethanol
series to absolute ethanol in 30-minute steps. Ethanol was
replaced with absolute methanol and H2O2 was added to a
final concentration of 0.5 % (v/v) to inactivate endogenous
peroxidases. After incubating for 45 minutes in the methanol-H2O2 solution, the tissues were rehydrated through a
decreasing ethanol dilution series (30-minute steps) into
PBS (150 mM NaCl, 20 mM phosphate, 0.5 % thimerosal,
pH 7.5). All further blocking, incubations, and washings
were performed at 4oC with agitation. Tissues were blocked
in PBS with 3% (v/v) heat-inactivated goat serum (Cappel,
Organaon Teknika Corp., Durham, NC) and 0.4% Triton
X-100 (PBS-GT) for 4 hours. Two different lots of rabbit
anti-serotonin antisera (IncStar, Stillwater, MN) were
used over the course of these experiments. Lot 8843026
was used at a final dilution of 1:1,000 in PBS-GT (PBS
with 0.25% Triton X-100). Subsequent lots of antisera from
different rabbits were used at final dilutions of 1:10,000 ,
1:15,000 in PBS-GT. Primary incubations lasted 72 hours.
Tissues were washed 24 to 72 hours (minimum 4 3 6 hour
washes) in PBS with 0.25% Triton X-100 (PBST). Staining
was accomplished by the avidin-biotin complex (ABC)
-horseradish peroxidase technique (Vectastain, Vector Labs,
Burlingame, CA) or by the peroxidase-antiperoxidase (PAP)
technique. Both ABC and PAP reactions produced similar
staining patterns of somata in the cerebropleural and
pedal ganglia. For ABC staining protocols, tissues were
incubated in biotinylated goat anti-rabbit IgG (Cappel) at
a final dilution of 1:200 in PBS-GT for 24 hours, washed for
24 to 72 hours in PBST, and incubated 24 hours in the ABC
reagent. For PAP reactions, tissues were incubated in goat
anti-rabbit IgG (Cappel) at a final dilution of 1:200 in
PBS-GT for 24 hours. Ganglia were washed for 24 to 72
hours (minimum 4 3 6 hour washes) and incubated in
rabbit PAP (Jackson ImmunoResearch, West Grove, PA) at
a final dilution of 1:100 for 24 hours. Tissues were then
washed for at least 48 hours in PBST. Tissues were washed
in PBS for 4 hours and transferred to 50 mM Tris-HCl
(pH 7.2) for 4 hours. In some experiments, the Tris-HCl
468
L.C. SUDLOW ET AL.
concentration was increased to 120 mM (pH 7.2). Tissues
were incubated at least 2 hours in 0.05% 3,38-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co., St.
Louis, MO) in Tris-HCl buffer at room temperature in the
dark. Final peroxidase reactions were performed by adding H2O2 to 0.05% to the DAB reaction solution. Progress
of the chromogenic reaction was monitored under subdued
lighting. Reaction times were typically 5–15 minutes.
Tissues were washed in Tris buffer (3 3 2 hours) and
dehydrated through absolute ethanol. Tissues were cleared
and mounted either in methyl salicylate or in Permount
(Fisher Scientific, Pittsburgh, PA). Soma diameters reported in this study were from the ,100-g animals.
For positive controls, a conjugate of bovine serum albumin (BSA) and 5-HT was synthesized after the method of
Steinbusch et al. (1983) for use in preabsorption controls.
In brief, 1 ml of 3 M sodium acetate (pH 7.2), 9.2 mg of
serotonin creatinine sulfate (Sigma) in 1 ml of distilled
water, 28.1 mg of BSA (BSA Fraction V, Sigma) in 1 ml of
distilled water, and 1 ml of 7.5% freshly made paraformaldehyde were sequentially added to 10 ml of distilled water
and reacted overnight in the dark at room temperature.
The 5-HT-BSA conjugation reaction was terminated by
dialysis (Spectra/Por 4; molecular weight cut-off, 12,000–
14,000; Spectrum Medical Industries, Los Angeles, CA)
against running deionized water for 24 hours. The 5-HTBSA conjugate was then aliquotted and lyophilized for
future use. Positive immunocytochemical control incubations used 1 mg of conjugate per 10 ml of antisera
overnight at 4oC with agitation. Serotonin immunoreactivity could be eliminated by preincubation of the primary
antisera with the 5-HT-BSA conjugate (n 5 3, data not
shown). Negative controls omitting the primary antisera
exhibited no staining of somata or neuropil (n 5 5, data not
shown).
For cryotome sectioning, fixed preparations were rinsed
in two to three changes of 0.5 M PBS, incubated at 4oC for
4–8 hours in 30% sucrose in 0.4 M Tris-HCl buffer solution
(pH 7.4), embedded in Tissue-Tec O.C.T. compound (Miles,
Elkhart, IN), and frozen on dry ice. Serial sections of four
preparations of the Pleurobranchaea CNS were cut at
30 µm with a cryotome, mounted on charged and precleaned microscope slides (ProbeOn Plus, Fisher), and
dried for 10–20 minutes at room temperature. The staining protocols were identical both for whole-mount preparations and cryotome sections. The nomenclature of ganglia
and ganglionic lobes is used as described earlier (Moroz
and Gillette, 1996).
RESULTS
The fixation and staining protocols preserved and defined immunoreactive elements in the CNS with a high
degree of resolution in both Pleurobranchaea and Tritonia.
The staining pattern of somata was generally replicable,
with slight differences mentioned further. Stained somata
and their unstained glial invaginations were distinct even
in the perinuclear region (Fig. 1A). Fine 5-HT-IR neuronal
processes could be traced in the neuropil (Fig. 1B) and
were often seen investing unstained somata (Fig. 1C,D).
2–6). Stained cell bodies of the dorsal surface of the
cerebropleural ganglion were distributed in three major,
bilaterally paired clusters (Figs. 2A, 3). A dorsal anterior
cluster consisted of the MCG (.240 µm diameter) and
three medium-sized neurons (150–190 µm) found in the
rhinophore lobe; soma positions were consistent among
preparations. In sectioned material, axons from the somata of the medium-sized cells were seen to enter the
neuropil and run posteromedially toward the cerebral
commissure (Fig. 6). One-to-two small 5-HT-IR somata
(70–80 µm) near the medium cells in the rhinophore lobe
were seen in sectioned material.
The second group was the dorsal medial cluster located
near the cerebral commissure (Figs. 2A, 3). This group of
5-HT-IR somata consisted of five small-diameter cells
(70–80 µm), identified elsewhere as As1, As2, As3, As4 and
As-rh, of which As1-4 are elements of an escape swimming
pattern generating network (Jing et al., 1997; Jing and
Gillette, 1998).
