localization of gaba-like immunoreactivity in the central nervous system ofaplysia californica

Localization of GABA-likeImmunoreactivity in the Central Nervous

System of Aplysia californica

MANUEL DIAZ-RIOS, ERIC SUESS, AND MARK W. MILLER*Institute of Neurobiology and Department of Anatomy, University of Puerto Rico,

San Juan, Puerto Rico 00901

ABSTRACTGamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia

californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supportedby pharmacological, biochemical, and anatomical investigations. In this study, the distribu-tion of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examinedby using wholemount immunohistochemistry and nerve backfill methods. GABAi neuronswere located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems werepresent in each of these ganglia, whereas more limited fiber systems were observed in theganglionic connectives. Some of the interganglionic fibers were found to originate from twounpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, eachof which exhibited bilateral projections. No GABAi fibers were found in the nerves thatinnervate peripheral sensory, motor, or visceral organs. Although GABAi cells were notobserved in the pleural or abdominal ganglia, these ganglia did receive limited projections ofGABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAineurons suggests that this transmitter system may be primarily involved in coordinatingcertain bilateral central pattern generator (CPG) systems related to feeding and locomotion.In addition, the presence of specific interganglionic GABAi projections also suggests a role inthe regulation or coordination of circuits that produce components of complex behaviors. J.Comp. Neurol. 413:255–270, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: molluscan neurotransmitters; molluscan feeding; molluscan locomotion; biocytin;

immunohistochemistry; g-aminobutyric acid

Neurotransmitter phenotype, one property that is typi-cally used in the classification of neural systems, can oftenprovide insight into the functional properties of neuralcircuits (Shepherd, 1998). Gamma-aminobutyric acid(GABA) is one neurotransmitter that is widely distributedin the mammalian central nervous system where it acts asa major mediator of synaptic inhibition (Roberts, 1960,1986; Florey, 1961). Substantial evidence suggests thatGABA also acts as a neurotransmitter in a wide range ofinvertebrate phyla, including arthropods (Kuffler and Ed-wards, 1958; Kravitz et al., 1963; Otsuka et al., 1967),echinoderms (Newman and Thorndyke, 1994), annelids(Ito et al., 1969; Cline, 1983, 1986), nematodes (del Castilloet al., 1964; Johnson and Stretton, 1987; McIntire et al.,1993), and planarians (Eriksson and Panula, 1994).

In molluscs, a neurotransmiter role for GABA wasinitially suggested by pharmacological studies in which itwas found to produce both excitatory and inhibitory re-sponses upon application to snail neurons (Gerschenfeldand Tauc, 1961; Walker et al., 1971, 1975). Biochemical

approaches demonstrated the presence of GABA, its syn-thesis, and its uptake in the central nervous systems ofseveral molluscan species (Osborne et al., 1971; Doleza-lova et al., 1973; Cottrell, 1974). The localization of GABAto specific neurons within molluscan nervous systems wasshown by using autoradiographic and immunohistologicaltechniques (Turner and Cottrell, 1978; Cooke and Gel-perin, 1988; Richmond et al., 1991; Arshavsky et al., 1993;Hernadi, 1994; Norekian, 1999).

GABA is present in each of the central ganglia of Aplysiacalifornica (Gastropoda, Opisthobranchia; Cottrell, 1974).

Grant sponsor: NSF CAREER Award; Grant number: IBN-9722349;Grant sponsor: RCMI Award (Division of Research Services, NIH); Grantnumber: RR-03051; Grant sponsor: NIGMS, NIH; Grant number: MBRSGM-08224; Grant sponsor: Puerto Rico EPSCoR (NSF); Grant sponsor:DoD Instrumentation (ONR); Grant number: N00014–93–1380.

*Correspondence to: Dr. Mark W. Miller, Institute of Neurobiology,University of Puerto Rico, 201 Blvd del Valle, San Juan, Puerto Rico 00901.E-mail: [email protected]

Received 1 April 1999; Revised 15 June 1999; Accepted 24 June 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 413:255–270 (1999)

r 1999 WILEY-LISS, INC.

Moreover, GABA produces characteristic responses whenapplied to specific neurons (Yarowsky and Carpenter,1977, 1978; King and Carpenter, 1987). Using sectionedganglia, Soinila and Mpitsos (1991) described a system ofGABA-immunoreactive (GABAi) neurons within the cen-tral nervous system of Aplysia. In this study, we haveexamined the distribution of GABAi neurons in Aplysiacalifornica using wholemount immunohistochemistry andnerve backfill methods. Our observations indicate thatGABAergic systems are confined to the central nervoussystem and that they appear to be specialized for the intra-and interganglionic transmission of information. Thesestructural features suggest that GABAergic neurons mayplay an important role in the organization of certaincomplex behaviors in Aplysia.

MATERIALS AND METHODS

Immunohistochemistry

Specimens of Aplysia californica (10–30 g) were pur-chased from the Aplysia Resource Facility and Experimen-tal Hatchery (University of Miami, Miami, FL) and main-tained in a refrigerated aquarium (14–16°C). The animalswere typically used within 2 weeks of their arrival. Theseprotocols were approved by the Institutional Animal Careand Use Committee (IACUC) of the University of PuertoRico Medical Sciences campus.

Standard wholemount immunohistochemical protocolswere followed (Longley and Longley, 1986; see Miller et al.,1991, 1992 for details regarding buffer composition, incuba-tion and wash procedures). Specimens were immobilizedwith an injection of isotonic MgCl2 equal to 50% of bodyweight. Ganglia were dissected, pinned out on Sylgard,and fixed (4 hours) by using cold 4% paraformaldehyde in80 mM phosphate buffer (PB: 24 mM KH2PO4, 56 mMNa2HPO4, pH 7.4) containing 24% sucrose. Ganglia werethen washed (53, room temperature with agitation) inPTA (PB containing 2% Triton X-100 and 0.1% sodiumazide). Following preincubation with normal donkey se-rum (0.8%), tissues were immersed (48 hours, room tem-perature) in the primary antibody (polyclonal; affinityisolated). The best results were obtained by using dilutionsranging from 1:50 to 1:200 in PTA. The primary antibody(rabbit host) was purchased from Sigma (St. Louis, MO,product no. A2052). Following repeated PTA washes, gan-glia were incubated in tetramethyl rhodamine (TRITC)-conjugated second antibody (donkey anti-rabbit IgG TRITC-conjugated F(ab’)2 fragment: Jackson ImmunoResearch,West Grove, PA) diluted 1:200 in PTA. Negative controlswere perfomed by omitting either the primary or secondantibodies. None of the immunopositive structures re-ported in this study exhibited autofluorescence within thewavelengths used for detection of rhodamine. Tissues werecleared in a glycerol:PB solution (1:6) prior to desheathing(see below). They were viewed and photographed on aninverted microscope equipped with epifluorescence (NikonOptiphot) or on a laser scanning confocal microscope(Odyssey, NORAN Instruments, Middleton WI). All im-ages shown in this article were originally photographedwith 35-mm film (Kodak T-MAX ASA 400). The negativeswere scanned (Nikon CoolScan) at 450 dpi and transportedas BMP files to Adobe Photoshop for adjusting overallcontrast and brightness. The images were then trans-ported as TIFF files to Corel Draw 8 for addition of labels

and organization of panels. For each ganglion, at least 25specimens were processed and examined.

