Nervous System Of Aplysia Californica Research Articles

Single-Neuron RNA Modification Analysis by Mass Spectrometry: Characterizing RNA Modification Patterns and Dynamics with Single-Cell Resolution.

The entire collection of post-transcriptional modifications to RNA, known as the epitranscriptome, has been increasingly recognized as a critical regulatory layer in the cellular translation machinery. However, contemporary methods for the analysis of RNA modifications are limited to the detection of highly abundant modifications in bulk tissue samples, potentially obscuring unique epitranscriptomes of individual cells with population averages. We developed an approach, single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS), that enables the detection and quantification of numerous post-transcriptionally modified nucleosides in single cells. When compared to a conventional RNA extraction approach that does not allow detection of RNA modifications in single cells, SNRMA-MS leverages an optimized sample preparation approach to detect up to 16 RNA modifications in individual neurons from the central nervous system of Aplysia californica. SNRMA-MS revealed that the RNA modification profiles of identified A. californica neurons with different physiological functions were mostly cell specific. However, functionally homologous neurons tended to demonstrate similar modification patterns. Stable isotope labeling with CD3-Met showed significant differences in RNA methylation rates that were dependent on the identity of the modification and the cell, with metacerebral cells (MCCs) displaying the fastest incorporation of CD3 groups into endogenous RNAs. Quantitative SNRMA-MS showed higher intracellular concentrations for 2'-O-methyladenosine and 2'-O-methylcytidine in homologous R2/LPl1 cell pairs than in MCCs. Overall, SNRMA-MS is the first analytical approach capable of simultaneously quantifying numerous RNA modifications in single neurons and revealing cell-specific modification profiles.

Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis-Trapped Ion Mobility Spectrometry Mass Spectrometry.

Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell-cell signaling peptides. The multiplicity of endogenous cell-cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE-TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.

Altered expression of ionotropic L-Glutamate receptors in aged sensory neurons of Aplysia californica.

The simplified nervous system of Aplysia californica (Aplysia) allows for detailed studies of physiological and molecular changes in small sets of neurons. Sensory neurons of the biting and tail withdrawal reflexes are glutamatergic and show reduced L-Glutamate current density in aged animals, making them a good candidate to study age-related changes in glutamatergic responses. To examine if changes in ionotropic L-Glu receptor (iGluR) transcription underlie reduced physiology, mRNA expression of iGluR was quantified in two sensory neuron clusters of two cohorts of Aplysia at both sexual maturity (~8 months) and advanced age (~12 months). Sensory neuron aging resulted in a significant overall decrease in expression of iGluR subunits in both sensory neuron clusters and cohorts. Although the individual subunits differentially expressed varied between sensory neuron clusters and different cohorts of animals, all differentially expressed subunits were downregulated, with no subunits showing significantly increased expression with age. Overall declines in transcript expression suggest that age-related declines in L-Glu responsiveness in Aplysia sensory neurons could be linked to overall declines in iGluR expression, rather than dysregulation of specific subunits. In both sensory neuron clusters tested the N-methyl-D-aspartate receptor subtype was expressed at significantly greater levels than other iGluR subtypes, suggesting an in vivo role for NMDAR-like receptors in Aplysia sensory neurons.

Highlighting manganese dynamics in the nervous system of Aplysia californica using MEMRI at ultra-high field

Exploring the pathways of manganese (Mn(2+)) transport in the nervous system becomes of interest as many recent studies use Mn(2+) as a neural tract tracer in mammals. In this study, we performed manganese enhanced MRI (MEMRI) at 17.2 T on the buccal ganglia of Aplysia californica. The main advantage of this model over mammalian systems is that it contains networks of large identified neurons. Using Mn(2+) retrograde transport along selected nerves, we first validated the mapping of motor neurons' axonal projections into peripheral nerves, previously obtained from optical imaging (Morton et al., 1991). This protocol was found not to alter the functional properties of the neuronal network. Second, we compared the Mn(2+) dynamics inside the ganglia in the presence or absence of chemical stimulation. We found that 2h of stimulation with the modulatory transmitter dopamine increased the extent of areas of intermediate signal enhancement caused by manganese accumulation. In the absence of dopamine, an overall decrease of the enhanced areas in favor of non-enhanced areas was found, as a result of natural Mn(2+) washout. This supports the hypothesis that, upon activation, Mn(2+) is released from labeled neurons and captured by other, initially unlabeled, neurons. However, the latter could not be clearly identified due to lack of sensitivity and multiplicity of possible pathways starting from labeled cells. Nonetheless, the Aplysia buccal ganglia remain a well-suited model for attempting to visualize Mn(2+) transport from neuron to neuron upon activation, as well as for studying dopaminergic modulation in a motor network.

