Localization of immunoreactive alpha-bag-cell peptide in the central nervous system of Aplysia

The bag cells of the marine mollusc Aplysia are well-characterized neuroendocrine cells that initiate egg laying, but the natural stimulus triggering bag-cell activity has not been determined. As a first step toward identifying central neurons that might provide synaptic or neurohormonal input onto the bag-cell network, antibodies specific for alpha-bag-cell peptide (alpha-BCP) were generated. This peptide belongs to a small family of structurally related peptides that can elicit bag-cell activity in vitro. Antibody specificity was established by immunodot assay and preabsorption studies: immunocytochemical labeling was abolished in each ganglion when the antibodies were preincubated with either alpha-BCP-thyroglobulin conjugate or alpha-BCP-(1-8) but was not affected by preincubation with thyroglobulin or thyroglobulin-thyroglobulin conjugate. The antibodies specifically labeled the bag cells in the abdominal ganglion and ectopic bag cells in both the abdominal and right pleural ganglia. The ectopic bag cells were similar to conventional bag cells in size and morphology, but varied in number and location among preparations. In the cerebral ganglion, the antibodies labeled a bilaterally symmetrical pair of cell clusters, containing approximately ten cells each, on the dorsal surface of the ganglion. The cerebral cells were smaller than bag cells, were constant in location, and sent their processes into the neuropil rather than the connective tissue sheath. Immunoreactive processes were observed in the neuropils of the cerebral, pleural, and pedal ganglia and among the axons of the cerebropedal, cerebropleural, and pleurovisceral connectives. No immunoreactive cell bodies were observed in the buccal or pedal ganglia. Identical patterns of labeling were observed in Aplysia californica, A. brasiliana, and A. dactylomela. The distribution of immunoreactive cell bodies within the circumesophageal ganglia of all three species thus parallels the distribution of receptive sites for the in vitro induction of bag-cell activity by atrial gland peptide B, a peptide structurally related to alpha-BCP. These observations suggest that the immunoreactive cells identified in these studies, or a subset of them, may be involved in the physiological induction of bag-cell activity. Since low doses of alpha-BCP have additional inhibitory actions on the bag cells, however, it is possible that the identified cells could play a more complex role in the regulation of bag-cell activity.     

Cyclic AMP regulates processing of neuropeptide precursor in bag cell neurons ofAplysia

The stimulation of a prolonged afterdischarge of action potentials in the bag cell neurons ofAplysia is accompanied by an elevation of cAMP levels in these cells. Such a discharge causes the release of egg-laying hormone (ELH) and several other neuroactive peptides, which are derived from a 32-kDa protein prohormone. We have examined the relationship between the elevation of cAMP levels and the processing of the 32-kDa ELH prohormone. The ELH prohormone was radiolabeled in bag cell clusters by incubation of abdominal ganglia in [³H]leucine and identified on SDS-PAGE by its specific localization to bag cell neurons and its immunoreactivity with antisera to ELH. After labeling the prohormone, further incorporation of [³H]leucine was blocked using either the protein synthesis inhibitor anisomycin or an excess of unlabeled leucine. The stimulation of an afterdischarge, or treatment of cells with the adenylate cyclase activator forskolin or a membrane permeant cAMP analog, resulted in the loss of radiolabeled 32-kDa ELH prohormone relative to that in control clusters. In the presence of tetrodotoxin (TTX), which prevents discharges and stimulation-evoked secretion in the bag cell neurons, forskolin also caused the depletion of labeled ELH prohormone, suggesting that secretionper se is not likely to be required for this effect. The decrease in intensity of the 32-kDa band was accompanied by an increase in a 29-kDa band within the somata. Occasionally, an increase in a group of faint bands with approximateM _r of 26-kDa was observed. Comparative peptide mapping indicated that the 29-kDa protein is likely to be derived from the 32-kDa ELH prohormone. Our findings suggest that elevations of cAMP accelerate and possibly alter the pattern of, processing of the 32-kDa ELH prohormone.     