During the course of this study, we found that moredefinitive staining was consistently obtained using gangliathat were not desheathed prior to the immunohistochemis-try process. This characteristic appears to be uniquelyassociated with GABA staining, as desheathing is gener-ally thought to be improve access of antibodies to neuronsin wholemount preparations (see Pearson and Lloyd, 1989;Giardino et al., 1996). This idiosyncrasy was particularlyevident in the pedal ganglia, where desheathed prepara-tions exhibited background levels of fluorescence thatcompletely obscured visibility of immunoreactive neuronsand fiber tracts. Ganglia were therefore desheathed follow-ing exposure to the antibodies. Because the rigidity ofprocessed ganglia was found to impede desheathing, prepa-rations were first cleared in glycerol. The glycerol reducedthe stiffness of the ganglia, making it possible to pin themto Sylgard. Although structural details and cues were lostfollowing clearing in glycerol, manual desheathing wasfacilitated due to the increased flexibility and the stabilityof the pinned ganglia.

Nerve and connective backfills

The advantages of the biotin-avidin system for investiga-tions combining nerve backfills with immunocytochemis-try have been demonstrated in a number of systems (seeColeman et al., 1992; Li and Chase, 1995). Recently, thesemethods were adapted for application to Aplysia by Dr.Y.-P. Xin (Center for Neurobiology and Behavior, ColumbiaUniversity College of Physicians and Surgeons) and Dr.Sven Vilim (Department of Physiology and Biophysics,Mount Sinai School of Medicine). Our protocol follows thatof Xin et al. (1999) with modifications based upon Johnsonet al. (1999). At least three specimens were examined foreach nerve or connective backfill.

Specimens were immobilized and nervous systems weredissected as described above. The ganglion of interest waspinned out near a small vaseline well that was formed onthe Sylgard surface. The nerve or connective being exam-ined was cut and drawn into the well. Care was taken toavoid contact between the end of the nerve and thevaseline. The tip of the nerve was cut one more time andthen the artificial seawater (ASW) inside the well waswithdrawn and replaced with a saturated aqueous solu-tion (1.6 mg / 30 µl) of biocytin (Sigma). The walls of thewell were then built up with successive layers of vaseline,forming an ‘‘igloo’’ that effectively isolated the biocytin poolfrom the ASW surrounding the ganglion. The preparationwas covered and incubated overnight at 4°C. The well wasthen removed, and ganglia were washed 3–5 times in ASW.They were then pinned out, and fixed in paraformaldehydeas described above. The fixed ganglia were transferred tomicrocentrifuge tubes, washed 5 times (30 minutes each)with PTA solution and incubated overnight (room tempera-ture, with shaking) in Rhodamine600 Avidin D (VectorLaboratories, Burlingame, CA) diluted 1:3,000 in PTA(24–48 hours, room temperature). Tissues were thenwashed 5 times with PTA and processed for GABA-likeimmunoreactivity, as described above. In the double-labeling experiments, a fluorescein isothiocyanate (FITC)-conjugated second antibody (donkey anti-rabbit IgG FITC-conjugated F(ab’)2 fragment: Jackson ImmunoResearch)diluted 1:200 in PTA was used to visualize labeled mate-rial. Ganglia were examined on a Nikon fluorescence

256 M. DIAZ-RIOS ET AL.

microscope or on the NORAN Odyssey scanning confocalmicroscope by using an argon laser and barrier filterssuitable for viewing FITC (band pass 520–560 nm) andrhodamine (long pass 590 nm).

RESULTS

Buccal ganglion

Each hemiganglion contained approximately 10 GABAicell bodies (Figs. 1; 2B; 3A2, 3B2, 3C2; 4A1, 4A2). A cluster of4 to 6 moderately sized (50–100 µm diameter) cells waslocated in the dorsolateral region of the ganglion, betweenthe origins of the esophageal nerve and buccal nerve 1(nomenclature of Gardner, 1971). The cells comprising this

cluster were heterogeneous in size, shape, and stainingintensity (Fig. 1A,B, arrowheads). They were usuallyequally distinguished, and slightly blurred, when viewingthe ganglion with the plane of focus on either the caudal orrostral surface (compare Fig. 1A and 1B), suggesting thatthis cluster occupies a deep position, beneath the mostsuperficial layer of cell bodies. Each cell projected a majorprocess in the ventromedial direction, i.e., toward thebuccal commissure. These fibers joined the major axontract that courses through the central region of the gan-glion. Individual fibers could not be followed beyond theirconvergence with this tract.

A bilateral cluster of two to three small (10–20 µmdiameter) GABAi neurons occupied a slightly more dorsal

Fig. 1. Gamma aminobutyric acid immunoreactivity (GABAi) inthe buccal ganglion. A: Plane of focus on the caudal surface of the righthemiganglion. A group of five moderately sized cells (arrowhead)is located in the lateral region of the ganglion. Fibers from these cellsprojected toward the midline. A second group of three smaller cells(arrow) occupied a more dorsal position, between the origins of theesophageal nerve (e n.) and buccal nerve 1 (b n.1). Prominent GABAifibers are present in the cerebral buccal connective (cbc). B: Rostralsurface of the buccal ganglion (same ganglion as A). The cluster oflarger cells (arrowhead) remains visible from this surface, whereas

the group of smaller cells can not be distinguished. A prominentGABAi cell (arrow) is located in the midline of the ganglion nearthe buccal commissure. C: Many of the large cells in the ventral motorneuron cluster were covered with fine GABAi fibers. Two suchcells, located in the medial region of the cluster (arrow), weretentatively identified as the B4/B5 cell pair. D: A bipolar unpairedGABAi neuron (arrow) was located in the midline of the buccalcommissure (b c.). r n., radula nerve. Scale bar 5 650 µm in A, B, andD; 250 µm in C.

