CHEMICAL, ENZYMATIC AND ULTRASTRUCTURAL CHARACTERIZATION OF 5-HYDROXYTRYPTAMINE-CONTAINING NEURONS FROM THE GANGLIA OF APLYSIA CALIFORNICA AND TRITONIA DIOMEDIA

Abstract— Several identified neurons in Aplysia and Tritonia ganglia were shown to contain measurable quantities (4–6 pmol/cell body) of 5-hydroxytryptamine (5-HT). A metabolic correlate for the limited distribution of 5-HT among the neurons of Tritonia is provided by the finding that the enzyme, aromatic acid decarboxylase (AAD), is 500 times more active in nerve cells containing 5-HT than in neurons devoid of the amine. Although all Aplysia neurons have some AAD activity, 5-HT cell bodies in this species are 10-fold more active than cell bodies which do not contain 5-HT. The cytoplasm of 5-HT cell bodies in Aplysia and Tritonia characteristically contains granules that have minimum diameters of approx. 1000 Å and eccentric opaque cores. This type of granule was not found in somata which did not contain measurable 5-HT. These data illustrate the metabolic and morphological specialization in 5-HT-containing neurons of molluscs.     

THE CHARACTERIZATION OF HISTIDINE DECARBOXYLASE AND ITS DISTRIBUTION IN NERVES, GANGLIA AND IN SINGLE NEURONAL CELL BODIES FROM THE CNS OF APLYSIA CALIFORNICA

Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C-2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ-cultured in the presence of [³H]histidine showed that every neuron accumulated labelled histidine, but only the HA-containing C-2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α-hydrazino-histidine but unaffected by concentrations of α-methyldopa or by 5-(3,4-dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15-fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C-2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA-containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA-rich neurons in Aplysia.     

Serotonin induces selective cleavage of the PKA RI subunit but not RII subunit in Aplysia neurons

PKA type I and type II are activated in Aplysia neurons by stimulation with serotonin (5-HT), which causes long-term facilitation (LTF). The proteolysis of the regulatory subunit (R) is thought important for the persistent activation of PKA, which is necessary to produce LTF. In this study, we report that the type I regulatory subunit (RI) and type II regulatory subunit (RII) are differentially regulated by proteolytic cleavage. RI, but not RII, was selectively cleaved after 5-HT treatment for 2h in Aplysia neurons. Interestingly, the proteasome inhibitor MG132 inhibited the cleavage of RI caused by 5-HT treatment in Aplysia neuron. Besides extracts from Aplysia ganglia treated with 5-HT cleaved (35)S-labeled RI synthesized in vitro, but not (35)S-labeled RII. This suggests that 5-HT induces the activation state of RI-specific proteolytic cleavage.     

Coexistence of FMRFamide, met-enkephalin and serotonin in molluscan neurons

1. Coexistence of FMRFamide, met-enkephalin and serotonin immunoreactivities was examined in Achatina fulica and Aplysia kurodai. 2. Coexistence of FMRFamide and serotonin was found in some neurons of the visceral, right parietal and pedal ganglia of Achatina fulica, and in the pedal ganglion of Aplysia kurodai. 3. In Achatina fulica, coexistence of FMRFamide and met-enkephalin was found in a neuron of the left parietal ganglion and that of met-enkephalin and serotonin was found in a giant neuron of the right parietal ganglion. 4. Based on these results, the biological significance of coexistence was discussed.     

Target-dependent morphological segregation of Aplysia sensory outgrowth in vitro.

The adult nervous system is characterized by partial or complete morphological segregation of terminals from different afferent neurons innervating the same postsynaptic target. This segregation is thought to result, in part, from competition between the afferent terminals. To explore the role of the target cell in the spatial distribution of presynaptic inputs, the sensory neurons of Aplysia were cultured either with or without a common target motor neuron. In the presence of a common target, the outgrowth from two different sensory neurons tends to occupy separate postsynaptic regions. When cultured without a target motor neuron, processes from different sensory neurons do not segregate, but rather grow freely along one another. Thus, morphological segregation of sensory outgrowth requires interaction with a target neuron and may reflect competition between presynaptic terminals for a limited number of synaptic sites on the motor neuron, or for a postsynaptic trophic factor.     

Modulation of a feeding neural circuit by microinjection of K+ channel expression genes into a single identified neuron in Aplysia kurodai

In Aplysia buccal ganglion expression genes for voltage-dependent K(+) channels (AKv1.1a) were injected into one of four electrically coupled multi-action (MA) neurons that directly inhibit jaw-closing (JC) motor neurons and may cooperatively generate their firing pattern during the feeding response. Following the DNA injection, the firing threshold increased and the spike frequency at the same current decreased in the current-induced excitation of the MA neuron; indicating a decrease in excitability of the MA neuron. This procedure also reduced the firing activity of MA neurons during the feeding-like rhythmic responses induced by the electrical nerve stimulation. Moreover, the firing pattern in JC motor neurons was remarkably changed, suggesting the effective contribution of a single MA neuron or electrically coupled MA neurons to the generation of the firing pattern in the JC motor neurons. This method appears useful for exploring the functional roles of specific neurons in complex neural circuits.     

Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process

1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.     

The metabolism of gamma aminobutyric acid in the lobster nervous system. Enzymes in single excitatory and inhibitory axons

γ-aminobutyric acid (GABA) is the inhibitory transmitter compound at the lobster neuromuscular junction. This paper presents a comparison of the enzymes of GABA metabolism in single identified inhibitory and excitatory axons from lobster walking legs. Inhibitory axons contain more than 100 times as much glutamic decarboxylase activity as do excitatory axons. GABA-glutamic transaminase is found in both excitatory and inhibitory axons, but about 50% more enzyme is present in inhibitory axons. The kinetic and electrophoretic behavior of the transaminase activity in excitatory and inhibitory axons is similar. Succinic semialdehyde dehydrogenase is found in both axon types, as is an unknown enzyme which converts a contaminant in radioactive glutamic acid to GABA. In lobster inhibitory neurons, therefore, the ability to accumulate GABA ultimately rests on the ability of the neuron to accumulate the enzyme glutamic decarboxylase.     

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.     

Alternative splicing in individual Aplysia neurons generates neuropeptide diversity

The neuron R15 is a peptidergic cell within the abdominal ganglion of Aplysia that participates in two neural circuits governing physiological and behavioral programs. We have cloned and characterized the major gene product expressed in this neuron. The R15 cDNA encodes a polyprotein precursor that is cleaved to yield a set of small neuropeptides. One peptide, R15 alpha 1, may act on different target cells to generate distinct but complementary physiological alterations that contribute to a program of cardiovascular changes in Aplysia. We have found that the RNA encoding the R15 polyprotein is spliced differently in different neurons. Our results suggest that alternative splicing of RNAs encoding polyproteins may provide a mechanism to generate distinct but overlapping sets of peptides that govern distinct but related physiological or behavioral programs.     

Stretch activation of the Aplysia S-channel

The S-channel, a receptor-mediated K+ channel of Aplysia sensory neurons which functions in neuromodulation, bears a strong resemblance to the ubiquitous stretch-activated channels of snail neurons. Snail neuron stretch channels are stretch sensitive only in the patch, not at the macroscopic level, a situation which leaves open the question of their physiological role. If S-channels resemble snail stretch channels because both belong to the same general class of channels, the S-channel, too, should display stretch sensitivity in the patch. We show, using single-channel recording, that the S-channel can be activated by stretch. Furthermore, we show that Aplysia neurons in general have stretch-activated K+ channels. We suggest that the stretch-sensitive K+ channels of molluscan neurons and other preparations (e.g., Drosophila muscle, snail heart) are S-like channels, i.e., receptor-mediated channels which adventitiously exhibit mechanosensitivity in the patch.     

Transfer of habituation in Aplysia: contribution of heterosynaptic pathways in habituation of the gill-withdrawal reflex

Habituation of the Aplysia gill-withdrawal reflex (and siphon-withdrawal reflex) has been attributed to low-frequency homosynaptic depression at central sensory-motor synapses. The recent demonstration that transfer of habituation between stimulation sites occurs in this model system has prompted the hypothesis that heterosynaptic inhibitory pathways also play a role in the mediation of habituation behavior. To test this hypothesis, the sites and mechanisms of neural plasticity which underlie transfer of habituation in Aplysia were examined. Transfer of habituation is a reduction in the reflex evoked at one stimulation site (siphon) due to repeated presentation of a stimulus to a second site (gill). Centrally mediated transfer of habituation, measured in a preparation lacking the siphon-gill peripheral nervous system (PNS), was associated with a reduced excitatory response in central motor neurons. Repeated tactile stimulation of the gill did not attenuate the gill response evoked by electrical stimulation of the branchial nerve nor the mechanoreceptor response recorded in LE sensory neurons. In contrast, repeated stimulation of siphon or gill at a site which was "off" the sensory field of a specific mechanoreceptor led to a diminution in synaptic transmission between that sensory neuron and its followers (motor neurons and inter-neurons). These data demonstrate that centrally mediated transfer of habituation results from heterosynaptic modulation of synaptic transmission at the sensory-motor (and sensory-interneuron) synapses. Therefore, habituation behavior in Aplysia is mediated through the conjoint action of homosynaptic and heterosynaptic inhibitory processes.     

Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro

To determine the influence that an appropriate target cell has on the axonal structure of a presynaptic neuron in vivo, we examined the morphologies of individual Aplysia sensory neurons in dissociated cell culture in the presence or absence of identified target motor neurons. We find that an appropriate target, the motor cell L7, regulates the morphological differentiation of the presynaptic sensory neurons in two ways: the target induces the axons of the sensory neurons to develop a more elaborate structure and to form active zones, and the target guides the outgrowth of the sensory neurons. The influence of the appropriate target, L7, on the morphological differentiation of sensory neurons appears to be related to the formation of chemical synaptic connections between the sensory neurons and L7, since sensory neurons co-cultured with an inappropriate target motor neuron do not exhibit a comparable elaboration of their axonal processes.     