Egg laying hormone inhibits a neuron (C-PR) involved in multiple manifestations of food-induced arousal in Aplysia

Egg laying behavior is known to suppress feeding in Aplysia, but both behaviors have common responses involving head movements and posture. Egg laying hormone (ELH) applied in vitro to the isolated nervous system of Aplysia reduces the spontaneous and the evoked activity of the C-PR, a neuron implicated in postural responses during feeding. The inhibitory effect of ELH on the C-PR appears to be mediated by interneurons primarily located in the pedal/pleural ganglia, which contain all the known direct follower cells of the C-PR. Our results do not support the idea that postural response during feeding and egg laying are mediated by the activation of a common arousal element, the C-PR. In fact, the C-PR seems to be a specific element for the food-arousal state, and the inhibition of the C-PR by ELH may contribute to the suppression of appetitive feeding responses during egg laying.     

Annexin-immunoreactive proteins in the nervous system and eye of the gastropods, Aplysia and Helix

We studied the distribution of annexin I- and annexin V-like proteins in the eye and central nervous system of the snails, Aplysia californica and Helix pomatia by immunocytochemistry. Annexin I-immunoreactive material in Aplysia californica was localized in sensory and corneal cells of the eye and in distinct neurons of the cerebral, buccal, and abdominal ganglia, where it was exclusively located in bag cells. Annexin V-immunoreactive neurons were restricted to the pleural ganglia of Aplysia californica. In Helix pomatia annexin I-immunoreactive neurons were present in the cerebral, buccal, visceral, and left and right parietal ganglia, whereas annexin V-immunoreactive neurons were present in left and right pleural, left and right parietal, visceral, and buccal ganglia. Annexin VI-immunoreactivity was absent in both gastropods studied. Our study shows a cell specific localization of annexin-like proteins in the central nervous system and eye of molluscs. The cell types containing the immunoreactive proteins suggests that the annexin-like proteins may be involved in intracellular signaling mechanisms, which ultimately may modulate egg-laying and circadian rhythmicity.     

Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study

The distribution of neurons reacting with an antibody raised against an insect neuropeptide, locustatachykinin I, was investigated in the CNS of the snail Helix pomatia. The localization of the neurons was compared with that of the substance P-like immunoreactive (SPLI) neurons in the different ganglia. Altogether, there are 800–1000 locustatachykinin-like immunoreactive (LomTKLI) neurons in the Helix CNS, occurring with an overwhelming dominancy (83.5%) in the cerebral ganglia. Within the cerebral ganglia, the majority of LomTKLI neurons were localized in the procerebrum. The number of SPLI neurons was high; 2000 SPLI nerve cells were found in the Helix CNS. The majority (44.5%) of SPLI neurons was also found in the cerebral ganglia and they were also concentrated in the procerebrum. The neuropils of all ganglia were densely innervated by both LomTKLI and SPLI fibers except the medullary mass of the procerebrum where only SPLI elements form an extremely dense innervation. In addition to the neuropil processes, LomTKLI neurons sent axon processes to the peripheral nerves. SPLI fibers also formed a dense network of varicose fibers in the connective tissue sheath around the ganglia where they innervated the blood vessel walls too. Immunolabeling on alternating cryostat sections revealed that LomTKLI and SPLI neurons are localized near each other in most cases; co-localization of the two immunoreactive materials could be seen in a very small number of neurons of the pedal and pleural ganglia. The present results show that the Helix CNS possesses distinct neuronal populations using different tachykinin-related peptides. It is suggested that the differential distribution of these neuropeptides also implies a diversity in their central and peripheral functions.     

Generation of new cerebral ganglion neurons in the snail Melampus: An ultrastructural study

Reports in the literature have established that reconnection of central neural tracts occurs following commissurotomy and cerebral ganglion excision in the primitive pulmonate snail Melampus bidentatus and have suggested the possibility that long-term regeneration might result in the appearance of new neurons in the ganglion bud. We have used electron microscopy to examine the ganglion buds that form by reconnection of cerebral nerves, commissure, and connectives following cerebral ganglion excision in adult Melampus. The buds were examined from 2.5 to 12 months postoperatively. By 2.5 months, ganglion buds consist of a mixture of axon tracts that travel through the bud region and some dendritic processes; a few synaptic contacts can be identified at this stage, scattered throughout the bud. By 5–6 months, some of the most advanced ganglia have undifferentiated cells that are distinct from glia. By 7 months, differentiated neurons with clear, small dense-core or neurosecretory vesicles are present, although these cells are not all concentrated in a rind on the ganglion surface. Another cell type, the pigment-sheath cell, is present by this stage. By 11–12 months, the most advanced regenerating ganglia have neurons which form a cell rind on the ganglion surface. The gross appearance of a regenerated ganglion at this stage is similar to that of the intact contralateral cerebral ganglion, although the regenerated ganglion is smaller. One 12-month ganglion was found to possess fairly normal intraganglionic morphology, with lobes and cell types that were recognizable. Hence, nerve cell regeneration can occur in the absence of body part regeneration in adult members of one species of pulmonate snail.     