GABA-LIKE IMMUNOREACTIVITY IN APLYSIA 257

Fig. 2. Buccal gamma aminobutyric acid-immunoreactive (GABAi)neurons with cerebral buccal connective (cbc) projections. A: Biocytinbackfill of the cerebral buccal connective visualized with rhodamine.The cbc associated with the hemiganglion on the right side of this fieldwas backfilled (connective is not visible within this image). Plane offocus is on the rostral surface of the ganglia. B: GABAi in the same

preparation as A, viewed with a fluorescein-conjugated second anti-body. Three double-labeled cells can be seen, including the medialrostral cells in both hemiganglia (arrows in A and B) and the unpairedmidline cell (arrowheads in A and B). b c., buccal commissure. Scalebar 5 350 µm.


position, near the edge of each hemiganglion between theesophageal nerve and buccal nerve 1 (Fig. 1A, arrow).These cells were clearly nearer the caudal surface of theganglion, because they were difficult to distinguish whenfocusing on the rostral surface (Fig. 1B). No fibers could beassociated with these small cells.

There was a single strongly immunoreactive neuron(60–80 µm diameter) on the rostral surface of each buccal

hemiganglion (Fig. 1B, arrow). These cells always had abipolar appearance, but some had a more elongated somathan others (Fig. 2A and B, arrows; compare left and righthemiganglia). They were usually located in the ventrome-dial region of the ganglion, near the identified multifunc-tion B4/B5 cell pair. However, in some preparations thesecells were positioned closer to the buccal commissure (e.g.,Fig. 1B). Each cell projected a fiber toward the buccal

Fig. 3. Buccal gamma aminobutyric acid-immunoreactive (GABAi)neurons with cerebral buccal connective (cbc) projections. A1: Biocytincbc backfill (labeled with rhodamine avidin) of unpaired bipolarneuron (arrow) in the buccal commissure (b c.). A2: Same field as A1.GABA immunoreactivity labeled with fluorescein second antibody.B1: Biocytin backfill (labeled with rhodamine avidin) of medial rostralcell (arrow) near the buccal commissure. B2: Same field as B1. GABA

immunoreactivity labeled with fluorescein second antibody. C1: Biocy-tin backfill (labeled with rhodamine avidin) of two neurons (arrow-head, arrow) in buccal ganglion contralateral to the backfilled cbc. Onebackfilled cell (arrowhead) had a stellate appearance, due to character-istic projections from its soma. C2: The stellate cell and an additionallarger backfilled cell (arrow) were also labeled with the GABAantiserum (fluorescein second antibody; C2). Scale bar 5 200 µm.


commissure. A second fiber extended laterally where itjoined the major ganglionic fiber bundle.

The central region of the buccal ganglion, including thebuccal commissure, contained a dense neuropil of fineGABAi fibers. A number of the large buccal cell bodieslocated in the ventral motor neuron cluster were coveredwith a network of varicose fibers, suggesting the presenceof axosomatic synapses in this system (Fig. 1C, arrow).Several large smooth fibers were present in the cerebralbuccal connective (cbc; Fig. 1A). No GABAi fibers wereobserved in the radula nerve, the esophageal nerve, orbuccal nerves 1–3.

A single unpaired GABAi neuron was also located in theregion of the buccal commissure (Fig. 1D, arrow). As thiscell was frequently shifted toward one hemiganglion or theother (see for example Fig. 2, arrowhead), its designationas an unpaired cell was initially equivocal. However, in afew preparations this neuron was located precisely in thecenter of the buccal commissure (Fig. 1D). It was viewedmore clearly from the caudal surface of the ganglion and italways had a characteristic bipolar form, projecting pro-cesses into each buccal hemiganglion (Fig. 1D).

Double-labeling experiments were conducted to deter-mine whether any of the GABAi buccal neurons project

fibers into the CBC. Connectives were backfilled withbiocytin, incubated in rhodamine-conjugated avidin, andthen processed for GABA-like immunoreactivity using aFITC-conjugated second antibody (see Materials and Meth-ods). In comparison to other substances that were testedfor this purpose (Lucifer yellow, DiI, Lucifer yellow-biocytin, 5–6 carboxyfluorescein), biocytin proved superiorin terms of migration distance, speed, brightness, andcompatibility with the immunohistochemistry procedure.However, we did encounter considerable interspecimenvariability by using this method, and it usually producedfewer stained neurons than are found with backfills byusing cobalt in this system (cf. Scott et al., 1991).

When CBC backfills and GABA immunoreactivity werecompared in single ganglia, double-labeling was observedin several neurons (Figs. 2, 3). Backfilling a single CBCwas found to label the unpaired medial GABAi neuron(Figs. 2A,B, arrowhead; 3A1, A2) and both of the rostralGABAi neurons (Figs. 2A,B, arrows; 3B1, B2). In addition,two neurons belonging to the caudal GABAi cluster in thecontralateral hemiganglion were double-labeled (Fig. 3C1,C2). One of these cells (Fig. 3C1, C2, arrows) was among thelargest and brightest neurons in the cluster, whereas theother (Fig. 3C1, C2, arrowheads) was considerably smaller

Fig. 4. Summary of gamma aminobutyric acid-immunoreactive(GABAi) neurons in the buccal and cerebral ganglia. A: Schematicrepresentation of GABAi neurons in the buccal ganglion viewed fromthe rostral surface (A1) and viewed from the caudal surface (A2). Cellswith bilateral projections to the cerebral buccal connectives (cbc; thetwo rostral cells and the unpaired midline cell) are shown withcross-hatched fill patterns and cells with unilateral projections to thecontralateral cbc (two neurons in each hemiganglion) are unshaded.Projections of the remaining cells (black fill pattern) are unknown.B: Schematic representation of GABAi neurons in the cerebral ganglionviewed from the ventral surface (B1) and viewed from the dorsal

surface (B2). Capital letters on the left side of ganglion in B1 denoteregions or clusters of the ganglion as designated in previous studies(Jahan-Parwar and Fredman, 1976; Rosen et al., 1979; Ono andMcCaman, 1980). Two pairs (one dorsal and one ventral) of GABAicerebral-buccal interneurons (cross-hatched fill patterns) are shownwith fibers projecting to the ipsilateral cbc. r n., radula nerve; sal n.,salivary nerve; e n., esophageal nerve; b n.1–3, buccal nerves 1–3,respectively; pt n., posterior tentacular nerve; o n., optic nerve; c-pl c.,cerebral-pleural connective; ul n., upper labial nerve; at n., anteriortentacular nerve; ll n., lower labial nerve; c-pd c., cerebral-pedalconnective.


and had a stellate appearance resulting from the presenceof a number of fine processes protruding from its soma.