Chemical neurostimulation using pulse code modulation (PCM) microfluidic chips

We report the implementation of a chemical neurostimulation technique using microfluidic devices. The microfluidic chip in this research is used for the in vitro study of the nervous system of Aplysia californica under localized chemical stimulation. The polydimethylsiloxane (PDMS) device is a one-bit pulse code modulator that digitally controls the concentration of the non-hydrolysable cholinergic agonist carbachol injected directly above a ganglion. The chip was successful in repeatedly and controllably inducing bursts of ingestive-like patterns. The ability of the chip to induce rhythmic activity through the sheath of the ganglion suggests that it could serve as the basis for an implantable, in vivo device to control neural activity and motor behavior using chemical stimulation.

Identification and characterization of homologues of vertebrate β‐thymosin in the marine mollusk Aplysia californica

The beta-thymosins have been known as actin-sequestering proteins, but now are recognized as molecules with multiple and diverse intracellular and extracellular functions. Two closely related proteins, beta-thymosin(His) and beta-thymosin(Gln), have been de novo sequenced by top-down mass spectrometry in the common neurobiology model, Aplysia californica. As determined by nanoelectrospray quadrupole-enhanced Fourier-Transform mass spectrometry with collisionally activated and electron-capture dissociations, both of these Aplysia beta-thymosins are acetylated and differ by a single residue in the central actin-binding domain. Profiling of individual cells and tissue by matrix-assisted laser desorption/ionization mass spectrometry reveals that these proteins are widely expressed in the Aplysia central nervous system, including in individual identified neurons, neuronal clusters, nerves and connective tissues. Newly identified beta-thymosin(His) and beta-thymosin(Gln) are also detected by mass spectrometry in hemolymph, and in releasates collected from whole ganglia. When applied exogenously, beta-thymosin proteins, purified from nerve cell extract, support the anchoring of neurons, and increase neurite sprouting and total neurite outgrowth in culture. These positive effects on neurite regeneration in cell culture suggest that the beta-thymosin proteins have an extracellular function in the central nervous system of Aplysia californica.

Localization of putative nitrergic neurons in peripheral chemosensory areas and the central nervous system ofAplysia 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.

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: Biochemical characterization and link to cGMP pathways

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5 mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65–92% with 500 μM of l-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5 mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l- N6-(1-iminoethyl)lysine dihydrochloride ( l-NIL, 5 mM), an iNOS inhibitor, by 78% and l-thiocitrulline (5 mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/μg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-( N, N-diethylamino) diazen-1-ium-1,2-diolate – 1 mM) or S-nitroso- N-acetylpenicillamine (1 mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1 mM) prior to the assay showed a 26–80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one – 1 mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.

Comparative localization of two serotonin receptors and sensorin in the central nervous system ofAplysia californica

Aplysia californica is a powerful model for understanding the cellular and molecular mechanisms underlying modulation of neuronal plasticity and learning. In the central nervous system of Aplysia, serotonin is associated with various behaviors. For example, it induces short-, intermediate-, and long-term synaptic changes in sensory neurons during learning and inhibits the afterdischarge of the bag cells that initiate egg-laying behavior. Little is known about the nature and contribution of serotonin receptors involved in the numerous serotonin-mediated physiological responses in Aplysia. Recently, two G(i)-coupled serotonin receptors (5-HT(ap1) and 5-HT(ap2)) were cloned. We now report that, by using in situ hybridization to express the profile of these receptors, we are able to gain critical insight into their roles in the behavior of Aplysia. We compared their distribution to that of sensorin-A, a peptide specifically found in sensory neurons. We wished to determine their involvement in some simple forms of behavioral modifications. 5-HT(ap1) and 5-HT(ap2) mRNAs are expressed in all ganglia of the Aplysia central nervous system. Stronger signal was observed with the 5-HT(ap2) antisense probe than with the 5-HT(ap1) antisense probe. Notably, mRNA coding for the receptors was found in several identified neurons, in the bag cells, in characterized serotonergic neurons, and in neurons of the mechanosensory clusters that expressed sensorin. We also observed heterogeneity of receptor expression between R2 and LPl1 and among neurons of a single cluster of sensory neurons. These results suggest that 5-HT(ap1) and 5-HT(ap2) receptors may regulate the response to serotonin and/or its release in several neurons.

Quantification of D/L-aspartic acids in Aplysia californica central nervous system by beta-cyclodextrin modified micellar electrokinetic chromatography.