Nonsynaptic characteristics of neurotransmission mediated by egg-laying hormone in the abdominal ganglion of Aplysia

The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.     

Serotonin-immunoreactive neurons and endogenous serotonin in the opisthosomal ventral nerve cord of the horseshoe crab,Limulus polyphemus

It has been suggested that serotonin serves as a neurotransmitter in the horseshoe crab, Limulus polyphemus. While some studies of identified groups of central neurons have been conducted, little is known concerning the neuronal organization in Limulus central ganglia. This study was undertaken to determine the localization of serotoninergic neurons in the opisthosomal ventral nerve cord of Limulus and to construct a basis for further comparative biochemical and pharmacological studies of the specific function of these neurons. Endogenous serotonin was, detected in the ventral nerve cord (chain of abdominal ganglia) by high performance liquid chromatography and electrochemical detection (HPLC-EC). Endogenous serotonin was quantified in the 9th through 13th ganglia, anterior (hemal) nerves, posterior (branchial) nerves, and connectives. The serotonin content in the abdominal ganglia was significantly reduced when the ganglia were incubated for 24 hours in Leibovitz's (L-15) medium containing reserpine or 5, 7-dihydroxytryptamine (5, 7-DHT), neurotoxins that block the uptake of serotonin into storage vesicles. The distribution of serotonin-immunoreactive neurons in the ventral nerve cord was determined by indirect immunocytochemistry. Treatment of the chain of ganglia with an anti-serotonin antiserum followed by treatment with a fluorescent-labeled antiserum raised against the primary antibody demonstrated specific staining in each, ganglion, the ganglionic roots, and connectives. Clusters of serotonin immunoreactive neurons were observed anteriolaterally and posteriorly in each ganglion. Processes from dense fiber bundles extended from these clusters of neurons to the central region of each ganglion. These results demonstrate that serotonin-immunoreactive neurons are present in the opisthosomal ventral nerve cord of the horseshoe crab and that serotonin may function as a neurotransmitter. © 1994 Wiley-Liss, Inc.     

Expression of the L11 neuropeptide gene in the Aplysia central nervous system

Neuron L11 in the abdominal ganglion of Aplysia californica is thought to be both cholinergic and peptidergic. In previous studies, we isolated a cDNA clone encoding the precursor for an L11 secreted protein(s) by differentially screening an abdominal ganglion cDNA library. We now report the isolation of genomic clones encoding the L11 cDNA sequences. Analysis of these clones reveals that the gene is present in a single copy per haploid genome. RNA blotting and cDNA cloning demonstrate that the L11 gene is expressed not only in the abdominal ganglion but in the head ganglia as well. To define the positions of cells expressing this gene and to follow their processes, we raised antibodies to synthetic peptides defined by the cDNA sequence. Histochemistry revealed about 100 neurons containing immunoreactive material. These cells arborize in the neuropil and are distributed throughout the central nervous system, representing about 0.5% of the Aplysia central neurons. In addition, cells in the abdominal ganglion send processes to the mantle floor at the base of the gill via the genital and branchial nerves. Our data suggest that this network of cells expresses the single L11 peptide gene.     

Distribution in the central nervous system of Aplysia of afferent fibers arising from cell bodies located in the periphery

The present study used autoradiography to determine the location of the projections of presumptive peripheral afferent neurons into the central nervous system of Aplysia. Selected peripheral tissues (with an empliasis on structures involved in feeding behavior) were exposed to radioactive amino acids, and the distribution of macromolecules transported into the nervous system via afferent fibers was determined by autoradiography. Different regions of the body exhibited different patterns of projections, and within the neuropil of the cerebral ganglion, there was a loose topographical prganization of projections from the head. For some regions of the body, the projection was largely limited to the ganglion from which the nerve enters; for other regions, the projection was very widespread. In some cases (e.g., rhinophore to eye), there was evidence of projections from one peripheral structure to another. Experiments with all peripheral tissues that were studied resulted in extensive labeling of central ganglia, indicating that afferents with peripheral cell bodies may provide a major source of sensory input to the central nervous system and suggesting that many or all of the numerous ultrafine axons visualized via electron microscopy in the nerves of Aplysia may originate from first- or second-order sensory afferents whose cell bodies are located in the periphery. © 1995 Wiley-Liss, Inc.     