Taken together, these observations indicate that at leastfive GABAi fibers in each CBC originate from neurons inthe buccal ganglion (shown schematically in Fig. 4). Threeof these cells, including the unpaired medial cell, projectbilaterally. Two GABAi cells in each hemiganglion appearto decussate and ascend exclusively into the contralateralCBC. The seven buccal GABAi cells with fibers in theCBCs are likely to function as buccal-cerebral interneu-rons, but their designation as such awaits further charac-terization. It is conceivable, for example, that some ofthese cells do not influence cerebral circuits at all, but thatthey project to more posterior ganglia via the cerebral-pedal connective (C-Pd c.) or the cerebral-pleural connec-tive (C-Pl c.; see below). This possibility remains to be tested indouble-labeling experiments in which the C-Pd and the C-Plconnectives are backfilled toward the buccal ganglion.

Cerebral ganglion

All of the cerebral GABAi neurons were located in theanterior and lateral regions of the ganglion (Figs. 4B,5A,C). The majority these cells were found in the E cluster(Fig. 4B1; nomenclature of Jahan-Parwar and Fredman,1976), i.e., within the angle formed by the cerebral-pedalconnective and the anterior tentacular nerve. AdditionalGABAi cell bodies were present in the ‘‘crotch’’ regionbetween the origins of the upper labial and the anteriortentacular nerve (the M cluster of Ono and McCaman,1980) and a few were located more medially in the D and Gclusters.

Approximately 30 GABAi cells were distributed in eachanterolateral quadrant of the ventral surface of the cere-bral ganglion (Fig. 5A). No asymmetries in neuron distribu-tion between the two hemiganglia could be establishedwith our methods. The GABAi neurons showed consider-

Fig. 5. Gamma aminobutyric acid-immunoreactivity (GABAi) inthe cerebral ganglion. A: Plane of focus on the ventral surface. Themajority of the GABAi neurons are located in lateral regions of theganglion. A major commissural fiber system crosses the midline of theganglion (arrow) and interganglionic fibers are present in the cerebralbuccal connective (cbc) and in the cerebral pedal connective (c-pd c.).B: Higher magnification of ventral surface of the cerebral ganglion inthe region of the anterior tentacular (at n.) and upper labial (ul n.)nerves. Clusters of six to eight neurons were present on each side of

the origin of the upper labial nerve (arrows). C: Plane of focus on thedorsal surface of another cerebral ganglion. The majority of the dorsalGABAi cells are also located in the E cluster region of the ganglion.D: Higher magnification of the area enclosed by dashed box in C. Thelarger dorsal GABAi cells occur as individuals, whereas smaller cellsappear as groups (arrow). Many of the dorsal GABAi cells give rise tofibers that project in a medial direction (arrowheads), where theyappear to contribute to the commissural system (see A). Scale bar 5500 µm in A and C; 200 µm in B and D.


able variation in size, shape, and staining intensity. Manyhad prominent initial segments, and it was possible tofollow their processes for considerable distances. Some ofthe ventral GABAi neurons were grouped. Two suchgroups were located on either side of the origin of the upperlabial nerve (Fig. 5B, arrows). Each contained 6 to 8neurons with varying shapes and sizes. The GABAi neu-rons on the dorsal surface of the cerebral ganglion weresomewhat smaller (range: 10–80 µm diameter) than theventral cells (Fig. 5C). Although the larger dorsal cellsappeared to be individuals, the smaller cells were grouped(Fig. 5D, arrow). It was often possible to distinguish thefibers of the dorsal GABAi cells, many of which wereoriented toward the medial region of the ganglion (Fig. 5D,arrowheads).

Many fine GABAi fibers crossed the midline of theganglion (Fig. 5A, arrow). Varicose fibers covered thesomata of several large neurons in the most lateral regionsof the ganglion. As detailed above, several strongly immu-noreactive fibers were present in each CBC. No fibers wereobserved in the anterior tentacular nerves, the upper orlower labial nerves, or the optic nerves. Two to four GABAifibers were present in each cerebral-pedal connective (Fig.

5A) and one to two were seen in each cerebral-pleuralconnective.

Several classes of neurons within the cerebral ganglionare known to project to the buccal ganglion via the CBC.These include the interganglionic cerebral-buccal mecha-nosensory afferents (ICBMs; Rosen et al., 1979, 1982), themodulatory serotonergic metacerebral cells (MCCs; Weisset al., 1978, 1982), and various cerebral-buccal interneu-rons circuits (CBIs) that regulate buccal circuits (Rosen etal., 1991; Perrins and Weiss, 1998; Xin et al., 1999;Hurwitz et al., 1999). Nerve backfill experiments wereconducted to determine whether GABAi fibers within theCBC were associated with identified cells of the cerebralganglion. Backfills using biocytin and visualized withrhodamine-conjugated avidin were in general agreementwith previous reports in which the CBC was filled withcobalt or nickel (Jahan-Parwar and Fredman, 1976; Rosenet al., 1982, 1991). A group of 5 to 9 cells was located in theregion of the ipsilateral J and K clusters (nomenclature ofRosen et al., 1979) on the ventromedial surface of theganglion (Fig. 6A1). Fewer (2 to 4) neurons were filled inthe contralateral J and K clusters. These cells correspondto the ICBMs, the subset of J and K cluster primary

Fig. 6. The majority of sensory neurons and interneurons in thecerebral ganglion with projections to the buccal ganglion do not exhibitgamma aminobutyric acid-immunoreactivity (GABAi). A1: Biocytinbackfill of the cerebral buccal connective visualized with rhodamine. Abundle of fibers (arrow) projects to the J and K clusters and terminatesin a cluster of small cells, corresponding to the interganglioniccerebral-buccal mechanoafferents (icbm). A2: Same field as A1. GABAimmunoreactivity labeled with rhodamine second antibody. No cells inthe region of the icbms were labeled. B1: Biocytin backfill of thecerebral buccal connective visualized with rhodamine. Several large

cells near the base of the cerebral buccal connective (cbc) were filled,including two posterior ovoid neurons (arrows), tentatively identifiedas the cerebral-buccal interneurons circuit (CBI)-5/6 pair (Perrins andWeiss, 1998) and a group of three anterior cells (arrowheads), tenta-tively identified as CBI-4 (Rosen et al., 1991) and CBI-8/9 (Xin et al.,1999). B2: Same field as B1. GABA immunoreactivity labeled withfluorescein second antibody. A single multipolar GABAi was locatednear the CBI-5/6 pair, but none of the identified CBIs were immunore-active. Scale bar 5 200 µm.


mechanoafferent neurons that project to the buccal gan-glion (Rosen et al., 1982). No GABAi cells were present inthe region of the backfilled ICBMs (Fig. 6A2). The absenceof GABAi neurons in the J and K clusters is consistentwith the observed lack of GABAi fibers in the peripheralnerves of the buccal and cerebral ganglia (see above),because the ICBMs and other mechanoreceptors in thisregion are known to project to their receptive fields viathese nerves (Rosen et al., 1979, 1982).