The third group was the dorsal lateral cluster found
laterally in each cerebropleural hemiganglion near the
roots of the anterior and posterior cerebropedal connectives (Figs. 2A, 3). The dorsal lateral cluster consisted of
five to seven small (70–80 µm), medium (160–180 µm), and
one giant somata. Last, one asymmetrical giant cell was
found on the dorsal posterior surface of the left cerebropleural ganglion near the left small body wall nerve (Figs.
2A, 3).
The ventral surface of the cerebropleural ganglia had
relatively few 5-HT-IR cells (Fig. 4). The MCGs were
visible because they occupy the edge of the anterior pole of
the cerebral lobe. Those few 5-HT-IR somata were located
in a single, ventral anterior cluster of four small (60–80 µm)
and three medium-sized (150–180 µm) somata arrayed
bilaterally along the medial border of each cerebropleural
hemiganglion on the ventral surface posterior to the MCGs
(Fig. 4). Finally, a single 5-HT-IR asymmetrical giant soma
was found on the ventral surface of the left cerebropleural
hemiganglion near the root of the left body wall nerve
(Fig. 4).
Neuropilar staining was also observed in the cerebropleural ganglion. Serotonin-immunoreactive fibers and varicosities were clearly visible both in whole-mounts (Fig. 2A)
and in sectioned material (Fig. 5). The strongest 5-HT-IR
neuropilar staining in the cerebropleural ganglia was
found lateral to the MCG somata (Figs. 2A, 5A, 6).
Additional dense regions of neuropilar staining were observed posterolaterally in the cerebropleural ganglion
(Fig. 2A). Serotonin-immunoreactive fibers crossed in the
anterior and posterior portions of the cerebral commissure
(Fig. 2A, 6). Numerous 5-HT-IR axons of various diameters
exited the ganglion from all major nerve roots with the sole
exception of the optic nerve, which had none (Fig. 2A).
There was an asymmetrical distribution of large-diameter
axons between the left and right cerebropleural hemiganglia: large-diameter axons were found in the left body wall
nerve, the left cerebrovisceral connective, and the left
small body wall nerve; the contralateral nerves lacked the
large-diameter axons.
Cerebropleural ganglion: Pleurobranchaea
Pedal ganglia: Pleurobranchaea
Serotonin-immunoreactive somata in the cerebropleural
ganglion were limited to four major clusters of cell bodies
distributed on both the dorsal and ventral surfaces (Figs.
The pedal ganglia of Pleurobranchaea exhibited the
most numerous 5-HT-IR somata and processes in the CNS.
In both left and right pedal ganglia, 5-HT-IR somata were
SEROTONIN IN MOLLUSCS
469
Fig. 1. Patterns of serotonin immunoreactivity in individual neurons of Pleurobranchaea (cryotome sections). Asterisks indicate 5-HTimmunonegative neuronal somata densely surrounded by serotoninimmunoreactive (5-HT-IR) terminals. A: Serotonin immunoreactivity
in two giant neurons (open and filled arrows) from the posterior lateral
lobe of the left pedal ganglion. Note a specific pattern of the serotonin
immunoreactivity in the larger neuron (open arrow). Perinuclear
areas (black curved arrow) stain homogeneously, whereas more distant cytoplasmic areas (arrowheads) show non-uniform staining due
to the presence of numerous glial processes in the cytoplasm of the
giant cell (see also Results section). B: Serotonin-immunoreactive
neuron in the anterior medial lobe of the right pedal ganglion. Note the
5-HT-IR soma (arrow) next to the immunonegative soma (asterisk).
C,D: Neighboring 40-µm sections from a giant neuron in the lateral
cerebropleural ganglion. Note the basket-like pattern of 5-HT-IR
neuronal terminals (arrow in C) around the neuronal somata.
n, nucleus. Scale bar 5 150 µm (applies to A–D).
found predominately at the dorsomedial lobes of the
ganglia (Figs. 2B, 3, 5B). This group of somata was termed
the ‘‘G’’ cells in earlier studies (Huang and Gillette, 1991,
1993; Sudlow and Gillette, 1995, 1997). When left and
right pedal ganglia from the same animal were compared
in whole-mount preparations, there were both similarities
and differences in the distribution of 5-HT-IR somata
(Fig. 3). Although there were similar numbers of largediameter (4) and medium-diameter (12–13) 5-HT-IR somata in the dorsomedial lobe of the pedal hemiganglia,
there were fewer small-diameter somata in the right pedal
than in the left pedal ganglion (Fig. 3). Also, somata by the
roots of the posterior and medial pedal nerves in the right
pedal ganglion were both fewer and larger than in the left
ganglion (Fig. 3). The dorsal surfaces of the left and right
posterior lateral lobes of the pedal ganglia had similar
numbers of 5-HT-IR somata. Lightly stained cells were
observed in the accessory lobes of the pedal ganglia. When
the ventral surfaces of pedal ganglia of the same animal
were compared, an asymmetry was observed in the distribution of medium and small-diameter 5-HT-IR somata
(Figs. 3, 4).
Serotonin-immunoreactive processes were observed exiting the pedal ganglia in all major nerve and connective
roots (Fig. 2B). Serotonin-immunoreactive axons ran in
the anterior and posterior cerebropedal connectives and in
the pedal and parapedal commissures. Tracts of fibers
coursed through the ganglia between the anterior and
posterior cerebropedal connectives and the posterior pedal
nerves (Fig. 2B). Axons arising from the 5-HT-IR somata at
the dorsomedial lobe coursed laterally in the pedal ganglion (Fig. 5B). Axons exited the pedal ganglia in all
peripheral nerves.
Buccal and visceral ganglia:
Pleurobranchaea
The buccal ganglion of Pleurobranchaea was devoid of
5-HT-IR somata (Figs. 7A, 8). One large-diameter, and
occasionally a small-diameter, axon entered the buccal
ganglion from each of the bilateral cerebrobuccal connectives. Processes of these fibers ramified in the buccal
ganglion and surrounded many of the somata (Figs. 7B, 8).
Because no 5-HT-IR somata were visible in the buccal
470
Fig. 2. Serotonin immunoreactivity in the central nervous system
of Pleurobranchaea (whole-mount preparation). Arrowheads point to
somata of the metacerebral giant neurons, immediately caudally
adjacent to which are neurons of the dorsal anterior cluster of
serotonin-immunoreactive (5-HT-IR) somata. A: Cerebropleural gan-
ganglion and only one large-caliber axon was present in
the cerebrobuccal connectives, the ramified 5-HT-IR processes were most likely associated with this large axon.
