Choline acetyltransferase in individual neurons of Aplysia californica

The activities of choline acetyltransferase in the various ganglia of the nervous system of Aplysia californica and in some of the individually identifiable neurons in these ganglia were measured. At least four of the neurons were characterized by an apparent absence of the enzyme. The neurons containing measurable amounts of the enzyme had reproducible levels from animal to animal. Individual neurons from the same animal were generally characterized by different levels of activity whether expressed on a cell or a protein basis. However, those pairs of neurons previously classified as ‘homologous’ because of their similar appearance, location and/or electrophysiological function, also contained the same total amounts of enzyme activity.     

Acetylcholine suppresses calcium current in neurons of Aplysia californica

1. The left upper quadrant neurons L2-L6 in the abdominal ganglion of Aplysia californica were voltage clamped in order to examine effects of acetylcholine on voltage-dependent Ca and Ca-dependent K currents. 2. "Puffed" application of 10-100 microM acetylcholine reduced both the early inward and late outward phases of the current elicited by depolarizing voltage steps. An identical effect of the peptide FMRFamide was previously found to result from a suppression of the Ca and Ca-dependent K currents. 3. This effect of acetylcholine was obscured by the simultaneous activation of a previously described K current resembling the "S" current. Extracellular tetraethylammonium (TEA) and 4-aminopyridine could not be used to eliminate this current, because both compounds also appeared to block the acetylcholine receptor mediating the putative suppression of Ca and Ca-dependent K currents. 4. The acetylcholine-induced "S"-like and other K currents could, however, be reduced or eliminated by injection of TEA+ or Cs+ into the cell, replacement of extracellular Ca2+ with Ba2+, and by shifting the K+ equilibrium potential so as to null K currents at the potential used to record Ca current, revealing in each case a partial (10-40%) suppression of the Ca (or Ba) current by acetylcholine. 5. The reduction of the outward phase of depolarization-activated current was confirmed to represent suppression of the Ca-dependent K current by acetylcholine. This effect was indirect, secondary to the suppression of Ca current, since acetylcholine had no effect on Ca-dependent K current elicited by direct injection of Ca2+ into the cell. 6. Activation of the "S"-like K current and suppression of the Ca current by FMRFamide are likely to be important in its proposed role as an agent of presynaptic inhibition in Aplysia. Since acetylcholine has identical effects, it too may have such a function.     

Premotor neurons in the feeding system of Aplysia californica

Central pattern generator (CPG) circuits control cyclic motor output underlying rhythmic behaviors. Although there have been extensive behavioral and cellular studies of food-induced feeding arousal as well as satiation in Aplysia, very little is known about the neuronal circuits controlling rhythmic consummatory feeding behavior. However, recent studies have identified premotor neurons that initiate and maintain buccal motor programs underlying ingestion and egestion in Aplysia. Other newly identified neurons receive synaptic input from feeding CPGs and in turn synapse with and control the output of buccal motor neurons. Some of these neurons and their effects within the buccal system are modulated by endogenous neuropeptides. With this information we can begin to understand how neuronal networks control buccal motor output and how their activity is modulated to produce flexibility in observed feeding behavior.     

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.     

Age-dependent behavioral changes and physiological changes in identified neurons in Aplysia californica

The gill withdrawal reflex (GWR) to direct gill stimulation was studied in sexually mature Aplysia and in those older by at least two months. The GWR threshold in old Aplysia was five- to sevenfold higher than that in mature animals. In the habituation paradigm, the GWR amplitude decremented rapidly to zero in old animals whereas in mature animals it persisted for at least ten trials. The GWR could not be dishabituated in old animals. The GWR is an age-dependent behavior in that parieto-visceral ganglion suppression of the GWR appears to increase with age. Also the electrophysiological properties of two neurons in the parieto-visceral ganglion were compared in the two age groups: L₇ a neuron which dishabituates the GWR in mature and not in old animals; and R₂ which manifests cytological changes with age. In old animals L₇′s input resistance was lower, the time constant was increased, and the size of the psp evoked by gill stimulation was smaller than those of mature L₇s. Similar membrane changes with age were measured in R₂. Soma size of L₇ was approximately the same in the two age groups as was that of R₂. The physiological parameters of neurons of known function continue to change during postmetamorphic life of Aplysia.     

Precursor and product processing in the bag cell neurons of Aplysia californica

Posttranslational processing in the biosynthesis of the egg-laying hormone (ELH) by the bag cell neurons of Aplysia californica was studied. The precursor (pro-ELH) to ELH was found to be resistant to solubilization in denaturant-free media throughout its lifetime. Its principle products show a similar insolubility for 3 h, but two of these, ca. 6,000 daltons, subsequently become readily recoverable in the low-speed supernatant of a homogenate of the cells. The remaining product shows no change in solubility characteristics. From studies employing ultracentrifugation and examination of axoplasmic transport, the solubility shift for the lower molecular weight products is interpreted to represent the liberation of secretory vesicles into the cytoplasm from larger membranous associations. This event is accompanied by, but does appear to be dependent upon, a 15% reduction in the molecular weight of one of the products. These findings are considered in the light of the extensively studied posttranslational processing regimen for the production of insulin in the pancreatic beta cell.     