Backfills of the CBC revealed several cells close to theorigin of the connective in the E-cluster (Fig. 6B1). Thesize, shape, and position of two of these cells (arrows)suggested that they correspond to the recently identifiedCBI-5/6 pair of interneurons that influence feeding rhythmsvia both buccal and cerebral connections (Perrins andWeiss, 1998). Although no GABAi was detected within theputative CBI-5/6 pair, a neighboring cell with a character-istic bipolar morphology did exhibit immunoreactivity(Fig. 6B2, arrow).

Additional backfilled cells were located slightly anteriorto the origin of the CBC (Fig. 6B1, arrowheads). Interneu-rons that have been localized to this region include CBI-4,a command-like element that evokes a continuous rhyth-mic buccal motor program (Rosen et al., 1991), and theCBI-8/9 cell pair, which are thought to specifically modu-late the protraction phase of the biting rhythm (Xin et al.,

1999). Although the transmitter(s) present in CBI-4 arepresently unknown, CBI-8/9 have been shown by immuno-cytochemical and biochemical methods to contain mem-bers of the myomodulin family of neuropeptides (Xin et al.,1999). No GABAi was detected in neurons in the region ofCBI-4 and CBI-8/9 (Fig.6B2).

The M cluster of the cerebral ganglion is comprised of6–10 cells that are located between the origins of the upperlabial and anterior tentacular nerves (Ono and McCaman,1980). Four cerebral-buccal interneurons (CBI-1, CBI-2,CBI-3, and CBI-12) have been localized to the M cluster(Rosen et al., 1991; Hurwitz et al., 1999). One neuron inthe posterior region of the M cluster on the ventral surfaceof the ganglion showed double-labeling, i.e., it stained withCBC backfills and exhibited GABA immunoreactivity (Fig.7A1, A2, arrows). In trying to identify this cell, it may bepossible to exclude CBI-1, the only cerebral-buccal interneu-ron in this group that projects bilaterally (Rosen et al.,1991). This exclusion is based upon the observation that asingle M cluster neuron was consistently backfilled fromthe contralateral CBC, and this cell was never found to beGABA-immunoreactive (not shown). Although the poste-rior location of the GABAi cell within the M-clustersuggests that it may correspond to CBI-3, its definitiveidentification will require further comparison to the knownphysiological and morphological characteristics of M clus-

Fig. 7. Gamma aminobutyric acid (GABA)-immunoreactive cere-bral-buccal interneurons. A1: Biocytin backfill of the cerebral buccalconnective (cbc) visualized with rhodamine. Two neurons in thisportion of the crotch region between the upper labial nerve (ul n.) andthe anterior tentacular nerve (at n.) were filled (arrow). A2: Same fieldas A1. GABA immunoreactivity labeled with fluorescein isothiocyanate(FITC)-conjugated second antibody. One of the immunoreactive cells(arrow) corresponds to the more posterior member of the pair that was

backfilled in A1. B1: Biocytin backfill of the cerebral buccal connectivevisualized with rhodamine. A moderately sized cell (arrow) is locatedin the G cluster in the region of of the initial segment of themetacerebral cell (mcc). B2: Same field as B1. GABA immunoreactivitylabeled with fluorescein second antibody. The G cluster neuronspecified in B1 exhibited GABA immunoreactivity (arrow). Scale bar 5200 µm.


ter CBIs (see Rosen et al., 1991; Wu-Morgan et al., 1998;Hurwitz et al., 1999).

An additional GABA-immunoreactive CBI was found inthe G cluster, in the region of the initial segment of theMCC (Fig. 7B1, B2, arrows). This cell typically had a ratherstout axon hillock which was oriented toward the CBC. Asingle CBI in this region was originally observed withnickel backfills (see Fig. 2 in Rosen et al., 1991) and wasrecently designated CBI-11 (Xin et al., 1999). The physi-ological properties of this neuron have not as yet beenreported.

Taken together, these results indicate that at least twoGABAi fibers in each cerebral-buccal connective belong toCBIs, i.e., they originate from neurons of the cerebralganglion that project to the buccal ganglion (Fig. 4B1, B2).No GABA-immunoreactive CBIs were found to projectbilaterally or to decussate prior to projecting to the buccalganglion. In this respect, the organization of the GABAiCBIs seems to differ from the GABAi BCI system (seeabove), in which all neurons appear to either decussateprior to projecting to the cerebral ganglion or to projectbilaterally. The unilateral organization of the GABAi CBIprojections does not imply that the influence of these cellsis confined to ipsilateral buccal targets, as many cerebral-buccal interneurons are known to reach contralateraltargets via the buccal commissure (Rosen et al., 1991;Perrins and Weiss, 1998).

Subesophageal ganglia

No strongly immunoreactive cell bodies were observedin the pleural or abdominal ganglia. However, these gan-glia were found to receive a limited number of GABAiprojections (see also Cleary and Li, 1990). Each of theseprojections appeared to originate in the pedal ganglia (seebelow).

The majority of the GABAi cells in the pedal gangliawere present in four bilateral clusters, designated GI–GIV(Fig. 8A). The largest cluster (GI) was located in theanteromedial region of the ganglion, within sector II ofHening et al. (1979). Upon close examination, considerablevariability could be seen in the sizes (range: 20 µm to 70µm diameter) and shapes of the neurons comprising thiscluster. It was difficult to obtain a focussed view of thisgroup of cells from either the dorsal or ventral surface ofthe ganglion, suggesting that it lies below the most super-ficial layer of cell somata.

Additional clusters (GII–GIV) were present more later-ally in each pedal ganglion. The cells in these groups weremore dispersed than in the GI cluster and were moreheterogeneous in size and shape. All four GABAi clusterswere associated with a compact immunoreactive fiberbundle that arched in a posteromedial direction toward thepedal commissure. This fiber tract originated in the regionof the lateral GABAi cluster (GIV) and passed slightlyposterior to the medial cluster (GI). It is likely to containaxons arising from neurons in all four of the clusters, butindividual fibers were never clearly associated with cellbodies. In the medial region of the ganglion, some of thefibers comprising this bundle appeared to depart andproject into the pedal commissure. The rest of the bundleremained in close apposition and curved back toward thecenter of the ganglion. As a result of this arch, the tracttypically exhibited an overall C-shaped appearance.