Because the axon of the MCG is the only giant, putatively
serotonergic axon identified in the cerebrobuccal connectives (Gillette and Davis, 1977), it is most likely that the
MCG is the source of the ramified 5-HT-IR processes.
Serotonin-immunoreactive processes could be observed
crossing to the contralateral buccal hemiganglion. Axons
exited the buccal ganglion through the buccal and stomatogastric roots, similar to the known branching pattern of
the MCGs in the buccal ganglion (Gillette and Davis,
1977). No 5-HT-IR somata were observed in the visceral
ganglion (Fig. 9); however, fibers were observed both
enmeshing somata and coursing through from the left and
right cerebrovisceral connectives to exit to the periphery.
L.C. SUDLOW ET AL.
glion, dorsal surface. Arrows indicate the five 5-HT-IR somata of the
A-cluster of the dorsal medial 5-HT-IR cluster (As1-4 and As-rh).
B: Pedal ganglion, dorsal surface. The arrow points to the G cells.
Scale bar 5 500 µm (applies to A,B).
Cerebropleural ganglia: Tritonia
Serotonin immunoreactivity in the cerebropleural ganglia of Tritonia is similar in many ways to Pleurobranchaea (Figs. 10–12). The MCGs in Tritonia were most
striking for their large size (.280 µm) relative to other
ganglion cells (Figs. 10, 12), but the dorsal anterior cluster
of smaller somata observed in Pleurobranchaea was not
present. There were few other stained somata on the
dorsal surface. In the dorsal medial cluster, five intensely
stained somata of small diameter (50–80 µm) were found
medially near the anterior border of the cerebral commissure (Figs. 10A, 12A). Of these, the three most anterolateral are likely to be the serotonergic ‘‘dorsal swim interneurons,’’ or DSIs identified in Tritonia’s swim network (Getting
et al., 1980; McClellan et al., 1994; Katz et al., 1994), and
SEROTONIN IN MOLLUSCS
471
Fig. 3. Schematic diagram of the distribution of serotoninimmunoreactive (5-HT-IR) neurons on dorsal surfaces of cerebropleural and pedal ganglia of Pleurobranchaea. Cerebropleural ganglion:
aCPC, anterior cerebropedal connective; BWN, body wall nerves (left
and right); CBC, cerebrobuccal connective; CC, cerebral commissure;
CVC, cerebrovisceral connective; MN, mouth nerve; ON, optic nerve;
OVN, oral veil nerve; pCPC, posterior cerebropedal connective; RhL,
rhinophore lobe; RN, rhinophore nerve; sBWN, left small body wall
nerve of cerebropleural ganglion; SCC, subcerebral commissure; TN,
tentacular nerve. Pedal ganglion: AccL, accessory lobe; aPN, anterior
pedal nerve; aLBWN, anterior lateral body wall nerve of the pedal
ganglia; DML, dorsomedial lobe; mPN, medial pedal nerve; PC, pedal
commissure; pLBWN, posterior lateral body wall nerve of the left
pedal ganglion; PLL, posterior lateral lobe; pPC, parapedal commissure; pPN, posterior pedal nerve.
homologs of the As1-3 neurons of Pleurobranchaea (Jing et
al., 1997). The remaining two most posteromedial cells
may correspond to the As-rh and As4 cells of Pleurobranchaea (Jing and Gillette, 1998). A second small cluster of
lightly stained, medium-sized (,120 µm) somata (shaded
cells illustrated in Fig. 12A) was located near the posterior
margin of the cerebral commissure, sometimes mingled
with the putative As-rh and As4 homologs.
The ventral surface of the Tritonia cerebropleural lobes,
as in Pleurobranchaea, exhibited relatively little serotonin
immunoreactivity (Fig. 12). All labeled somata were found
in the anterior cerebral lobes. Two medium-sized (,110–
140 µm) and 11–14 small-sized (50–70 µm) somata, the
ventral anterior cluster, were observed just caudal to the
MCGs. The posterior lobes of the cerebropleural ganglion
did not exhibit consistent serotonin immunoreactivity. An
occasional 5-HT-IR soma was found in these lobes. How-
ever, these oddly located 5-HT-IR somata were inconsistent, suggesting that their occurrence in the posterior
lobes of the cerebropleural ganglion was a developmental
vagary. The ventral neuropil of the Tritonia cerebropleural
ganglion had one area in each hemiganglion showing a
higher density in neuropil labeling. This lateral region in
the cerebral lobes, near the roots of cerebral nerve 4 (Figs.
10A, 12B), is an area associated with mechanoreceptive
inputs to the cerebral ganglion (Audesirk, 1979). A similar
area in Pleurobranchaea was located near the roots of the
oral veil, tentacular, and mouth nerves (Fig. 2A).
Pedal ganglia: Tritonia
Serotonin-immunoreactive somata on the dorsal surface
of the Tritonia pedal ganglia were clustered as a group
posterolaterally (Figs. 11, 12). There was some variation in
the sizes and distributions of somata between the left and
472
L.C. SUDLOW ET AL.
Fig. 4. Schematic diagram of the distribution of serotonin-immunoreactive neurons on the ventral
surface of the cerebropleural and pedal ganglia of Pleurobranchaea. Pedal abbreviation: VML, ventromedial lobe. For other abbreviations, see the legend to Figure 3.
right ganglia, as well as between specimens. The dorsal
surface of the left pedal ganglion exhibited a characteristic
giant soma (250–280 µm) near the root of the cerebropedal
connective (Figs. 11, 12), possibly the previously identified
‘‘left pedal 1 neuron’’ (Willows et al., 1973). In some
specimens, additional giant somata could be observed
posterior and slightly ventral to the medial giant. These
may have been ventral somata that migrated to a slightly
more medial location, and hence could be readily observed
from the dorsal surface at nearly the same plane of focus as
the medial giant. In whole-mounts of the left ganglion, 3–5
medium-sized (90–220 µm) and 20–24 small-diameter somata
(30–90 µm) were observed (Figs. 11, 12). The dorsal surface of
the right pedal ganglion lacked the medial giant found in the
contralateral ganglion. Three asymmetrical medium-sized
and several small (30–35 µm) somata were slightly posterior
in the posterolateral region of the right pedal ganglion.
