Characterization of Neuronal Protein Phosphatases in Aplysia californica

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.     

Ultrastructural correlates of interneuronal function in the abdominal ganglion of Aplysia californica

The synaptic inputs and outputs of the major interneuron L10 of the abdominal ganglion of Aplysia were studied using an intracellular staining technique for the electron microscope. The sites of both the chemical synaptic input and output of L10 are localized to the dendritic arborizations that arise from the axon in the ganglion neuropil. Thus, the interneuronal functions are mediated at the dendritic processes and could occur in the absence of spiking in the axon and cell body. The sites of L10 synaptic output are presumed to be at. aggregations of vesicles and mitochondria in the dendrites. The synaptic vesicle content of L10, a cholinergic neuron, with many large dense vesicles resembles that described for serotonergic cells in Aplysia, making distinction of synaptic pharmacology by ultrastructure difficult. Focal membrane specializations with a clear synaptic cleft were not observed between L10 and its large population of postsynaptic cells. In contrast, clear focal input sites were frequently found on L10. Gap junctions, sites of probable electrical coupling between L10 and other neurons, were also found. These observations are discussed as evidence that many synapses do not have focal specializations.     

Neural structures in the receptive field of pleural ganglion mechanosensory neurons of Aplysia californica

The peripheral processes of the mechanoafferents that, when stimulated, initiate the much-studied tail withdrawal reflex of Aplysia californica have not been characterized. We show that immunofluorescence staining for class III -tubulin highlights neurons and reveals nerve tracts and fine neuronal processes in Aplysia tissue. Coupled with transmission and scanning electron microscopy, class III -tubulin immunofluorescence is consistent with the possibility that mechanoafferents in the receptive field of pleural ganglion mechanosensory neurons penetrate the tail epidermis and terminate as ciliated endings. This view is reinforced by comparisons among neuronal processes in several mechanosensory epidermal regions and in a chemosensory epidermis.     

Ultrastructural correlates of interneuronal function in the abdominal ganglion of Aplysia californica.

The synaptic inputs and outputs of the major interneuron L10 of the abdominal ganglion of Aplysia were studied using an intracellular staining technique for the electron microscope. The sites of both the chemical synaptic input and output of L10 are localized to the dendritic arborizations that arise from the axon in the ganglion neuropil. Thus, the interneuronal functions are mediated at the dendritic processes and could occur in the absence of spiking in the axon and cell body. The sites of L10 synaptic output are presumed to be at aggregations of vesicles and mitochondria in the dendrites. The synaptic vesicle content of L10, a cholinergic neuron, with many large dense vesicles resembles that described for serotonergic cells in Aplysia, making distinction of synaptic pharmacology by ultrastructure difficult. Focal membrane specializations with a clear synaptic cleft were not observed between L10 and its large population of postsynaptic cells. In contrast, clear focal input sites were frequently found on L10. Gap junctions, sites of probable electrical coupling between L10 and other neurons, were also found. These observations are discussed as evidence that many synapses do not have focal specializations.     

Ultrastructure of the osphradium of Aplysia californica

Summary The osphradium of Aplysia californica, a sensory organ, is a small yellow-brown epithelial patch located in the mantle cavity immediately anterior to the rostral attachment of the gill. Scanning electron microscopy reveals a round ellipsoid structure of 0.6–1 mm in diameter with a central, occasionally folded, sensory epithelium. The central area is covered with microvilli and surrounded by a densely ciliated epithelium. Transmission electron micrographs show that the columnar supporting cells in the sensory epithelium contain an abundance of apical pigment granules and microvilli. Between the epithelial-supporting cells, the putative sensory elements consist of thin neurites (0.4–1.5 m in diameter) that reach the sea-water side of the osphradium. The neurites contain many neurotubules, mitochondria, vesicles and cilia in their apices. The nerve endings originate from cell bodies up to 40 m below the epithelium or in the osphradial ganglion itself, as revealed by electron microscopy and retrograde labeling with Lucifer yellow. There appear to be two populations of putative sensory cells, a large population of heavily stained cell bodies 4–10 m in diameter and a few scattered cells of large diameter (25–60 m). Following lanthanum impregnation, septate junctions can be seen between all types of cells in the epithelium, 3–5 m below the sea-water surface. This study provides new information for further investigation of osmo- and mechanosensation in Aplysia californica.     