The majority of GABAi fibers exiting each pedal gan-glion projected to the contralateral ganglion via the pedal

commissure (Fig. 8A,B, pd c.). Exceptions to this patternincluded two fibers originating from a pair of medium-sized dorsal GABAi cells (Fig. 8B, arrowheads) located inthe posteromedial region of each pedal ganglion. It wassometimes possible to follow these axons (Fig. 8A, arrow)as they coursed ventrally and anteriorly toward the pleural-pedal connective. The fibers passed through the pleural-pedal connective as a pair and then appeared to anasta-mose within the pleural ganglion. They gave rise to manyfine varicose secondary and tertiary branches that termi-nated in the central region of the pleural ganglion. Thesefibers did not appear to continue into any of the pleuralnerves or connectives.

An additional large (80–100 µm diameter) GABAi cellwas located in the posteromedial quadrant of the rightpedal ganglion (Fig. 8B, arrow). This unpaired cell gaverise to two fibers, both of which appeared to emanate fromnear the cell soma. One of these fibers could be traced tothe ipsilateral pleural-pedal connective, whereas the sec-ond was directed toward the pedal commissure. Theipsilateral fiber passed into the pleural ganglion, where itcould be followed to the pleural abdominal connective. Nobranching of this fiber was observed within the pleuralganglion. No GABAi fibers exited the pedal ganglia via themotor nerves, the statocyst nerve, or the parapedal commis-sure.

A single strongly immunoreactive fiber descended ineach pleural-abdominal connective (Fig. 8D). Each fiberremained within its hemiganglion of entry, where it pro-jected toward the posterior portion of the ganglion. Bothaxons could be followed at least two-thirds of the waythrough the ganglion, giving rise to many short secondaryand tertiary varicose branches along their entire length.

Backfills of pleural-abdominal connectives resulted instaining of both ipsilateral and contralateral neurons inthe pedal ganglia. One cell in the posteromedial quadrantof the right pedal ganglion appeared to fill with backfills ofeither the ipsilateral (n 5 3) or the contralateral (n 5 3)pleural-abdominal connective. Double-labeling experi-ments conducted with both ipsilateral and contralateralfills demonstrated that this cell corresponds to the largeGABAi neuron described above (Fig. 9A1, A2, B). It ap-pears, therefore, that both of the GABAi inputs to theabdominal ganglion originate from this unpaired pedalneuron (Fig. 9B, cross-hatched cell).

DISCUSSION

GABAergic systems in molluscs

A considerable amount of data have been amassedsupporting the role of GABA as a neurotransmitter inmolluscs (see Walker, 1986). The presence of GABA in themolluscan central nervous system was first shown in thecephalopod, Eledone cirrhosa (Bradford et al., 1969; Coryand Rose, 1969), and subsequently demonstrated in thepulmonates Helix pomatia (Osborne et al., 1971; Doleza-lova et al., 1973), Helix aspersa (Osborne, 1971), Achatinafulica (Takeuchi et al., 1977), and Helisoma trivolvis(Richmond et al., 1991). Of particular relevance to thepresent study are the findings of Cottrell (1974) whomeasured GABA levels in the various ganglia of Aplysiadactylomela. In that study, the role of GABA as a neuro-transmitter substance was supported by the demonstra-tion of its differential distribution within the central


Fig. 8. Gamma aminobutyric acid (GABA) immunoreactivity inthe subesophageal ganglia. A: Ventral surface of the right pedalganglion. Four clusters (designated GI–GIV) were distributed along amajor immunoreactive fiber tract coursing from the pedal commissure(pd c.) in an anterior-lateral direction through the ganglion. A pair oflarger cells is located in the posterior region of the ganglion (arrow)give rise to fibers that project in an anterior direction toward thepleural-pedal connective (pl -pd c.). B: Dorsal surface of right pedalganglion. Same preparation as in A. The cell bodies of the neurons thatgive rise to the fibers indicated in A are visible in this plane of focus(arrowheads). A larger strongly immunoreactive cell (arrow) is locatedin the posteromedial region of the ganglion. C: Posterior region of the

pleural ganglion. A pair of closely apposed fibers (small arrow) entersthe ganglion from the pl-pd c. These fibers terminate in varicosebranches within the pleural ganglion. An additional smooth fiber(large arrow) enters the ganglion from the pl-pd c. and courses throughthe ganglion to the pleural abdominal connective (not shown) withoutbranching. D: Right upper quadrant of the abdominal ganglion. Asingle strongly immunoreactive fiber (arrow) enters the ganglion fromthe pleural abdominal connective (pl-a c.) and extends in the posteriordirection giving off a few fine branches along its course. c-pd c.,cerebral-pedal connective. Scale bars 5 500 µm in A and B; 200 µm inC and D.

nervous system (CNS). The highest concentrations weremeasured in the buccal and cerebral ganglia, an observa-tion that appears to be consistent with the cellular localiza-tion of GABA-like immunoreactivity in Aplysia californicaobserved in the present investigation.

Previous studies using immunohistochemical tech-niques to map GABAergic systems in gastropod molluscshave been conducted by using three pulmonate species; theterrestrial slug Limax maximus (a stylommatophoran;Cooke and Gelperin, 1988), the pond snail Helisomatrivolvis (a basommatophoran; Richmond et al., 1991), andthe land snail Helix pomatia (a stylommatophoran; Her-nadi, 1994). Within the opisthobranch subclass, previousinvestigators have examined Clione limacina (a pteropod,Arshavsky et al., 1993), Pleurobranchaea californica (anotaspid, Soinila and Mpitsos, 1991), and the subject of the

present study, Aplysia californica (an anaspid, Soinila andMpitsos, 1991). Although our observations lend generalsupport to the findings of these previous workers, theapplication of wholemount procedures and the combinedbackfill methods provide additional information that shouldfacilitate the identification of specific GABAergic neuronsin Aplysia.