The ventral surfaces of left and right pedal ganglia were
the richest in serotonin immunoreactivity of the Tritonia
CNS (Fig. 12). The somata were located near the anterior
and the posterior margins of each ganglion, leaving a zone
between the margins relatively devoid of 5-HT-IR somata
(Fig. 12). At least 20 giant somata could be observed in
each pedal ganglion, mostly near the posterior margin.
Medium-diameter and small-diameter somata were found
predominately near the anterior margin of the pedal ganglia.
Buccal ganglion: Tritonia
As in Pleurobranchaea, the buccal ganglion of Tritonia
lacked 5-HT-IR somata (not shown). Stained fibers coursed
through the buccal ganglion to branch and exit in nerves,
and processes were also observed enmeshing the somata of
the buccal ganglion.
DISCUSSION
Localization of identified 5-HT-IR
somata in Pleurobranchaea
The present morphologic results can be combined in
several cases with physiologic data on identified neurons
and with previous data on the peripheral distribution of
SEROTONIN IN MOLLUSCS
473
Fig. 5. Serotonin immunoreactivity in the somata and neuropil in
the central nervous system of Pleurobranchaea. A: Giant metacerebral
serotonin (5-HT) -containing cells (long black arrows) and serotoninimmunoreactive (5-HT-IR) somata of the dorsal anterior cluster (short
black arrows) in the anterior lobe of cerebropleural ganglion (horizontal cryotome sections). Curved open arrows indicate areas of intense
neuropilar 5-HT-IR staining (asterisks) in the anterior cerebral and
rhinophore lobes of the cerebropleural ganglion; small open arrow
indicates 5-HT-IR neurons of the dorsal lateral cluster of the cerebro-
pleural ganglion; arrowhead indicates a couple of small 5-HT-IR
neurons in the medial part of the ganglion. B: Identified pedal 5-HT-IR
neurons. Medial G-cluster (black arrows) in the right pedal ganglion
with associated group of smaller moderately 5-HT-IR neurons in the
posterior part of the same lobe (small open arrow). Asterisk indicates
5-HT-IR in the neuropil area; large open arrow indicates the cluster of
5-HT-IR neurons in the posterior lateral lobe of the ganglion. Scale
bars 5 500 µm in A, 400 µm in B.
5-HT to yield a fuller picture of serotonergic function in
Pleurobranchaea. In particular, we found three identified
groups of 5-HT-IR cells whose members already have been
identified and characterized to varying degrees: (1) the G
neurons of the dorsomedial lobes of the pedal ganglia; (2)
the As1-4 and As-rh neurons of the dorsal medial cluster in
the cerebropleural ganglion; and (3) the MCG and some
members of the dorsal anterior cluster of the cerebropleu-
ral ganglion. Each of these clusters may compose a functional group of cells with specific roles in different behaviors.
The G neurons of the pedal ganglion are already known
to form a functional group whose members are extensively
electrically coupled (J. Jing and R. Gillette, unpublished
observations) and send peripheral axons to the foot through
the pedal nerves (Jing et al., 1993). By analogy to the
474
L.C. SUDLOW ET AL.
Fig. 6. Organization of serotonin-immunoreactive (5-HT-IR) neuropil in the cerebropleural ganglion of Pleurobranchaea (cryotome
sections). Nomarksi interference contrast image of 5-HT-IR somata in
the anterior cerebral lobe. An open arrow indicates the cluster of
5-HT-IR neurons sending processes to the central neuropil areas.
Small black arrow indicates 5-HT-IR somata of the dorsal lateral
cluster. Asterisk indicates 5-HT-IR in the neuropil area. cc, cerebral
commissure. Scale bar 5 80 µm.
apparently homologous neurons of Tritonia (Audesirk et
al., 1979) and Lymnaea (Syed et al., 1988), they may act as
effectors of ciliary locomotion. Additionally, the present
demonstration that the G neurons are 5-HT-IR suggests
that this group may also compose an intrinsically modulatory motor network; it has been shown that they themselves respond sensitively to 5-HT with depolarization
(Sudlow and Gillette, 1995, 1997). Serotonergic activation
of the G neurons is mediated by receptors coupled to adenylyl
cyclase, whose cyclic AMP product activates a cyclic AMPgated cation current, INa,cAMP (Sudlow and Gillette, 1995, 1997).
The elements of the As1-4 group also form a functional
group. These neurons are members of the escape swimming pattern generating network in which they provide
neuromodulatory effects that promote the swim episode
(Jing et al., 1997; Jing and Gillette, 1998). They are both
electrically and chemically coupled, and fire synchronously; their outputs descend to the pedal ganglion. These
neurons express INa,cAMP (Jing et al., 1997), as do many
feeding cells (Green and Gillette, 1983), the G neurons
(Sudlow and Gillette, 1995, 1997), and the MCG (K. Huang
and R. Gillette, unpublished observations). Whether
INa,cAMP in As1-4 responds to 5-HT stimulation is not yet
known, but their reciprocal long-lasting EPSPs are consistent with such neuromodulation.
Some evidence suggests that the dorsal anterior cluster
of 5-HT-IR cells is also a functional group of coupled,
coactive elements. The MCG neurons were previously
found to be electrically coupled to multiple adjacent neurons with which they fire synchronously during generation
of the feeding rhythm (Gillette and Davis, 1977); it is likely
Fig. 7. Serotonin immunoreactivity in the buccal ganglion of
Pleurobranchaea (whole-mounts). A: Serotonin-immunoreactive (5-HTIR) process can be seen coursing through the buccal ganglion.
B: Nomarksi interference contrast image of somata of buccal ganglion
exhibit basketlike enmeshing by 5-HT-IR termini. Scale bar 5 500 µm
in A, 200 µm in B.
SEROTONIN IN MOLLUSCS
475
Fig. 8. Serotonin immunoreactivity in the buccal ganglion of
Pleurobranchaea (cryotome sections). A: General dorsal view. Open
arrows indicate intensely stained neuropil areas in the medial parts of
the ganglion lobes. B: Central neuropil area. Asterisks indicate the
serotonin (5-HT) -immunonegative neuronal somata surrounded by 5-HTimmunoreactive terminals. Scale bars 5 500 µm in A, 80 µm in B.
that the other serotonergic neurons of this cluster are
those coupled cells, by analogy with the electrically coupled
networks of the G neurons and the As1-4 group (Jing et al.,
1997; J. Jing and R. Gillette, unpublished observations).