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.     

Localization of myomodulin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.

The distribution of myomodulin-like peptides in the nervous system of Aplysia californica was examined by using immunocytochemical techniques. Neurons and cell clusters containing immunoreactive material were located in each of the major central ganglia. Myomodulin-like immunoreactivity was also present in fibers in each of the connectives between the ganglia and in peripheral nerves. Varicosities containing immunoreactive material were located on specific regions of peripheral tissues associated with the feeding, digestive, cardiovascular, and reproductive systems. Double-labeling experiments were used to demonstrate myomodulin-like immunoreactivity in two identified neurons, the motor neuron B16 in the buccal ganglion and the widely acting interneuron L10 in the abdominal ganglion. Structures in the eye and cerebral ganglion that may correspond to the optic circadian pacemaker system were also stained. The central and peripheral distribution of myomodulin-like immunoreactivity indicates that this family of neuropeptides is present in specific efferent, afferent, and interneuronal elements that participate in a diversity of neural circuits in Aplysia.     

Activation of neurosecretory cells enhances their synthesis of secretory protein

We have asked whether neurosecretory cells respond to activation by selectively augmenting their synthesis of secretory protein, using the bag cells ofAplysia, which produce and secrete a peptide egg-laying hormone, ELH. Exposure of bag cell organs to 100 mM K⁺ for 4 h increased their incorporation of tritiated leucine into ELH precursors by about 25%, an effect which persisted for at least 8 h after the stimulus. Since the high potassium treatment had no effect on the rate of loss of label from previously-synthesized ELH proteins, we conclude that the enhanced labeling represents enhanced synthesis. Elevated external potassium did not enhance ELH synthesis in clusters of bag cell somata which had been surgically isolated from their neurites, suggesting that the augmentation of synthesis is not mediated by direct effects of the high potassium solution. Mediation by a secretion-linked process is indicated by the fact that low Ca²⁺/high Mg²⁺ media blocked the effect of high K⁺. Whether the regulatory signal involves receipt of presynaptic transmitter or hormone secretion remains to be determined.     

Localization of GABA-like immunoreactivity in the central nervous system of Aplysia californica.

Gamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supported by pharmacological, biochemical, and anatomical investigations. In this study, the distribution of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examined by using wholemount immunohistochemistry and nerve backfill methods. GABAi neurons were located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems were present in each of these ganglia, whereas more limited fiber systems were observed in the ganglionic connectives. Some of the interganglionic fibers were found to originate from two unpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, each of which exhibited bilateral projections. No GABAi fibers were found in the nerves that innervate peripheral sensory, motor, or visceral organs. Although GABAi cells were not observed in the pleural or abdominal ganglia, these ganglia did receive limited projections of GABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAi neurons suggests that this transmitter system may be primarily involved in coordinating certain bilateral central pattern generator (CPG) systems related to feeding and locomotion. In addition, the presence of specific interganglionic GABAi projections also suggests a role in the regulation or coordination of circuits that produce components of complex behaviors.     

Localization of putative nitrergic neurons in peripheral chemosensory areas and the central nervous system of Aplysia californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Aplysia californica was studied by using NADPH-diaphorase (NADPH-d) histochemistry in the CNS and peripheral organs. Chemosensory areas (the mouth area, rhinophores, and tentacles) express the most intense staining, primarily in the form of peripheral highly packed neuropil regions with a glomerular appearance as well as in epithelial sensory-like cells. These epithelial NADPH-d-reactive cells were small and had multiple apical ciliated processes exposed to the environment. NADPH-d processes were also found in the salivary glands, but there was no or very little staining in the buccal mass and foot musculature. In the CNS, most NADPH-d reactivity was associated with the neuropil of the cerebral ganglia, with the highest density of glomeruli-like NADPH-d-reactive neurites in the areas of the termini and around F and C clusters. A few NADPH-d-reactive neurons were also found in other central ganglia, including paired neurons in the buccal, pedal, and pleural ganglia and a few asymmetrical neurons in the abdominal ganglion. The distribution patterns of NADPH-d-reactive neurons did not overlap with other known neurotransmitter systems. The highly selective NADPH-d labeling revealed here suggests the presence of NOS in sensory areas both in the CNS and the peripheral organs of Aplysia and implies a role for NO as a modulator of chemosensory processing.     