Parallel processing in an identified neural circuit: the Aplysia californica gill-withdrawal response model system

The response of the gill of Aplysia calfornica Cooper to weak to moderate tactile stimulation of the siphon, the gill-withdrawal response or GWR, has been an important model system for work aimed at understanding the relationship between neural plasticity and simple forms of non-associative and associative learning. Interest in the GWR has been based largely on the hypothesis that the response could be explained adequately by parallel monosynaptic reflex arcs between six parietovisceral ganglion (PVG) gill motor neurons (GMNs) and a cluster of sensory neurons termed the LE cluster. This hypothesis, the Kupfermann-Kandel model, made clear, falsifiable predictions that have stimulated experimental work for many years. Here, we review tests of three predictions of the Kupfermann-Kandel model: (1) that the GWR is a simple, reflexive behaviour graded with stimulus intensity; (2) that central nervous system (CNS) pathways are necessary and sufficient for the GWR; and (3) that activity in six identified GMNs is sufficient to account for the GWR. The available data suggest that (1) a variety of action patterns occur in the context of the GWR; (2) the PVG is not necessary and the diffuse peripheral nervous system (PNS) is sufficient to mediate these action patterns; and (3) the role of any individual GMN in the behaviour varies. Both the control of gill-withdrawal responses, and plasticity in these responses, are broadly distributed across both PNS and CNS pathways. The Kupfermann-Kandel model is inconsistent with the available data and therefore stands rejected. There is, no known causal connection or correlation between the observed plasticity at the identified synapses in this system and behavioural changes during non-associative and associative learning paradigms. Critical examination of these well-studied central pathways suggests that they represent a 'wetware' neural network, architecturally similar to the neural network models of the widely used 'Perceptron' and/or 'Back-propagation' type. Such models may offer a more biologically realistic representation of nervous system organisation than has been thought. In this model, the six parallel GMNs of the CNS correspond to a hidden layer within one module of the gill-control system. That is, the gill-control system appears to be organised as a distributed system with several parallel modules, some of which are neural networks in their own right. A new model is presented here which predicts that the six GMNs serve as components of a 'push-pull' gain control system, along with known but largely unidentified inhibitory motor neurons from the PVG. This 'push-pull' gain control system sets the responsiveness of the peripheral gill motor system. Neither causal nor correlational links between specific forms of neural plasticity and behavioural plasticity have been demonstrated in the GWR model system. However, the GWR model system does provide an opportunity to observe and describe directly the physiological and biochemical mechanisms of distributed representation and parallel processing in a largely identifiable 'wetware' neural network.     

Identification of an abdominal ganglion neuron antagonizing L7-driven gill contraction in Aplysia

Gill motor neuron L7-induced longitudinal shortening of the gill in Aplysia kurodai and A. juliana was suppressed when extracellular stimuli were applied to a restricted dorsal central region of the abdominal ganglion. We found a neuron there which antagonized the L7-driven contraction. Since the contraction was suppressed when the identified neuron was activated simultaneously with L7, we refer to the newly found neuron as “Anti-L7”. Anti-L7 did not change the L7 impulse generation in the abdominal ganglion. No direct synaptic connection from L7 to Anti-L7 was detected. A fluorescent dye injected into the soma of Anti-L7 revealed that the neuron sent axonal branches to the branchial nerve. These results may show that Anti-L7 antagonizes L7 at the periphery inside the gill, rather than in the abdominal ganglion. EJPs induced by L7 were unaffected by Anti-L7. Activation of Anti-L7 alone did not induce any change in tone or membrane potential of the gill musculature. The suppressive effect of Anti-L7 lasts many seconds after the cessation of a train of Anti-L7 impulses. The results may suggest that the suppression is mediated through an inhibitory neuromodulatory mechanism without inhibition of L7 itself. Accepted: 1 April 1999     

Identification, Purification, and Characterization of the Molecular Forms of Aplysia californica Peptidylglycine α-Amidating Enzyme

