An autoradiographic analysis of neurogenesis in juvenile Aplysia californica

In developing Aplysia californica, a dramatic proliferation of new neurons occurs throughout the central nervous system (CNS) surprisingly late in juvenile development (Cash and Carew, 1989). In the present study, we investigated the source of these new neurons. Using tritiated thymidine autoradiography, we examined two different juvenile stages: stage 11 (before the large-scale proliferation) and stage 12 (at the peak of proliferation). Previous results implicated the body wall as a source for neurons in developing Aplysia (McAllister, Scheller, Kandel, and Axel, 1983; Jacob, 1984). Thus, we focused our attention on the body wall adjacent to a specific central ganglion, the abdominal ganglion. We found that in stage 11 there was uniform labelling of cells across the entire body wall. However, in stage 12 there was significantly more labelling in the body wall region immediately adjacent to the abdominal ganglion compared to flanking regions. Thus, at the time of neuronal proliferation, specific and highly localized regions of the body wall immediately opposite their target in the CNS show a significant increase in cell division. We also examined the distribution of labelled cells in the abdominal ganglion at survival times of 1 and 7 days after thymidine injection. In both stage 11 and stage 12, the fraction of labelled cells on the surface of the ganglion decreased over time, with a corresponding significant increase in the fraction observed on the inside. Our results support the hypothesis that specific regions of body wall are significantly up-regulated in juvenile Aplysia development, giving rise to widespread neuronal proliferation. These neurons then migrate from the body wall to their target ganglion, and from there continue migrating into the ganglion to achieve their final position.     

Stages in the post-hatching development of Aplysia californica

In order to study the development of the nervous system of the marine mollusc, Aplysia californica, it is necessary objectively to assess the maturity of individual specimens. This can be done by defining stages in the life cycle. The post-hatching development can be divided into four phases: planktonic, metamorphic, juvenile, and adult. These phases can be further subdivided into 13 stages on the basis of behavioral and morphological characteristics visible in living specimens: Stage 1, newly hatched; Stage 2, eyes develop; Stage 3, the larval heart beats; Stage 4, maximum shell size is reached; Stage 5, the propodium develops; Stage 6, red spots appear; Stage 7, the velum is shed; Stage 8, eyebrows appear; Stage 9, pink color develops; Stage 10, white spots appear; Stage 11, rhinophores grow; Stage 12, the genital groove forms; Stage 13, egg laying begins. Reconstructions from serial sections taken from specimens fixed at each of these stages reveal the sequence of formation of the major organ systems. The nervous system develops gradually. The cerebral and pedal ganglia are present at Stage 1, the optic ganglia develop at Stage 2, the abdominal, pleural, and osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and the genital ganglion at Stage 13. Because Aplysia develops gradually, it is possible to analyze the contribution which gastropod torsion makes to the different phases of the life cycle. The Aplysia embryo undergoes 120 degrees torsion prior to Stage 1. The major visceral organs, the digestive system, heart, gill, and visceral nervous system, develop sybsequently in their post-torsional positions. After metamorphosis, there is a partial de-torsion which involves only the digestive system. Torsion of the digestive system may therefore be beneficial only to the pre-metamorphic larva, and not to the postmetamorphic juvenile.     

Localization of glutamate-like immunoreactive neurons in the central and peripheral nervous system of the adult and developing pond snail, Lymnaea stagnalis

We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50-60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.     

Serotonin immunoreactivity of neurons in the gastropod Aplysia californica

Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation.     

The transcriptome of the early life history stages of the California Sea Hare Aplysia californica

Aplysia californica is a marine opisthobranch mollusc used as a model organism in neurobiology for cellular analyses of learning and behavior because it possesses a comparatively small number of neurons of large size. The mollusca comprise the second largest animal phylum, yet detailed genetic and genomic information is only recently beginning to accrue. Thus developmental and comparative evolutionary biology as well as biomedical research would benefit from additional information on DNA sequences of Aplysia. Therefore, we have constructed a series of unidirectional cDNA libraries from different life stages of Aplysia. These include whole organisms from the egg, veliger, metamorphic, and juvenile stages as well as adult neural tissue for reference. Individual clones were randomly picked, and high-throughput, single pass sequence analysis was performed to generate 7971 sequences. Of these, there were 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. A significant number (497) of ESTs did not match existing Aplysia ESTs and are thus potentially novel sequences for Aplysia. GO and KEGG analyses of these novel sequences indicated that a large number were involved in protein binding and translation, consistent with the predominant biosynthetic role in development and the presence of stage-specific protein isoforms.     