In presence of an organic modifier (e.g. methanol), separation of amino acid enantiomers tagged with naphthalene-2,3-dicarboxaldehyde by beta-cyclodextrin modified micellar electrokinetic chromatography was dramatically improved. Coupled with laser-induced fluorescence detection, the method was well suited for analysis of D/L-amino acid enantiomers present in mass/volume-limited biological samples such as cell clusters. The five major ganglia dissected from the central nervous system of Aplysia californica, a widely used neuronal model, were analyzed to determine D- and L-aspartic acid enantiomers both free and bound in proteins/tissue matrix. The analyses revealed high levels of free D-aspartic acid ranging from 0.13 to 0.82 micromol/g wet tissue (or 6.0-21.2% of the total free aspartic acid) in all of the five ganglia. However, no D-aspartic acid was detected bound in protein/tissue matrix. The content of free D-aspartic acid in the liver tissue was also found below the detection limit of the method, which was 1 x 10(-8) M.

Regulation of eukaryotic initiation factor 4E phosphorylation in the nervous system of Aplysia californica.

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.

Positive injury signals induce growth and prolong survival in Aplysia neurons.

Injury to a peripheral nerve initiates changes that can lead to regeneration of the damaged axons. How information about a distant injury is communicated to the cell body is not clear. Using the nervous system of Aplysia californica, we tested the idea that some of this information is conveyed via positive injury signals-axoplasmic proteins that are activated at the injury site and transported to the cell soma. We collected these proteins by crushing pedal nerves and then placing a ligation proximal to the ligation. The contralateral nerves were ligated as controls. Twenty h later, axoplasm was extruded from the nerve segment just distal to the ligation on the crushed nerves (cr/lig) and on the control nerves (lig). The total proteins were rhodaminated and injected into the cytoplasm of neurons in vitro to look for nuclear import. Punctate fluorescence was detected in the nucleus of all seven neurons injected with the cr/lig axoplasm. Only two of five neurons injected with lig axoplasm had any fluorescence. Equal amounts of cr/lig and lig axoplasm were then injected directly into the cell bodies of neurons maintained in vitro. The cells injected with cr/lig axoplasm exhibited renewed growth and significantly longer survival: 25.9 +/- 2.1 days (mean +/- SEM: n = 22) relative to the cells injected with lig axoplasm (15.3 +/- 1.2 days; n = 14) and to those that were not injected (12.2 +/- 1.7 days; n = 24). Fractionation of the cr/lig axoplasm indicated that different factors are responsible for growth and survival.

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

Axonal regeneration in the central nervous system of aplysia californica determined by anterograde transport of biocytin.

Rhythmic biting, a component of consummatory feeding behavior in the sea hare Aplysia californica, is eliminated following bilateral cerebral-buccal connective (CBC) crushes and recovers within 14 days postlesion. To assess axonal regeneration after CBC lesions, we used biocytin backfills of CBCs followed by fluorescence labeling with streptavidin-lissamine rhodamine. Anterograde transport of biocytin showed up to 1 mm of outgrowth by regenerating axons at 3 days postlesion. At 7 days postlesion, the regenerated axons approached or had entered the ipsilateral buccal neuropil and exhibited numerous varicosities; the average rate of axonal growth was 326 microm/day for the longest, most rapidly growing axons labeled in the CBC. The number of varicosities on labeled axons, suggestive of intercellular interactions, was increased dramatically at all times postlesion. At 14 and 20 days postlesion, regenerated axons branched extensively in the ipsilateral buccal neuropil, entered the contralateral buccal neuropil, and entered peripheral nerves on both sides of the midline. At these later times postlesion, some labeled axons encircled unlabeled buccal cell bodies and exhibited branches containing numerous varicosities, indicative of axosomatic contacts. Some regenerating axons were observed in the sheath of the CBC, but the vast majority of labeled axons remained confined to the connective core, as in control preparations. The bilateral projections within the buccal ganglia of labeled cerebral-to-buccal axons and the large number of varicosities present on these processes are indicative of regenerating axons and synapses that likely contribute to the functional recovery of rhythmic biting.

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.

Ag, a novel protein secreted from Aplysia glia.

We have isolated a cDNA (ag for Aplysia glial) corresponding to an mRNA specific to the nervous system of Aplysia californica. In this study, we characterized the ag cDNA sequence and the distribution of ag mRNA and protein in the Aplysia nervous system. The ag cDNA contains an open reading frame that encodes a novel 29 kD protein. In situ hybridizations demonstrate that ag mRNA is conspicuously absent from the cell bodies of the large neurons constituting the external layer of the ganglia. Instead, it is largely confined to a subset of small, apparently non-neuronal cells surrounding the neurons at the border of the neuropil, is sparsely scattered within the neuropil, and is widespread within the connective nerves, a pattern consistent with glial localization. Polyclonal anti-ag antiserum recognizes a protein between 27 and 29 kD that is more broadly distributed, especially within the neuropil. The distributions of ag mRNA and protein, together with the presence of a putative signal peptide, suggest that ag protein is secreted. Two findings support this hypothesis: first, ag protein is detectable by western blot in Aplysia hemolymph. Second, full length ag protein expressed in COS cells is secreted, but ag lacking the putative signal peptide is not. Secretion from glia raises the possibility that this abundant protein may affect neighboring neurons.