Abstract: Peptidylglycine α-amidating enzyme (PAM; EC 1.14.17.3) is responsible for the conversion of peptides with a COOH-terminal glycine into α-amidated peptides, a posttranslational modification often required for biological activity and/or increased stability. Such an activity able to convert the model peptide d -Tyr-Val-Gly into d -Tyr-Val-amide was found to be present in the marine mollusk Aplysia californica . Examination of this amidating activity as well as its immunoreactivity demonstrates that (1) it can be found mainly in the atrial gland, heart, and CNS but is barely detectable in the hepatopancreas and gonads, (2) it requires as essential cofactors copper, molecular oxygen, and ascorbate, and (3) it exists in at least two molecular forms, a soluble and a membrane-bound form. Purification of this activity from the atrial gland was accomplished using Cu²⁺-chelating Sepharose, gel permeation, and hydroxyapatite chromatography. In addition, using polyclonal antibodies raised against various parts of the rat amidating enzyme, we demonstrate that numerous immunologically recognized regions are conserved in both the soluble and membrane-bound Aplysia californica PAM.     

Sensitizing stimulation causes a long-term increase in heart rate in Aplysia californica

These studies show that cutaneous stimulation that evoked body wall contraction elicited a concurrent disruption of cardiovascular function. A pinch or test shock to the tail caused a 10- to 30-s increase in diastolic pressure and variability in pulse pressure. Sensitizing cutaneous stimulation which produced enhancement of the tail withdrawal reflex caused no enhancement of the evoked cardiovascular responses. At 20 min post-sensitization training a gradual increase in heart rate was observed and at 60 min post-sensitization training, heart rate was 111 ± 4.3% presensitization values. These long-term changes in cardiovascular function appear to be mediated by the central nervous system. Chemical blockade of conduction at P9 or the pleural-abdominal connectives prevented the sensitization-induced increase in heart rate. Accepted: 21 May 1999     

Development of neurons in the abdominal ganglion of Aplysia californica. I. Axosomatic synaptic contacts.

The development of mariculture techniques for the raising of Aplysia californica in the laboratory from fertilized egg to reproductively mature adult permits the study of the developmental program whereby individual identified neurons in the abdominal ganglion acquire their specific adult properties. In this paper, we describe one of the early steps of this developmental program: the outgrowth of axonal processes by neurons of the abdominal ganglion. Axonal outgrowth is correlated with and may be triggered by the transient appearance of morphologically identifiable axosomatic contacts between the as yet undifferentiated cell body of specific neurons and an axon terminal from an incoming nerve fiber from the pleuroabdominal connective. The evidence that transient axosomatic contacts may signal neuronal differentiation is the following: (1) Axosomatic contacts have not been observed in the abdominal ganglion of adult animals, whereas they are commonly observed during the early stages of development. (2) Cells that receive axosomatic contacts are undifferentiated morphologically and do not as yet have axons. By contrast, cells with axons do not have soma contacts. (3) Individual cells that can be identified from animal to animal in the same and succeeding developmental stages receive axosomatic contacts on similar topographic postions of the cell body at one point in development. Axon outgrowth then occurs at the site of contact. Later in development, with further axon extension, these cells no longer have synaptic contacts on the cell body or axon.     

Cerebrin prohormone processing, distribution and action in Aplysia californica

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.     

Determination of Glutathione and ATP in Ganglia and Individual Neurons of Aplysia californica

The levels of two gamma-glutamyl cycle substrates, glutathione and ATP, were determined in single identified nerve cell bodies from the CNS of Aplysia californica. The glutathione content of single cells averaged 30 +/- 4.9 mumol/g protein. Glutathione levels were similar in identified cholinergic, serotonergic, and histaminergic cells, as well as in neurons whose transmitters are not yet identified. The abdominal rostral white cells, which are enriched in glycine, a component amino acid of glutathione, did not possess distinctively higher glutathione concentrations. The ATP content of single Aplysia nerve cell bodies averaged 15.0 +/- 1.5 mumol/g protein. Despite the vast chemical, anatomical, and functional heterogeneity between Aplysia central neurons, no cells were found that contained unusually high or low ATP levels.     