Regional and segmental differences in the embryonic expression of a putative leech Hox gene,Lox2, by central neurons immunoreactive to FMRFamide-like neuropeptides

We performed immunofluorescence experiments using a rat polyclonal antibody on formaldehyde-fixed whole-mount embryos to characterize the expression of a putative leech Hox gene, Lox2, during embryonic development. The main goal was to determine whether the differentiation of subsets of FMRFamide-like immunoreactive (FLI) neurons coincide with the expression domain of Lox2. The earliest expression of Lox2 was detected in relatively large, prominent nuclei in the posterior region at embryonic day 4, a very early stage. Lox2 expression was also detected in subsets of central neurons (neurons located in the CNS) located in midbody ganglia 6 (M6)–M21. In addition, Lox2 was expressed by a number of segment-specific and segmentally repeated central FLI neurons. Lox2-positive FLI neurons of interest included some of those previously identified: the rostral most ventral (RMV) neurons, the circular ventral (CV) neurons, and cell 261. The paired RMVs, which are located in all midbody ganglia, expressed Lox2 only in M7–M19. The CV neurons, specialized motor neurons that innervate the circular ventral muscles of the body wall, expressed Lox2 in M7–M19. The putative cell 261 expressed Lox2 in M7–M12, where Lox1 is also expressed. FMRFamide staining in putative segmental homologs of cell 261 was not detected in other segmental ganglia. Our results suggest a role for Lox2 in very early embryonic development (before the formation of the CNS), and in the differentiation of segmentally repeated and region-specific FLI neurons.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

Postembryonic development of the mushroom bodies in the ant, Camponotus japonicus

Mushroom bodies (MB) are insect brain centers involved in learning and other complex behaviors and they are particularly large in ants. We describe the larval and pupal development of the MB in the carpenter ant, Camponotus japonicus. Based on morphological cues, we characterized the stages of preimaginal development of worker ants. We then describe morphological changes and neurogenesis underlying the MB development. Kenyon cells are produced in a proliferation cluster formed by symmetrical division of MB neuroblasts. While the duration of larval instars shows great individual variation, MB neuroblasts increase in number in each successive larval instar. The number of neuroblasts increases further during prepupal stages and peaks during early pupal stages. It decreases rapidly, and then neurogenesis generally ceases during the mid pupal stage (P4). In contrast to the larval period, the MB development of individuals is highly synchronized with physical time throughout metamorphosis. We show that carpenter ants (C. japonicus) have approximately half as many MB neuroblasts than are found in the honey bee Apis mellifera. Mature MBs of carpenter ants and honey bees reportedly comprise almost the same number of neurons. We therefore suggest that the MB neuroblasts in C. japonicus divide more often in order to produce a final number of MB neurons similar to that of honey bees.     

METABOLISM OF PUTATIVE TRANSMITTERS IN INDIVIDUAL NEURONS OF APLYSIA CALIFORNICA: AROMATIC AMINO ACID DECARBOXYLASE

Abstract— The activities of aromatic amino acid decarboxylase (EC 4.1.1.26) in various ganglia, nerve trunks, and individual identifiable neurons of Aplysia culifornica were measured. The distribution of the decarboxylase enzyme is ubiquitous throughout the central nervous system of the Aplysia . Every Aplysia neuron tested contained some decarboxylase activity. The presence of this particular synthetic enzyme in an Aplysia neuron, therefore, cannot be used to classify these neurons as 'aminergic'.     

Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.     

Catecholamine neurons in Aplysia: improved light-microscopic resolution and ultrastructural study using paraformaldehyde and glutaraldehyde (FaGlu) cytochemistry

We have modified the formaldehyde-glutaraldehyde (FaGlu) histofluorescence method of Furness, Costa, and Blessing (1977a) and Furness, Costa, and Wilson (1977b) to examine wholemounts and sections of both juvenile and adult ganglia as well as peripheral tissues of Aplysia californica. FaGlu fluorescence is the result of a reaction between formaldehyde and tissue catecholamines to produce water-insoluble (fixed) fluorophores. In serially sectioned cerebral ganglia, 70-80 positive neurons were observed (many in clusters of 10-20 cells), many more than were found using the glyoxylic acid technique. Catecholamine-containing varicosities were densely packed in localized portions of the neuropil of all central ganglia. Exclusive localization in the neuropil of presumed dopamine release sites is similar to that previously found for the neuropeptide SCP but differs from the widespread ramification of varicose neurites containing 5-HT, FMRFamide, and ELH. The FaGlu technique also enabled us to study the ultrastructure of catecholamine-containing neurons. In contrast to the larger vesicles found in serotonergic and histaminergic neurons, these dopaminergic neurons contain 70 nm dense-cored vesicles.     

Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly

Summary Neurons immunoreactive with antibodies to serotonin (5-HT) were mapped in the thoracico-abdominal ganglia of the blowfly, Calliphora erythrocephala, during postembryonic development. Reconstructions from serial sections of tissue processed with a preincubation PAP-method permitted a detailed analysis of the morphological changes occurring in 5-HT-immunoreactive (5-HTi) neurons.All the 5-HTi cell bodies in the thoracico-abdominal ganglia of the 3rd instar larva, except two in the metathoracic ganglion, retain their immunochemical phenotype throughout pupal development. Hence, all the adult 5-HTi neurons in these ganglia differentiate during embryonic development. The finer processes of the larval 5-HTi neurons undergo a substantial regression during the first 24 h of pupal development, and thereafter new branches form on the primary processes of the same cell bodies. The slight change in relative position of 5-HTi cell bodies and the reorganization of the neuropil into an adult pattern occur during the first half of pupal development. The neuropil mass and extent of 5-HTi processes continue to increase during the following days and appear to be fully developed two days (80% of pupal development) before hatching.On the basis of the presented data, some of the basic processes are discussed that lead to the transformation of the larval nervous system into its adult form.     

Post embryonic development of the central nervous system of the spider Argiope aurantia (Lucas)

Volumetric and histological changes of the central nervous system were studied during post embryonic development of a spider, Argiope aurantia. The neural mass of Argiope grows allometrically with respect to volume of the cephalothorax and body weight. In the first instar 46% of the cephalothoracic volume constitutes the neural mass and this is reduced to 4% in the female (9th stage) and 12% in the male (7th stage) spider. Growth curves for the cephalic ganglion, measured at all stages, represent a straight line. The neural mass of females is two and a half times larger than that of the males. The ganglion increased 24 fold in female and 10 fold in male spiders. Addition of neural mass occurs in all stages. The brain volume is greater than that of the subesophageal ganglion in the first two instars. In subsequent stadia, the subesophageal ganglion grows faster, and in females it is finally three times and in males two times larger than the brain. Growth of cortex and neuropile depict exponential curves. Comparison of growth patterns of these shows an inverse relationship during development. While the volume of the cortex is higher in the first two or three stages, the volume of the neuropile is higher in the remaining stadia. The causes for this growth pattern are discussed. Counts of cell numbers show that there is a constant population of neurons throughout the post-embryonic development. The number of nerve cells in females is higher than in males, 11% in the subesophageal ganglion and 58% in the brain. The growth of the cortex is partly accomplished by an increase in cell volume. In male and female spiders the increase in Type-B cells is 20 and 50 fold, while that of large motor neurons is 200 and 600 fold respectively. The motor neurons of 20 μ and above number 63 in male and 916 in female adult spiders. The growth of neuropile occurs through an increase of dendritic arborization and axonal branching. The largest axons measure 1 μ in the first and 16 μ in adult stages. An increase of incoming sensory fibers is also noticed during development. Invasion of neural lamella into cortex and neuropile increases during development. Neural lamella which are 1-2 μ in the first stage grow to 40–100 μ thickness in adult female spiders, near the origin of the main nerves. One type of astral cells, counted in neuropile, increases 10 fold. The appearance of a central body and the beginning of web construction coincide during the second instar. The relationship between these two is discussed.     