Kandel (1979) proposed that investigation of neuralcircuits underlying homologous behaviors in closely re-lated molluscan species could provide insight into whichproperties of these networks remain invariant and whichare modified over time. The similarities in the stainingpatterns between Aplysia and the distantly related pulmo-nates suggest that the GABAergic systems in molluscs areancient and highly conserved. In all cases examined todate, the distribution of GABA-immunoreactive cell bodies

Fig. 9. Interganglionic projections originating in gamma aminobu-tyric acid-immunoreactive (GABAi) pedal neurons. A1: Biocytin back-fill of the left (contralateral) pleural abdominal connective visualizedwith rhodamine. One cell (arrow) on the dorsal surface of the rightpedal ganglion was filled. A2: Same field as A1. GABA immunoreactiv-ity labeled with fluorescein second antibody. One immunoreactive cell(arrow) corresponded to the backfilled neuron in A1. A nearby immuno-reactive cell (arrowhead) was not labeled by the backfill. Calibrationbar 5 200 µm. B: Schematic summary of interganglionic projections

originating in GABAi pedal neurons. The cross-hatched cell corre-sponds to the unpaired dorsal neuron shown in A. This cell hasbilateral projections that course through the pleural ganglion and intothe pleural-abdominal connective. Also shown are two pairs of ventralinterneurons (black) that project to their respective ipsilateral pleuralganglia. L. Pleuroal G., leftpleural ganglion; R. Pedal G., right pedalganglion; c-pd c., cerebral-pedal connective; pl-a c., pleural-abdominalconnective.


is restricted to the buccal, cerebral, and pedal ganglia.Moreover, with the exception of a pair of fibers in thebuccal nerves of Helisoma (Richmond et al., 1991), and alimited innervation of the lips in Helix (Hernadi, 1994), theGABAergic systems of molluscs appear to be restricted tothe central nervous system. This organization contrastssharply with those of other invertebrate phyla, includingthe arthropods (Usherwood, 1977; Atwood, 1982; Mulloneyand Hall, 1990), nematodes (Johnson and Stretton, 1987),and annelids (Cline, 1986) where GABA acts as a majorperipheral neurotransmitter.

GABAergic cells and circuits

Although the localization of GABAergic immunoreactiv-ity was predominantly bilateral and symmetrical, therewere at least two instances in which it was found inunpaired cells. The unpaired buccal cell was occasionallyfound in the mid-line between the two buccal hemiganglia(Fig. 1D), but its position was more often skewed in onedirection or the other. Unpaired buccal neurons have beendescribed previously in the buccal system of Aplysia (Lloydet al., 1987; Rathouz and Kirk, 1988; Kabotyanski et al.,1998), but none have been physiologically characterized todate. In the pond snail Lymnaea stagnalis, the unpairedcholinergic slow oscillator (SO) interneuron has beenintensively studied and is thought to act as a modulatoryelement of the feeding circuit (Rose and Benjamin, 1981;Elliott and Benjamin, 1985; Yeoman et al., 1993).

The second unpaired GABAi cell was located on thedorsal surface of the right pedal ganglion. Richmond et al.(1991) described a similar unpaired GABA-immunoreac-tive neuron on the dorsal surface of the right pedalganglion in Helisoma. In both Helisoma and Aplysia, theunpaired pedal neuron projects bilaterally to the visceralganglion, where it appears to act as the predominantsource of GABAergic innervation. Such a limited GABAer-gic input to the abdominal ganglion is consistent withobservations of Yarowsky and Carpenter (1977) who notedthat most abdominal neurons did not respond to ionopho-retic application of GABA. Cells that were found to re-spond included the left upper quadrant (LUQ) cell L3, thegiant neuron R2, and the parabolic burster R15. Each ofthese cells appears to be located within regions of theganglion that are innervated by the branches of theunpaired pedal neuron.

The presence of axosomatic contacts on specific cellbodies has been demonstrated in several neurotransmittersystems in Aplysia (Kistler et al., 1985; Pearson and Lloyd,1989; Soinila et al., 1990; Miller et al., 1992; Kabotyanskiet al., 1998). Using iontophoretic techniques, Yarowskyand Carpenter (1977, 1978) found that some Aplysianeurons responded to somal application of GABA, whereasothers only responded to activation of neuropil receptors.In those cases where receptors were present on the cellbody, it was suggested that these ‘‘extrajunctional’’ recep-tors were likely to be similar to the synaptic receptors inthe neuropil. The presence of axosomatic contacts thatmay correspond to release sites suggests that, at least insome cases, somatic GABA receptors may in fact beinvolved in synaptic signalling.

Because many instances of cotransmission have beendemonstrated within identified neurons of Aplysia (seeCropper et al., 1987; Church and Lloyd, 1991; Kupfer-mann, 1992), it is of interest to compare the pattern ofGABAi neurons with maps obtained for other neurotrans-

mitters and modulators. Within the buccal ganglion, thereare notable similarities between the distribution of GABAineurons and the staining pattern observed by using histo-logical methods to detect catecholamines. An unpaireddopaminergic medial cell (Rathouz and Kirk, 1988; Gold-stein and Schwartz, 1989) with projections in both CBCs(Kabotyanski, 1998) and a bilateral pair of rostral dopamin-ergic interneurons with bilateral CBC projections (the B20cell pair; Teyke et al., 1993) have been described. With thepossible exception of these cells and the two immunoreac-tive cerebral-buccal interneurons (see below), the map ofGABAi cells does not appear to include neurons that havebeen characterized previously.

Recent studies have produced a complete description ofthe population of cerebral neurons that project an axoninto the CBC (Rosen et al., 1991; Xin et al., 1999; Hurwitzet al., 1999). Four CBIs are located within the M cluster,including CBI-1, CBI-2, CBI-3 (Rosen et al., 1991), andCBI-12 (Hurwitz et al., 1999). In attempting to identify theGABA-immunoreactive CBI, it is possible to exclude thebilaterally projecting dopaminergic CBI-1 neuron, becauseno contralateral GABAi CBIs were observed. The largesize and posterior position of the GABAi CBI suggests thatit may correspond to CBI-3, a neuron that expresses theneuropeptide APGWamide and that appears to be criti-cally involved in selecting between ingestive and egestivebuccal motor programs (Wu-Morgan et al., 1998).

Functional considerations

A role for GABA in circuits that mediate molluscanfeeding behaviors was initially suggested by pharmacologi-cal studies. Arshavsky et al. (1991, 1993) found thatinjection of GABA into the hemocoel of Clione could evokeessential elements of feeding behavior. Electrophysiologi-cal experiments revealed a variety of GABAergic actions,including activation of the feeding CPG and excitation oftentacular motor neurons. It was proposed that the ClioneCNS contains GABAergic ‘‘command’’ neurons that acti-vate various components of the consummatory phase offeeding (including protraction of the tentacles, catchingthe prey, extracting the prey, and swallowing), a role whichthey termed ‘‘functional synergy.’’ Consistent with thisnotion, Norekian and Satterlie (1993) observed coordi-nated effects of GABA on motor neurons that resulted inextrusion of the buccal cones of Clione. A similar activationof buccal rhythms by GABA has been reported in Helisoma(Richmond et al., 1986), whereas an inhibitory effect wasobserved on the buccal feeding program of Limax maximus(Cooke et al., 1985). In three preliminary experiments, inwhich GABA (up to 1 mM) was applied to isolated buccalganglia of Aplysia californica, we have not observed activa-tion of rhythms (not shown). In one case, GABA was foundto inhibit a spontaneously active rhythmic program, butthese experiments are difficult to interpret in view of theknown complexity of the control of buccal ganglion rhyth-micity, the likelihood that cotransmitters are present inGABAergic interneurons (see above), and the critical roleof buccal-cerebral corollary discharge signals in the genera-tion of feeding rhythms in Aplysia (see Kirk, 1989; Chiel etal., 1990; Rosen, 1991; Teyke et al., 1993; Perrins andWeiss, 1996, 1998; Kabotyanski et al., 1998; Wu-Morgan etal., 1998; Xin et al., 1999; Hurwitz et al., 1999).