The MCG itself is activated by touch, chemosensory stimuli
and activity in the feeding oscillator motor network, of
which it is an element. This neuron provides most or all of
the serotonergic innervation of both the feeding network in
the buccal ganglion and the feeding musculature of the
buccal mass and mouth area; its coupled partners may
serve analogous areas in cerebropleural ganglion neuropil
and in the musculature of the head region. The MCG also
476
L.C. SUDLOW ET AL.
expresses INa,cAMP in its soma (K. Huang and R. Gillette,
unpublished observations); the autosensitivity of this cell
and its coupled partners to 5-HT is not yet tested.
Origin of peripheral innervation
The peripheral 5-HT-IR staining in the ciliary layer of
the foot noted previously (Moroz et al., 1997) must originate in the G cells of the medial lobe of the pedal ganglia.
Similarly, axons of the MCG must provide the extensive
serotonin immunoreactivity of the buccal mass and esophagus through its branches known to innervate these areas
(Gillette and Davis, 1977). For the most part, the central
neurons providing the extensive 5-HT-IR innervation of
the reproductive system and chemosensory areas remain
to be identified through correlation of the present maps
with nerve backfills and double-labeling experiments.
However, one notable exception is the finding of serotonin
immunoreactivity in As-rh and swim interneuron As4 (J.
Jing and R. Gillette, unpublished observations): these cells
provide the 5-HT-IR innervation of the rhinophore and
tentacle and are thus likely to be the source of serotonergic
innervation of the chemosensory epithelium (Moroz et al.,
1997). Identification of these cells may enable rigorous
testing of the role of 5-HT in modulating chemosensory
pathways at the level of receptors, primary afferents, or
both.
Comparative analysis of serotonin
immunoreactivity in gastropods
Fig. 9. Serotonin-immunoreactive (5-HT-IR) processes in the visceral ganglion of Pleurobranchaea (whole-mount preparation). Scale
bar 5 200 µm.
Fig. 10. Serotonin immunoreactivity in the central nervous system
of Tritonia (whole-mounts). The large-diameter stained somata in both
A and B are the metacerebral giant somata. A: Dorsal view. The long
white arrows point to the three dorsal swim interneurons (DSIs) and
two additional serotonin-immunoreactive somata, putatively As-rh
and As4 homologs of the dorsal medial cluster. The small arrowheads
Cerebropleural 5-HT-IR somata in Pleurobranchaea were
confined to four primary clusters. Similar clustering has
been noted in cerebral ganglia of other gastropod molluscs
(Kistler et al., 1985; Land and Crow, 1985; Longley and
Longley, 1986; Kemenes et al., 1989; Diefenbach and
Goldberg, 1990; Soinila and Mpitsos, 1991; Satterlie et al.,
1995). The dorsal and ventral anterior clusters including
the symmetrical 5-HT-IR MCG somata, perhaps the most
well studied pair of homologous molluscan serotonergic
neurons (Weinreich et al., 1973; Weiss and Kupfermann,
1976; Sakharov, 1976; Gillette and Davis, 1977; Granzow
and Rowell, 1981; Croll, 1987), and several adjacent smallto medium-sized 5-HT-IR somata near the MCG are clearly
recognizable here in Pleurobranchaea and in other species,
whereas Tritonia lacks the smaller diameter dorsal anterior cluster cells. Similar symmetrical groups of 5-HTcontaining medium-sized cells and MCGs were also described earlier in Tritonia sp. by Manokhina and Kuz’mina
point to lightly staining somata (shaded cells in Fig. 12A) near
putative As-rh and As4 homologs. B: Ventral view. The open arrow
points to the densely staining neuropil enmeshing the somata in the
lateral region of the cerebral ganglion. The small arrowheads point to
the ventral anterior cluster. Scale bar 5 200 µm (applies to A,B).
SEROTONIN IN MOLLUSCS
Fig. 11. Serotonin immunoreactivity in the pedal ganglia of Tritonia (whole-mount preparation). The view is of the dorsal surface of the
left pedal ganglion. The large-diameter soma is located near the root of
the cerebropedal connective. Scale bar 5 200 µm.
(1971) with the formaldehyde-induced fluorescence technique. The locations of the homologous clusters, as identified by the MCG, vary somewhat across species. The dorsal
anterior cluster was found at the anterior pole of the
cerebropleural ganglia in Pleurobranchaea (Figs. 2, 3, 10,
12) and in the cerebral ganglion of the pteropod Clione
(Satterlie et al., 1995). In contrast, in Aplysia (Ono and
McCaman, 1984; Longley and Longley, 1986; Soinila and
Mpitsos, 1991), Lymnaea (Kemenes et al., 1989), and
Helisoma (Diefenbach and Goldberg, 1990), the likely
homologous cluster is found somewhat caudally on the
dorsal ganglion surface.
A likely homologous dorsal medial 5-HT-IR cerebral
cluster was found near the cerebral commissures in all the
above species (Longley and Longley, 1986; Kemenes et al.,
1989; Diefenbach and Goldberg, 1990; Nolen and Carew,
1994; Satterlie et al., 1995), but this cluster was not
described in earlier formaldehyde-induced fluorescence
studies on Tritonia (Manokhina and Kuz’mina, 1971). In
Aplysia, the left and right CB1 5-HT-IR interneurons
involved in the modulation of the gill-siphon withdrawal
circuit in Aplysia belong to this dorsal medial cerebral
cluster (Mackey et al., 1989). These cells in Pleurobranchaea and Tritonia (named the As1-3 and the DSI-A-C
cells, respectively) are components of a homologous escape
swimming network and potentiate its output (Getting et
al., 1980; Lennard et al., 1980; McClellan et al., 1994; Katz
et al., 1994; Jing et al., 1997; Jing and Gillette, 1998).
477
Furthermore, we surmise that the dorsal lateral cerebropleural cluster found in Pleurobranchaea is homologous to
the lightly stained cluster found at the posterior margin of
the Tritonia cerebral commissure.
The presence and distribution of giant 5-HT-IR somata
in the posterior lobes of Pleurobranchaea’s cerebropleural
ganglion is an unusual finding. These lobes were presumably pleural lobes in origin, based on their position relative
to the cerebral commissure. No such staining has been
reported for the pleural ganglia or pleural lobes of any of
the gastropod or pteropod species studied thus far (Goldstein et al., 1984; Ono and McCaman, 1984; Longley and
Longley, 1986; Jahan-Parwar et al., 1987; Kemenes et al.,
1989; Nolen and Carew, 1994). In contrast, putative serotonergic neurons have been identified in the parietal
(intestinal or abdominal) ganglia of Lymnaea (Kemenes et
al., 1989) and Aplysia (Ono and McCaman, 1984; Longley
and Longley, 1986; Jahan-Parwar et al., 1987; Soinila and
Mpitsos, 1991). Because Pleurobranchaea lacks distinct or
separate parietal ganglia, it is likely that these 5-HT-IR
somata in the posterior cerebropleural lobes represent the
remnants of the parietal (intestinal) ganglia found in other
molluscs (cf. Bullock and Horridge, 1965). Because Tritonia does not have routinely staining 5-HT-IR somata in its
pleural lobes, the remnants of Tritonia’s parietal lobes may
have been fused into the pedal ganglia.