Embryogenesis of the histaminergic system in the pond snail, Lymnaea stagnalis L.: an immunocytochemical and biochemical study

Embryogenesis of the histaminergic system in the pond snail, Lymnaea stagnalis, was investigated by means of immunocytochemistry and HPLC assay. From the earliest onset of the of histamine-immunoreactive (HA-IR) elements, the labelled neurons were confined to the pedal, cerebral and buccal ganglia, whereas no IR cells within the pleural, parietal and visceral ganglia were detectable during the embryogenesis. Peripheral projections of the embryonic HA-IR neurons were missing. No transient HA-IR neurons could be found either inside or outside the CNS. The first HA-IR elements appeared at about E55% of embryonic development, at the beginning of metamorphosis, and were represented by three pairs of neurons located in the cerebral ganglia. Following metamorphosis, four pairs of HA-IR neurons were added; two of them occurred in the pedal (E65% stage of development) and two in the buccal (E90% stage of development) ganglia. During embryogenesis, HA-IR fibers were present in the cerebro-pedal connectives and in the cerebral, pedal and buccal commissures, whereas only little arborization could be observed in the neuropil of the ganglia. HPLC measurements revealed a gradual increase of HA content in the embryos during development, corresponding well to the course of the appearance of immunolabeled elements. It is suggested that the developing HAergic system plays a specific role in the process of gangliogenesis and CNS plasticity of embryonic Lymnaea.     

Embryogenesis of GABAergic elements in the nervous system of Eisenia fetida (Annelida, Oligochaeta)

The appearance and development of the GABA-immunoreactive nervous elements in the central nervous system of Eisenia fetida were studied by immunocytochemistry. The nervous system originates from the neuroectoderm situated on the ventral side of the embryo. The organization of the circumpharyngeal ring starts earlier than that of the ventral cord. In the elementary ring the first GABA-immunopositive neurons can be observed (E1 stage) around the mouth. Later the cell number gradually increases and parallel to this process the elementary ring is separeted into a superficial and a deeper portion. The brain and the subesophageal ganglion will be organized from the superficial ring, while the nervous elements of the deeper ring will give rise for the first GABA-immunoreactive elements of the stomatogastric nervous system. In the early stages of the embryogenesis the immunoreactive cells of the developing brain appear solitary, while from the stage E4 they gradually are observed in groups. According to their position, these cell groups are similar to those observed in the brain of the adult earthworms. During embryogenesis the level of the ventral cord ganglia depends on their position in the ectodermal germ bands. It means, that the more organized ganglia are near the circumpharyngeal ring, mean while less developed ganglia are situated caudally from them. By the end of the embryogenesis all ganglia of the ventral cord will be equally well organized. The nerve tracts of the ganglia are built up from contra- and ipsilateral by projected fibres. From E3 stage the medial tracts, mean while from the E4 stage the lateral tracts begin to be formed. During the next stages, more and more fibres connect to the both tracts. At hatching, the development of the central nervous system of Eisenia fetida is not completed, the process is continued during the postembryonic development.     

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.     

Trophic effects of androgen: development and hormonal regulation of neuron number in a sexually dimorphic vocal motor nucleus.

In Xenopus laevis, the laryngeal motor nucleus (n. of cranial nerves IX-X) is part of a sexually differentiated, androgen sensitive neuromuscular system devoted to vocalization. Adult males have more n. IX-X neurons than females; however, during development of n. IX-X, the rate of neurogenesis does not appear to differ between the sexes. In this study, we explored the role of naturally occurring cell death in the development of this nucleus and asked whether cell death might be involved in establishing the sex difference in neuron number. Counts of n. IX-X neurons reveal that at tadpole stage 56, males and females have similar numbers of n. IX-X neurons, but by stage 64 male neuron numbers are greater. This sex difference arises owing to a greater net loss of neurons in females-males lose approximately 25% of their n. IX-X neurons between stages 56 and 64, while females lose approximately 47%. Sexual differentiation of n. IX-X neuron number coincides with a period of developmental cell death, as evidenced by terminal transferase-mediated dUTP nick-end labeling and the presence of pyknotic nuclei in n. IX-X. A role for gonadal hormones in controlling cell number was examined by treating tadpoles with exogenous androgen and determining the number of n. IX-X neurons at stage 64. Dihydrotestosterone (DHT) treatment from the beginning of the cell death period (stage 54) until stage 64 had no effect on the number of n. IX-X neurons in males but did significantly increase n. IX-X neuron number in females. This increase was sufficient to abolish the sex difference normally observed at stage 64. Although DHT induced increases in female neuron number, it did not induce increases in cell proliferation or addition of newly born neurons to n. IX-X. DHT may therefore have increased neuron number by protecting cells from death. We conclude that androgens can influence the survival of n. IX-X neurons during a period of naturally occurring cell death, and that this action of androgen is critical to the development of sex differences in n. IX-X neuron number.     

Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions

Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay (RIA) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends an axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in "split-foot" preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.