The presence of GABA-immunoreactive buccal-cerebralinterneurons and cerebral-buccal interneurons supportsthe proposed role of this neurotransmitter in the regula-


tion of feeding-related behaviors in Aplysia. A comparisonof GABAi neurons in the feeding circuits of the variousmolluscan species studied to date suggests that this sys-tem may play a very fundamental role, especially in viewof the wide range of feeding strategies and behaviors thatthese species employ. Helisoma and Limax, two speciesthat are classified as ‘‘raspers’’ according to Audesirk andAudesirk (1985), each have three strongly GABAi neuronsin each buccal hemiganglion (Cooke and Gelperin, 1988;Richmond et al., 1991). In Helisoma, one of these cellsprojects into the ipsilateral CBC and two project into thecontralateral CBC (Richmond et al., 1991). Interestingly,Clione limacina, a distantly related ‘‘hunter’’with a highlycomplex predatory feeding behavior, also has three GABAineurons in each buccal ganglion, at least one of whichprojects to the cerebral ganglion (Arshavsky et al., 1993).Our results and those of Soinila and Mpitsos (1991)indicate that the number of GABAi neurons in the buccalganglion of Aplysia is somewhat greater (approximately 10cells in each ganglion), and that at least seven GABAibuccal neurons project to the cerebral ganglion. AlthoughAplysia is generally classified as a ‘‘browser,’’ it has beenknown for some time that specimens will exhibit grazingbehavior (i.e., biting against the substrate while locomot-ing) depending upon the type of food available (Kupfer-mann and Carew, 1974; see also Hurwitz et al., 1999).Interestingly, we have observed an indistinguishable pat-tern of GABAi staining in the buccal ganglion of thetropical marine mollusc Bursatella leachii (unpublishedobservations), an aplysiid that normally feeds by grazing,but which will browse when constrained by available foodtypes (Ramos et al., 1995; unpublished observations). Theincreased number of GABAergic buccal neurons found inthe aplysiids may be in some way related to the flexibilityof their feeding strategies (see Hurwitz et al., 1999).

The observation that GABAi fibers are restricted to thecentral ganglia indicates that this transmitter system isinvolved in some aspect of premotor integration, ratherthan in motor or sensory projections. The majority ofGABAi fibers were found within the commissures betweenthe paired buccal, cerebral, and pedal ganglia (see alsoRichmond et al., 1991), suggesting that they are involvedin regulating or coordinating bilateral sytstems. Theseganglia contain the principal central pattern generatorcircuits underlying rhythmic feeding-related behaviors(Kupfermann, 1974; Perrins and Weiss, 1996) and locomo-tion (Jahan-Parwar and Fredman, 1978, 1980; Hening etal., 1979). In the pedal system, Jahan-Parwar and Fred-man (1979) found that sectioning the pedal commissurecaused the motor activity of each hemifoot to becomedesynchronized. From this observation and additionallesion experiments, they concluded that each pedal gan-glion contains the locomotor pattern generator controllingthe ipsilateral hemifoot and that coordination of thesecontrol centers is maintained by the pedal commissure(Jahan-Parwar and Fredman, 1979). The more intensivelystudied buccal system appears to be organized similarlyand is exceptionally well integrated, because we know ofno reports in which the two buccal hemiganglia wereshown to produce distinct rhythms or phase relationships.Many neurons are known to exert bilateral synaptic effectsin this system (Plummer and Kirk, 1990; Rosen et al.,1991; Miller et al., 1994). Recently, the predominantsynaptic actions of two buccal neurons (B63 and B34) were

found to occur in contralateral interneurons and motorneurons, leading to the proposal that these cells play asignificant role in producing the strong synchrony betweenthe two buccal hemiganglia (Hurwitz et al., 1997).

In addition to the coordination of the rhythmic feedingand locomotor CPGs, there are a number of additionalpossible roles for the commissural GABAi systems. Theseinclude certain reflexes, such as tentacular withdrawal,which are known to occur bilaterally in response to aunilateral stimulus (Fredman and Jahan-Parwar, 1977)and the crossed proprioceptive and tactile reflexes thatoccur in the foot (Jahan-Parwar and Fredman, 1978b).More complex head movements such as those associatedwith egg laying (Cobbs and Pinsker, 1982) and the appeti-tive responses to food (Teyke et al., 1990) also appear torequire bilateral coordination of central programs. Finally,commissural systems are known to be involved in coordi-nating bilateral motor responses responsible for maintain-ing spatial orientation in molluscs (see Orlovsky, 1991;Deliagina et al., 1998). In this regard, the proximity of themedial pedal GABAi clusters to the statocysts may benoted, although we have no evidence that they are in factassociated with these gravitational sensory organs. Inter-estingly, Jahan-Parwar and Fredman (1979) reported thatAplysia with pedal commissure lesions did not exhibitimpairments in their ability to right themselves.

In summary, this study supports the hypothesis thatGABAergic neurons are involved in the regulation ofcomplex behavior patterns in Aplysia. The observationthat the GABAi system is confined to the central nervoussystem, and its localization to specific neurons and cir-cuits, should facilitate future investigations of GABAergicsynapses and the role of individual GABAergic neurons inthe generation and regulation of behavior.

ACKNOWLEDGMENTS

This work was supported, in part, by NSF CAREERaward IBN-9722349, RCMI award RR-03051 (Division ofResearch Services, NIH), MBRS GM-08224 (NIGMS, NIH),Puerto Rico EPSCoR (NSF), and DoD Instrumentationgrant N00014–93–1380 (ONR). The students and facultyof the Tropical Neuroethology for Undergraduates summerprogram (1998) were instrumental in developing the meth-ods used in this study. Professor Yuri Arshavsky providedcommentary on an earlier version of the manuscript andSixto Quiles helped in the preparation of the figures.

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localization of gaba-like immunoreactivity in the central nervous system ofaplysia californica

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