Development of neurons in the abdominal ganglion of Aplysia californica. II. Nonneural support cells.

The release by nonneural support cells of a diffusable chemical substance into the local environment in which sympathetic neurons develop is thought to play a crucial role in their differentiation. In this paper, we describe a novel class of nonneural support cells associated with a central ganglion of Aplysia californica during the premetamorphic stages of development. These support cells contain secretory granules whose contents are primarily released at metamorphosis. The release of these contents may signal the burst of neuronal growth and maturation that occurs following metamorphosis. The evidence in support of this notion is the following: (1) Spontaneous release of the granule material at metamorphosis coincides with an increase in cell body growth and a more marked increase in the density of synapses of the abdominal ganglion. (2) Premature release of the granule material before metamorphosis with artificial seawater containing a high concentration of potassium results in a burst in cell body growth and a premature increase in synapse density. (3) Premature release of granule material also results in a precocious increase in the number of spines formed and synaptic contacts received by specific identified cells. Based on the findings in this and the preceding paper, we propose a two-stage model of the developmental program for differentiation of neurons in the abdominal ganglion. First, axosomatic contacts trigger axonal outgrowth. Second, material released from the granules of the support cells stimulates further steps in neuronal differentiation, including cell growth, spine development, and synapse formation.     

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.     

D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system

The content, synthesis and transport of d ‐aspartate (d ‐Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser‐induced fluorescence and radionuclide detection. Millimolar concentrations of d ‐Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different d ‐Asp levels; for example, in the F‐ and C‐clusters, up to 85% of the free Asp is present in the d ‐form. Heterogeneous distribution of d ‐Asp is also found in the individual identified neurons tested, including the optical ganglion top‐layer neurons, metacerebral cells, R2 neurons, and F‐, C‐ and G‐cluster neurons. The F‐cluster neurons have the highest percentage of d ‐Asp (～58% of the total Asp), whereas the lowest value of ～8% is found in R2 neurons. In pulse‐chase experiments with radiolabeled d ‐Asp, followed by CE with radionuclide detection, the synthesis of d ‐Asp from l ‐aspartate (l ‐Asp) is confirmed. Is d ‐Asp in the soma, or is it transported to distantly located release sites? d ‐Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both d ‐Asp and l ‐Asp are transported in the pleuroabdominal connectives in a colchicine‐dependent manner, whereas several other amino acids are not. Finally, d ‐Asp produces electrophysiological effects similar to those induced by l ‐Asp. These data are consistent with an active role for d ‐Asp in cell‐to‐cell communication.     

Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles

Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain–Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones’ previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry–based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.     

Ingestion motor programs of Aplysia are modulated by short-term synaptic enhancement in cerebral-buccal interneuron pathways

In the sea slug Aplysia, buccal synapses of cerebral-buccal interneurons (CBIs) CBI-2 and CBI-12 exhibit short-term synaptic enhancement (STE), including frequency-dependant facilitation and augmentation/post-tetanic potentiation (AUG/PTP). The STE that results from driving CBI-2 or CBI-12 is associated with significantly decreased latency to burst onset in buccal premotor neurons and motor neurons, increased cycle frequency of ingestion buccal motor programs (iBMPs) and increased intraburst firing frequency of buccal neurons during iBMPs. Tests of paired-pulse facilitation during AUG/PTP suggest that the locus for this plasticity is presynaptic. The AUG/PTP is not elicited by heterosynaptic pathways, indicating that its origin is homosynaptic. At low CBI-2 and CBI-12 firing frequencies, STE is likely to contribute to iBMP initiation, while at higher firing frequencies, STE is correlated with increased cycle frequency of iBMPs. Thus, STE is an important component of the mechanisms whereby cerebral neurons regulate cyclic feeding programs and likely contributes to observed variations in behavioral responses, including feeding arousal. Electronic Publication     

Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica

Summary Polyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85000 and 67500 molecular weight.     

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica

Abstract: The metabolism of polyamines was investigated by injecting purified [³H]putrescine directly into the soma of the giant neuron R2 of Aplysia . Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [³H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10-⁶ M , metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10⁻⁴ M , relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S -adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of ³H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.