The buccal ganglia in Pleurobranchaea and Tritonia
both lack 5-HT-IR somata even though varicosities and
fibers in their buccal ganglia were stained. This finding is
consistent with other species (Ono and McCaman, 1984;
Longley and Longley, 1986; Jahan-Parwar et al., 1987;
Kemenes et al., 1989; Nolen and Carew, 1994; Satterlie et
al., 1995) and suggests that the MCGs are the major and
perhaps sole source of 5-HT innervation. The investment
of the buccal somata by numerous 5-HT-IR varicosities
suggests axosomatic synapses similar to MCG innervation
in Aplysia (Goldstein et al., 1984).
An interesting twist in the centralization of the CNS has
left the majority of giant 5-HT-IR somata (the G cells) on
the ventral surface of Tritonia’s pedal ganglia, whereas
they reside dorsally in Pleurobranchaea (Figs. 3, 12).
Presumably, the half-twist in the cerebropedal connectives
of the notaspids (the likely ancestral condition) is straightened out by a rotation of the ganglia in the nudibranch as
they fuse to the cerebropleural complex, leaving the cell
clusters in the ventral position. Pedal 5-HT-IR somata,
similar in location to the Pleurobranchaea G cells, are
found in a wide variety of opisthobranch, anaspidean, and
pteropod molluscs (Ono and McCaman, 1984; Land and
Crow, 1985; Longley and Longley, 1986; Kemenes et al.,
1989; Diefenbach and Goldberg, 1990; Satterlie et al.,
1995). Outside of Tritonia and Pleurobranchaea in which
they may effect ciliary locomotion, they act in Aplysia
brasiliana and Aplysia californica as modulators of the
parapodial muscles (McPherson and Blankenship, 1991a,
1991b, 1991c, 1992) and similarly in Clione limacina
(Satterlie, 1995).
CONCLUSIONS AND FUTURE DIRECTIONS
Serotonin has been proposed to modulate the general
arousal state associated with feeding, locomotor, defensive, and other behaviors in gastropods and diverse invertebrates (Sakharov, 1990; Weiger, 1997) based on its
abilities to up-regulate spontaneous activity and reactivity
in both neural networks and muscle. In general, our
478
L.C. SUDLOW ET AL.
Fig. 12. Schematic diagram of the distribution of serotoninimmunoreactive neurons in the central nervous system of Tritonia
diomedea. A: Dorsal view. B: Ventral view. Empty circles represent
somata that stained in a small number of preparations. Shaded circles
represent lightly stained somata. Dark circles represent heavily
stained somata. Shaded areas on the ventral lateral region of the
cerebral ganglion represent the intensely stained neuropil in Fig. 10B.
Nerve notations are based on the anatomical scheme of Willows et al.
(1973): CBC, cerebrobuccal connective; CC, cerebral commissure;
CeN, cerebral nerve; CPC, cerebropedal connective; PdN, pedal nerve;
PlN, pleural nerve.
emerging picture of a serotonergic arousal network in
Pleurobranchaea bears similarities to the compartmental
system of serotonergic cells proposed to modulate swimming in Clione (Satterlie and Norekian, 1996) in which the
component clusters can act either in concert and perhaps
differentially as well. For Pleurobranchaea, the different
serotonergic clusters are associated with aspects of feeding
behavior (the cerebral anterior cluster, including the MCG),
SEROTONIN IN MOLLUSCS
escape swimming (dorsal medial cluster), and creeping
locomotion (pedal medial lobe cluster, or G neurons). Our
observations indicate that neurons within clusters are
coupled electrically, chemically, or both, and are coactive,
and that clusters may interact with each other (Jing et al.,
1997; J. Jing and R. Gillette, unpublished observations).
Clusters are also known to act in different states of
coordination: during the escape swim the As1-4 fire in
intense bursts locked to the swim rhythm, and during
feeding the MCG firing pattern is itself locked to the
feeding rhythm, whereas the As1-4 group is silent (J. Jing
and R. Gillette, unpublished observations). Thus, the
putatively serotonergic clusters have distinct target neural networks, and they potentially coordinate behavior
through actions either in concert or semi-independently.
It is also notable that some of the putatively serotonergic
neurons identified here, including the MCGs and the G
cells of the pedal ganglia, may produce another potent
neuromodulator, nitric oxide. These neurons coexpress
NADPH-diaphorase staining, a marker for nitric oxide
synthase (Moroz and Gillette, 1996). The colocalization of
serotonin immunoreactivity and NADPH-diaphorase staining in these neurons suggests possible interactions of the
two signaling molecules on target cells.
In conclusion, in locating the putatively serotonergic
somata of the CNS, we have established a reference base
for the further study of the regulation of central and
peripheral physiologic activities in Pleurobranchaea and
Tritonia, and for comparative studies in other invertebrates.
ACKNOWLEDGMENTS
We thank Dr. J.M. Ding for technical assistance.
LITERATURE CITED
Audesirk, G. (1979) Oral mechanoreceptors in Tritonia diomedea: I. Electrophysiologic properties and locations of receptive fields. J. Comp. Physiol.
130:71–78.
Audesirk, G., R.E. McCaman, and A.O.D. Willows (1979) The role of
serotonin in the control of pedal ciliary activity by identified neurons in
Tritonia diomedea. Comp. Biochem. Physiol. 62C:87–91.
Bullock, T.H. and G.A. Horridge (1965) Structure and Function in the
Nervous Systems of Invertebrates. San Francisco and London: W.H.
Freeman.
Croll, R.P. (1987) Identified neurons and cellular homologies. In M.A. Ali
(ed): Nervous Systems in Invertebrates. NATO ASI Series, Series AL
Life Sciences, Vol. 141. New York: Plenum Press, pp. 41–59.
Diefenbach, T.J. and J.I. Goldberg (1990) Postembryonic expression of the
serotonin phenotype in Helisoma trivolvis: Comparison between laboratory-reared and wild-type strains. Can. J. Zool. 68:1382–1389.
Getting, P.A., P.R. Lennard, and R.I. Hume (1980) Central pattern generator mediating swimming in Tritonia: I. Identification and synaptic
interactions. J. Neurophysiol. 44:151–164.