Specific, water-soluble polypeptides in identified neurons of Aplysia californica.

Application of an ethylene glycol lysis technique to extract water-soluble, low molecular weight polypeptides in Aplysia neurons, was used in conjunction with microgradient gel electrophoresis and micro-isoelectric focusing, to identify unique polypeptides in specific, identified neurons. The polypeptides found in neurons R15, R3-13, R14, and the bag cells were particularly abundant, consistent with the previously suggested neurosecretory role for these cells. Water extraction of the strongly basic polypeptides (pI 10.7) in R3-13 and R14 required an acidic lysis medium.     

Activation of cerebral neurons by static nerve inputs in Aplysia californica

A number of cerebral B-neurons of Aplysia californica were activated by tactile stimulation of the statocysts and by electrical stimulation of the static nerve. Types of responses recorded included antidromic spike, monosynaptic EPSP with or without spike and polysynaptic EPSPs. Some of the B-neurons were inhibited by trains of electrical stimulation of the static nerve. Hyperpolarization was mostly preceded by a short increase of spiking, usually during stimulation. Some A-neurons also responded with inhibition. The statocyst nerve contained axons carrying information not only from the statocysts to the cerebral ganglion but also from the cerebral ganglion to the statocyst. The latter pathway could be activated by tactile stimulation of the tentacles. Activation of either static nerves resulted in the increase of activity of the other static nerve through the cerebral ganglion, suggesting interaction between the two statocysts.     

Characterization of protein-linked glycoconjugates produced by identified neurons of Aplysia californica

The biosynthetic capabilities of individual neurons of the abdominal ganglion of the marine mollusc Aplysia californica have been analyzed after intrasomatic injection of 3H-monosaccharides. Glycopeptides prepared from the metabolically labeled cells were fractionated using serial lectin affinity and gel filtration chromatography. The fractionation procedure yielded eight populations of glycopeptides, and comparison of two different neurons (R2 and R14) showed that the quantity of the individual species produced is cell-dependent. Structural analysis indicated that the glycoconjugates produced by the Aplysia neuron constitute both O- and N-linked structures as well as an unusual class of oligosaccharide whose linkage to protein is unknown. The O-linked units are small and consist only of N-acetylglucosamine or N-acetylgalactosamine attached to protein. High-mannose-type asparagine-linked units are produced by the neurons, and some of these appear to be processed to biantennary complex-type units that bind to lentil lectin-agarose. Overall, although the Aplysia neurons produce oligosaccharides of a nature similar to that produced by higher eucaryotes, the N- and O-linked structures produced by the neurons do not achieve the complexity of the comparable structures produced by mammalian cells. The results provide a basis for further studies aimed at understanding the role of glycoconjugates in the development of the nervous system.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 or 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

Circadian organization in Aplysia californica.

Substantial progress has been made in unraveling the organization of the circadian system of Aplysia californica. There are at least three circadian pacemakers in Aplysia. One has been localized in each eye and a third lies outside the eyes. Removal of the eyes disrupts the free-running locomotor activity rhythm; however, an extraocular oscillator can mediate a free-running rhythm in some eyeless animals. Although photoreceptors sufficient for entrainment of the ocular oscillator have been localized in the retina, photoreceptors outside the eyes are capable of "driving" a diurnal rhythm of locomotor activity and may also influence entrainment of ocular pacemakers. Finally, attention has been focused on the optic nerve as a coupling pathway between various parts of the system. The evidence suggests that information transmitted in the optic nerves is involved in entrainment of the ocular pacemaker by light, and in ocular control of the locomotor activity rhythm.     

Regional differences in processing of locally translated prohormone in peptidergic neurons of Aplysia californica

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.     

Biosynthesis and processing of presumed neurosecretory proteins in single identified neurons of Aplysia californica.

The biosynthesis and processing of low molecular weight protein (presumed neurosecretory protein) in cells R15, R14 and L11 of Aplysia californica was studied at high resolution by polyacrylamide slab gel electrophoresis in sodium dodecylsulfate. The number of low molecular weight proteins detected in each cell ranges from 3 in R14 and L11 to 5 to 6 in R15. In each of the cells studied, the low molecular weight protein consists of a primary precursor of ca. 12,000 daltons, and its proteolytic processing products. In each cell, the smallest protein, or in the case of R14, one of the two smallest proteins, accumulates to a significant extent, suggesting that it might correspond to a final processed neurohormone. In cell R15, the biosynthesis of the primary precursor and its subsequent processing to smaller peptides is largely unaffected by removal of extracellular calcium, by replacement of calcium with cobalt or by inhibition of spontaneous bursting via stimulation of the brachial nerve.     

Batrachotoxin inhibits axonal transport without affecting membrane potential in single neurons of Aplysia californica

Intrasomatic injection of 0.25 ng of batrachotoxin (BTX) caused no depolarization of giant cerebral neurons (GCN) of Aplysia californica within 4 hr at 23° C. The amplitude of action potential and the input resistance were slightly and reversibly decreased. The same dose of BTX caused a 68% inhibition of fast axonal transport.     

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.     

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.