Helix peptide immunoreactivity pattern in the nervous system of juvenile aplysia

Distribution of neurons immunopositive to antibody against the small peptides encoded by the Helix Command-Specific 2 (HCS2) gene in the central nervous system of juvenile Aplysia californica was investigated. The HCS2 gene is specifically expressed in the withdrawal behavior neurons of the terrestrial snail Helix lucorum. In Aplysia, 20-25 immunopositive neuronal somata were observed on dorsal surface of each pleural ganglion (including a giant pleural neuron). The HCS2-encoded peptide immunopositive fibers were observed in neuropiles of all ganglia and in many nerves. Functional significance of Aplysia immunopositive cells is discussed.     

Gangliogenesis in the prosobranch gastropod Ilyanassa obsoleta

We determined that the larval nervous system of Ilyanassa obsoleta contains paired cerebral, pleural, pedal, buccal, and intestinal ganglia and unpaired apical, osphradial, and visceral ganglia. We used a modified form of NADPH diaphorase histochemistry to compare the neuroanatomy of precompetent (including specimens 6, 8, and 12 days after hatching), competent, and metamorphosing larvae with postmetamorphic juveniles. This method highlighted ganglionic neuropils and allowed us to identify individual ganglia at various stages of development, thereby laying a foundation for concurrent histochemical studies. The first ganglia to form were the unpaired apical and osphradial ganglia and the paired cerebral and pedal ganglia. In larvae 6 days after hatching, the neuropil had already appeared in the apical and osphradial ganglia. Neuropil began to be apparent in the cerebral and pedal ganglia 2 days later. At that time, the pleural and buccal ganglia were identifiable and adjacent to the posterior edge of the cerebral ganglia. The ganglia of the visceral loop were concurrently recognizable, although the supraintestinal ganglion developed slightly earlier than the subintestinal and visceral ganglia. By 12 days after hatching, all of the major adult ganglia were discernible. The apical ganglion was retained by newly metamorphosed juveniles, but not by juveniles 2 days later. After metamorphosis was complete, the central nervous system (CNS) was consolidated into its juvenile form with ipsilateral cerebral and pleural ganglia being partially fused. The metamorphic translocation of ganglia, which included a caudal relocation of the cerebrals and the migration of the buccals from above the esophagus to a position below it, correlated with the movement of the proboscis to the dorsal part of the head. © 1996 Wiley-Liss, Inc.     

Neuropeptide proctolin (H-Arg-Try-Leu-Pro-Thr-OH): immunocytochemical mapping of neurons in the central nervous system of the cockroach

Proctolinlike immunoreactivity was mapped in the central nervous system of the cockroach, Periplaneta americana, by using whole-ganglion immunoreacted preparations. The procedure for this immunohistochemical staining of whole-mounts is described. Immunoreactivity was confined to neuronal cell bodies and and processes. These were found in all ganglia of the CNS. The cells varied in the consistency and intensity of their staining. The occurrence and variability of staining is described in detail. Cell bodies were found in the dorsal, ventral, and lateral regions of the ganglia. The highest number of cell bodies was found in the terminal ganglion and the lowest number in the cerebral ganglion. Those in the cerebral ganglion occurred mainly in the tritocerebral lobes. The distribution of immunoreactive cell bodies correlated with results previously obtained by radioimmunoassay. Immunoreactive processes were detected in all interganglionic connectives and many ganglionic nerve roots. Dense ramifications of immunoreactive processes and variocosities were detected in many of the ganglia. The widespread presence of immunoreactivity suggests that proctolin has diverse central and peripheral functions. The mapping immunoreactive neuronal somata provides a valuable step in the identification of putative proctolin-containing neurons suitable for further biochemical, anatomical, and physiological analysis.