Gillette, R. and W.J. Davis (1977) The role of the metacerebral giant
neurone in the feeding behaviour of Pleurobranchaea. J. Comp. Physiol.
116:129–159.
Gillette, R., L.L. Moroz, R. Fuller, P. Floyd, and J.V. Sweedler (1997) 5-HT
and NO co-localized in neurons of feeding and locomotor networks may
interact to regulate hunger state in Pleurobranchaea. Soc. Neurosci.
Abstr. 23:978.
Glanzman, D.L., S.L. Mackey, R.D. Hawkins, A.M. Dyke, P.E. Lloyd, and
E.R. Kandel (1989) Depletion of serotonin in the nervous system of
Aplysia reduces the behavioral enhancement of gill withdrawal as well
as the heterosynaptic facilitation produced by tail shock. J. Neurosci.
9:4200–4213.
Goldstein, R., H.B. Kistler, H.W.M. Steinbusch, and J.H. Schwartz (1984)
Distribution of serotonin-immunoreactivity in juvenile Aplysia. Neuroscience 11:535–547.
479
Granzow, B. and C.H.F. Rowell (1981) Further observations on the serotonergic cerebral neurones of Helisoma (Mollusca, Gastropoda): The case
for homology with the metacerebral giant cells. J. Exp. Biol. 90:283–
305.
Green, D.J. and R. Gillette (1983) Patch- and voltage-clamp analysis of
cyclic AMP-stimulated inward current underlying neurone bursting.
Nature 306:784–785.
Huang, R.-C. and R. Gillette (1991) Kinetic analysis of cAMP-activated Na1
current in the molluscan neuron: A diffusion-reaction model. J. Gen.
Physiol. 98:835–848.
Huang, R.-C. and R. Gillette (1993) Coregulation of cyclic AMP-activated
Na1 current by Ca21. J. Physiol. 462:307–320.
Jacobs, B.L. and C.A. Fornal (1993) 5-HT and motor control: A hypothesis.
Trends Neurosci. 16:346–352.
Jahan-Parwar, B., K.S. Rozsa, J. Salankia, M.L. Evans, and D.O. Carpenter
(1987) In vivo labeling of serotonin-containing neurons by 5,7dihydroxytryptamine in Aplysia. Brain Res. 426:173–178.
Jing, J. and R. Gillette (1995) Neuronal elements that mediate escape
swimming and suppress feeding behavior in the predatory sea slug
Pleurobranchaea. J. Neurophysiol. 74:1900–1910.
Jing, J. and R. Gillette (1998) The central pattern generator forescape
swimming in Pleurobranchaea californica. J. Neurophysiol. in press.
Jing, J., L.C. Sudlow, and R. Gillette (1993) Neural organization of feeding
and aversive behavior in the predatory seaslug Pleurobranchaea. Soc.
Neurosci. Abstr. 19:581.
Jing, J., L.C. Sudlow, and R. Gillette (1997) Mechanisms of pattern
generation of escape swimming and potential role of cAMP-gated cation
current in the sea slug Pleurobranchaea. Soc. Neurosci. Abstr. 23:1047.
Kandel, E.R. and J.H. Schwartz (1982) Molecular biology of learning:
Modulation of transmitter release. Science 218:433–443.
Katz, P.S. and W.N. Frost (1995a) Intrinsic neuromodulation in the Tritonia
swim CPG: Serotonin mediates both neuromodulation and neurotransmission by the dorsal swim interneurons. J. Neurophysiol. 74:2281–
2294.
Katz, P.S. and W.N. Frost (1995b) Intrinsic neuromodulation in the Tritonia
swim CPG: The serotonergic dorsal swim interneurons act presynaptically to enhance transmitter release from interneuron C2. J. Neurosci.
15:6035–6045.
Katz, P.S., P.A. Getting, and W.N. Frost (1994) Dynamic neuromodulation
of synaptic strength intrinsic to a central pattern generator circuit.
Nature 367:729–731.
Kemenes, G.Y., K. Elekes, L. Hiripi, and P.R. Benjamin (1989) A comparison
of four techniques for mapping the distribution of serotonin and
serotonin-containing neurons in fixed and living ganglia of the snail,
Lymnaea. J. Neurocytol. 18:193–208.
Kistler, H.B., R.D. Hawkins, J. Koester, H.W.M. Steinbusch, E.R. Kandel,
and J.H. Schwartz (1985) Distribution of serotonin-immunoreactive cell
bodies and processes in the abdominal ganglion of mature Aplysia. J.
Neurosci. 5:72–80.
Kupfermann, I. and K.R. Weiss (1981) The role of serotonin in arousal of
feeding behavior of Aplysia. In B.L. Jacobs and A. Gelperin (eds):
Serotonin Neurotransmission and Behavior. Cambridge: MIT Press, pp.
255–287.
Kyriakides, M.A. and C.R. McCrohan (1989) Effect of putative neuromodulators on rhythmic buccal motor output in Lymnaea stagnalis. J.
Neurobiol. 20:635–650.
Land, P.W. and T. Crow (1985) Serotonin immunoreactivity in the circumesophageal nervous system of Hermissenda crassicornis. Neurosci.
Lett. 62:199–205.
Lennard, P.R., P.A. Getting, and R.I. Hume (1980) Central pattern generator mediating swimming in Tritonia: II. Initiation, maintenance, and
termination. J. Neurophysiol. 44:165–173.
Longley, R.D. and A.J. Longley (1986) Serotonin immunoreactivity of
neurons in the gastropod Aplysia californica. J. Neurobiol. 17:339–358.
Mackey, S.L., E.R. Kandel, and R.D. Hawkins (1989) Identified serotonergic
neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce
presynaptic facilitation of siphon sensory neurons. J. Neurosci. 9:4227–
4235.
Manokhina, M.S. and L.V. Kuz’mina (1971) Distribution of biogenic amines
in the central nervous system of the pacific nudibranch mollusk
Tritonia sp. Zh. Evol. Biokhim. Fiziol. 7:357–361 (in Russian).
McClellan, A.D., G.D. Brown, and P.A. Getting (1994) Modulation of
swimming in Tritonia: Excitatory and inhibitory effects of serotonin. J.
Comp. Physiol. 174A:257–266.
480
McPherson, D.R. and J.E. Blankenship (1991a) Neural control of swimming
in Aplysia brasiliana: I. Innervation of parapodial muscle by pedal
ganglion motoneurons. J. Neurophysiol. 66:1338–1351.