Type II regulatory subunits of cAMP-dependent protein kinase and their binding proteins in the nervous system of Aplysia californica.

Two type II regulatory (R) subunits of cAMP-dependent protein kinase (PKA) of 50 and 47 kDa have been identified in Aplysia neurons by several criteria which include phosphorylation by the catalytic subunit of PKA and nanomolar affinity for a peptide fragment of the human thyroid protein Ht 31, properties that in mammals distinguish type II from type I R subunits. The neuronal type II R subunits are differentially localized within cells. For example, the 50-kDa polypeptide is enriched in taxol-stabilized microtubules. In addition, at least seven high molecular mass neuronal RII-binding proteins ranging in mass from 110 to 420 kDa have been demonstrated by a blot overlay technique, which uses 32P-labeled bovine RII alpha as a probe. The RII-binding proteins also exhibit discrete patterns of subcellular localization. For example, the 420 kDa species is enriched in taxol-stabilized microtubules and therefore may serve to anchor the 50-kDa RII subunit. The localization of PKA through the association of RII subunits with the binding proteins may anchor the multifunctional kinase close to key substrates and thereby contribute to the spatial organization required to mediate the orderly phosphorylation events that underly neuronal modulation.

Characterization of two isoforms of protein kinase C in the nervous system of Aplysia californica.

By molecular cloning Kruger et al. (Kruger, K. E., Sossin, W. S., Sacktor, T. C., Bergold, P. J., Beushausen, S., and Schwartz, J. H. (1991) J. Neurosci. 11, 2303-2313) provided evidence for three isoforms of protein kinase C (PKC) in Aplysia, two of which, Apl I and Apl II, are expressed abundantly in the nervous system. We resolve two major kinase activities from nervous tissue by column chromatography on DEAE-cellulose and hydroxylapatite (HAP), one Ca(2+)-activated and the other Ca(2+)-independent. We show that these two activities correspond to the previously cloned Apl I and II. These two isoforms appear to be the only major PKCs present in nervous tissue. The Apl I kinase is strongly activated by cis-fatty acids but only in the presence of Ca2+. Thus functionally, Apl I is more like the alpha and beta isoforms of vertebrate PKC, than to the vertebrate neural gamma isoform. The Apl II kinase is Ca(2+)-independent and resembles vertebrate PKC epsilon. The simplicity of the PKC isoform distribution in Aplysia makes this mollusc an attractive animal for understanding the differential regulation and physiological activities of Ca(2+)-activated and Ca(2+)-independent PKCs.

The morphological localization and biochemical characterization of a synapsin I-like antigen in the nervous system of Aplysia californica.

Synapsins are a well-characterized class of phosphoproteins found at synapses in the mammalian nervous system. One member of this family, synapsin I, has been extensively studied and shown to associate in a phosphorylation-dependent manner with both small synaptic vesicles and cytoskeletal elements. Though the characteristics of synapsin I suggest an important function in synaptic transmission, its definitive role is still in question. In an effort to find a model system in which to test directly the function of synapsin I, we have looked in the nervous system of the marine mollusc Aplysia californica for synapsin I-like antigens (SILA). Light microscope immunocytochemical studies using polyclonal and monoclonal antibodies to bovine brain synapsin I demonstrate Aplysia SILA in neuronal somata, in the neuropil, and at some identified synapses. Though SILA were exclusively associated with neuronal structures in Aplysia, the pattern of staining suggested that they are not present at all synaptic terminals. This interpretation was corroborated by ultrastructural studies in which SILA were present at some synaptic terminals but absent, or in low abundance, in adjacent terminals. In axons, SILA were associated with vesicles of 120-150 nm diameter, as well as with filamentous structures. Biochemical studies identified small amounts of SILA of 40 and 50 kD molecular weight that are recognized by several antibodies to mammalian synapsin I, and are acid extractable, collagenase-sensitive phosphoproteins; these are criteria used to define synapsin I homologues in other species. Our studies indicate that SILA are present in neurons in Aplysia californica but suggested that they represent only a small percentage of the total protein within the nervous system.

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.