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.     

Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.     

A Qualitative Assessment of Neuron Populations Expressing the Enzymes of Dopamine Synthesis in the Rat Accurate Nucleus during Ontogeny

The ratio of neuron populations expressing either tyrosine hydroxylase or aromatic L-amino acid decarboxylase, which are enzymes of dopamine synthesis, was estimated quantitatively in the accurate nucleus of male and female rats on the 21st day of intrauterine development, the 9th day of postnatal development, and in adult animals. The enzymes in neurons were revealed by double immunocytochemical labeling, followed by identification under a fluorescence microscope. At all the developmental stages, three neuron populations differing in the expression of these enzymes were revealed. By the end of the prenatal period, most of the neurons (99%) contained only one of the enzymes, and the proportion of neurons expressing both enzymes (dopaminergic neurons) did not exceed 1%. During postnatal development, the proportion of neurons with one enzyme proved to decrease, whereas that of dopaminergic neurons increased. However, the latter proportion, even in adult animals, did not exceed 50% of the total number of neurons expressing the enzymes of dopamine synthesis. Thus, the population of neurons expressing both enzymes increases during rat ontogeny, whereas the number of neurons expressing only one enzyme decreases.     

Structure, bioactivity, and cellular localization of myomodulin B: a novel Aplysia peptide.

Important insights into mechanisms by which neuromuscular activity can be modulated have been gained by the study of experimentally advantageous preparations such as the ARC neuromuscular system of Aplysia. Previous studies have indicated that one source of modulatory input to the ARC muscle is its own two motor neurons, B15 and B16. Both of these neurons synthesize multiple peptide cotransmitters in addition to their primary neurotransmitter acetylcholine (ACh). Peptides present in the ARC motor neurons include SCPA, SCPB, buccalin A and B, and myomodulin A. We have now purified a novel neuropeptide, myomodulin B, which is structurally similar to myomodulin A. Myomodulin B is present in two identified Aplysia neurons that contain myomodulin A; the ARC motor neuron B16 and the abdominal neuron L10. Ratios of myomodulin A to myomodulin B are approximately 6:1 in both cells. Like myomodulin A, myomodulin B potentiates ARC neuromuscular activity; it acts postsynaptically, and increases the size and relaxation rate of muscle contractions elicited either by motor neuron stimulation or by direct application of ACh to the ARC. When myomodulin A is applied to the ARC in high doses (e.g., at about 10(-7) M), it decreases the size of motor neuron-elicited muscle contractions. This inhibitory effect is never seen with myomodulin B. Thus, despite the structural similarity between the two myomodulins, there exists what may be an important difference in their bioactivity.     

Conopressin G, a molluscan vasopressin-like peptide, alters gill behaviors in Aplysia.

Superfusion of an invertebrate vasopressin structural analogue, conopressin G, over the abdominal ganglion of an in vitro preparation of Aplysia californica has significant neurophysiological and behavioral effects. Both the amplitude of the siphon-evoked gill withdrawal reflux and concomitant activity in gill motor neurons are reduced in the presence of conopressin G. Moreover, the frequency of spontaneous gill movements and their neural correlate, interneuron II activity, are increased. These behavioral modifications strongly resemble those that occur during the food-aroused behavioral state in intact Aplysia. In addition, conopressin G superfusion reduces both the excitability of gill motor neurons and the strength of gill contractions in response to gill motor neuron discharges elicited by direct depolarizing current. A role for conopressin G or a similar peptide in the modulation of gill behaviors associated with the food-aroused state is suggested.     

Neural mechanism generating firing patterns in jaw motoneurons during the food-induced response in Aplysia kurodai

1. In each right and left buccal ganglia of Aplysia kurodai, we identified 4 premotor neurons impinging on the ipsilateral jaw-closing and -opening motoneurons. Three of them (MA1 neurons) had features of multifunctional neurons. Current-induced spikes in the MA1 neurons produced excitatory junction potentials (EJPs) in the buccal muscle fibers. In addition, tactile stimulation of the buccal muscle surface produced a train of spikes in the MA1 neurons without synaptic input. The other neuron (MA2) had only a premotor function. 2. The MA1 and MA2 neurons had similar synaptic effects on the jaw-closing and -opening motoneurons. Current-induced spikes in the premotor neurons gave rise to monosynaptic inhibitory postsynaptic potentials (IPSPs) in the ipsilateral jaw-closing motoneurons. Simultaneously, spikes in one of the MA1 neurons and the MA2 also gave rise to monosynaptic excitatory postsynaptic potentials (EPSPs) in the ipsilateral jaw-opening motoneuron. 3. The IPSPs and the EPSPs induced by spikes in the premotor neurons were reversibly blocked by d-tubocurarine and hexamethonium, respectively, suggesting that the MA1 and MA2 neurons are cholinergic. 4. When depolarizing and hyperpolarizing current pulses were passed into one premotor neuron, attenuated but similar potential changes were produced in another randomly selected premotor neuron in the same ganglion, suggesting that they are electronically coupled.