McPherson, D.R. and J.E. Blankenship (1991b) Neural control of swimming
in Aplysia brasiliana: II. Organization of pedal motoneurons and
parapodial motor fields. J. Neurophysiol. 66:1352–1365.
McPherson, D.R. and J.E. Blankenship (1991c) Neural control of swimming
in Aplysia brasiliana: III. Serotonergic modulatory neurons. J. Neurophysiol. 66:1366–1379.
McPherson, D.R. and J.E. Blankenship (1992) Neuronal modulation of foot
and body-wall contractions in Aplysia californica. J. Neurophysiol.
67:23–28.
Moroz, L.L. (1991) Monoaminergic control of the respiratory behaviour in
freshwater pulmonate snail, Lymnaea stagnalis (L.). In W. Winlow, O.V.
Vinogradova, and D.A. Sakharov (eds): Signal Molecules and Behaviour. Manchester: Manchester University Press, pp. 101–123.
Moroz, L.L. and R. Gillette (1996) NADPH-diaphorase localization in the
CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea
californica. J. Comp. Neurol. 367:607–622.
Moroz, L.L., L.C. Sudlow, J. Jing, and R. Gillette. (1997) Serotoninimmunoreactivity in peripheral tissues of the opisthobranch molluscs
Pleurobranchaea californica and Tritonia diomedea. J. Comp. Neurol.
382:176–188.
Nolen, T.G. and T.J. Carew (1994) Ontogeny of serotonin-immunoreactive
neurons in juvenile Aplysia californica: Implications for the development of learning. Behav. Neural Biol. 61:282–295.
Ono, J.K. and R.E. McCaman (1984) Immunocytochemical localization and
direct assays of serotonin-containing neurons in Aplysia. Neuroscience
11:549–560.
Palovick, R.A., B.A. Basberg, and J.L. Ram (1982) Behavioral state changes
induced in Pleurobranchaea and Aplysia by serotonin. Behav. Neural
Biol. 35:383–394.
Panchin, Y.V., Y.I. Arshavsky, T.G. Deliagina, G.N. Orlovsky, L.B. Popova,
and A.I. Selverston (1996) Control of locomotion in the marine mollusc
Clione limacina: XI. Effects of serotonin. Exp. Brain Res. 109:361–365.
Sakharov, D.A. (1976) Nerve cell homologies in gastropods. In J. Salanki
(ed): Neurobiology of Invertebrates. Gastropoda Brain. Budapest: Acad.
Kiado, pp. 27–39.
Sakharov, D.A. (1990) Integrative function of serotonin common to distantly related invertebrate animals. In M.K.S. Gustafsson and M.
Reuter (eds): The Early Brain. Abo: Abo Akademy Press. pp. 73–88.
Satterlie, R.A. (1995) Serotonergic modulation of swimming speed in the
pteropod mollusc Clione limacina: II. Peripheral modulatory neurons.
J. Exp. Biol. 198:905–916.
Satterlie, R.A. and T.P. Norekian (1996) Modulation of swimming speed in
the pteropod mollusc, Clione limacina: Role of a compartmental serotonergic system. Invert. Neurosci. 2:157–165.
L.C. SUDLOW ET AL.
Satterlie, R.A., T.P. Norekian, S. Jordan, and C.J. Kazilek (1995) Serotonergic modulation of swimming speed in the pteropod mollusc Clione
limacina: I. Serotonin immunoreactivity in the central nervous system
and wings. J. Exp. Biol. 198:895–904.
Senseman, D. and A. Gelperin (1974) Comparative aspects of the morphology and physiology of a single identified neuron in Helix aspersa, Limax
maximus, and Ariolimax californica. Malacol. Rev. 7:51–52.
Soinila, S. and G.J. Mpitsos (1991) Immunohistochemistry of diverging and
converging neurotransmitter systems in mollusks. Biol. Bull. 181:484–
499.
Steinbusch, H.W.M., A.A.J. Verhofstad, H.W.J. Joosten (1983) Antibodies to
serotonin for neuroimmunocytochemical studies: Methodological aspects and applications. In A.C. Cuello (ed): Immunocytochemistry. New
York: John Wiley & Sons, pp. 193–214.
Sudlow, L.C. and R. Gillette (1995) Cyclic AMP-gated sodium current in
neurons of the pedal ganglion of Pleurobranchaea californica is activated by serotonin. J. Neurophysiol. 73:2230–2236.
Sudlow, L.C. and R. Gillette (1997) Cyclic AMP levels, adenylyl cyclase
activity, and their stimulation by serotonin quantified in intact neurons. J. Gen. Physiol. 110:243–255.
Syed, N.I., D. Harrison, and W. Winlow (1988) Locomotion in Lymnaea: Role
of serotonergic motoneurones controlling the pedal cilia. A-cluster
neurones. In J. Salanki and K. S-Rozsa (eds): Neurobiology of Invertebrates: Transmitters, Modulators and Receptors. Symp. Biol. Hungarica. Volume 36. Tihany: Acad Kiado, pp. 387–402.
Weiger, W.A. (1997) Serotonergic modulation of behaviour: A phylogenetic
overview. Biol. Rev. 72:61–95.
Weinreich, D., M.W. McCaman, R.E. McCaman, and J.E. Vaughn (1973)
Chemical, enzymatic, and ultrastructural characterization of 5-hydroxytryptamine-containing neurons from the ganglia of Aplysia californica and Tritonia diomedea. J. Neurochem. 20:969–976.
Weiss, K.R. and I. Kupfermann (1976) Homology of the giant serotonergic
neurons (metacerebral cells) in Aplysia and pulmonate molluscs. Brain
Res. 117:33–49.
Weiss, K.R., J.L. Cohen, and I. Kupfermann (1978) Modulatory control of
buccal musculature by a serotonergic neuron (metacerebral cell) in
Aplysia. J. Neurophysiol. 41:181–203.
Willows, A.O.D., D.A. Dorsett, and G. Hoyle (1973) The neuronal basis of
behavior in Tritonia: I. Functional organization of the central nervous
system. J. Neurobiol. 4:207–237.
Yeoman, M.S., M.J. Brierley, and P.R. Benjamin (1996) Central pattern
generator interneurons are targets for the modulatory serotonergic
cerebral giant cells in the feeding system of Lymnaea. J. Neurophysiol.
75:11–25.
Zakharov, I.S., V.N. Ierusalimsky, and P.M. Balaban (1995) Pedal serotonergic neurons modulate the synaptic input of withdrawal interneurons of
Helix. Invert. Neurosci. 1:41–52.