Synaptic actions of identified peptidergic neuron R15 in Aplysia. II. Contraction of pleuroabdominal connectives mediated by motoneuron L7

The purpose of this study was to determine the synaptic actions of the bursting peptidergic neuron R15 in Aplysia. R15 is known to be excited by the neuroendocrine bag cells, which trigger egg laying. In the two companion papers, we show that R15 mediates some of the effects of the bag cells on respiratory and reproductive organs. In this paper, we demonstrate that R15 excites L7, a multimodal motoneuron located in the abdominal ganglion. Although L7 excites several types of muscle fibers as well as neurons, the excitation of L7 by R15 is probably strong enough to cause contraction only of the sheath muscle of the pleuroabdominal connectives, which has an exceptionally low threshold for activation. The excitatory actions of R15 on L7, which desensitize profoundly, appear to be mediated by R15 alpha 1 peptide. The synaptic action of R15 on L7 and on the respiratory pumping system (Alevizos et al., 1991a) can be fully expressed only if R15 is first silenced for 2 hr by injection of hyperpolarizing current. A similar protocol for eliminating desensitization may prove to be generally useful for revealing the synaptic actions of other spontaneously active neurons that have rapidly desensitizing postsynaptic actions.     

Localization of catecholamines in the buccal ganglia of Aplysia californica

The distribution of catecholamines and serotonin in the buccal ganglia and buccal nerves of Aplysia californica was examined using glyoxylic acid-induced histofluorescence. Three identifiable, medium-sized cell bodies, two paired and one unpaired, and 5 smaller cells fluoresced blue-green indicating the presence of catecholamines. Numerous fluorescent axons were observed in the neuropil and peripheral nerves, including a network of catecholaminergic processes emerging from the esophageal nerve and surrounding the base of the esophagus. The presence of catecholaminergic cells and processes in the buccal system suggests that these transmitters are used in the control of feeding behavior and digestion.     

Characterization of glycolipids synthesized in an identified neuron of Aplysia californica

Because radioactive precursors can be injected directly into the cell body or axon of R2, a giant, identified neuron of the Aplysia abdominal ganglion, it was possible to show that glycolipid is synthesized in the cell body, inserted into membranes along with glycoprotein, and then exported into the axon within organelles that are moved by fast axonal transport. After intrasomatic injection of N-[3H]-acetyl-D-galactosamine, five major 3H-glycolipids were identified using thin layer polysilicic acid glass fiber chromatography. At least two of the lipids are negatively charged. Analysis of 32P-labeled lipid from the abdominal ganglion revealed the presence of 2-aminoethylphosphonate, indicating that these polar substances are sphingophosphonoglycolipids. The major 3H-glycolipids synthesized in R2 are similar to a family of phospholipids isolated from the skin of A. kurodai, previously characterized by Araki et al. (Araki, S., Y. Komai, and M. Satake (1980) Biochem J. 87: 503-510). Since sialic acid is absent in Aplysia as in other invertebrates, these polar glycolipids may function like gangliosides in vertebrates. The polar 3H-glycolipids are synthesized and incorporated into intracytoplasmic membranes solely in the cell body. Direct injection of the labeled sugar into the axon revealed no local synthesis or exchange of glycolipid. Moreover, there was no indication for transfer from glial cells into axoplasm. Although the incorporation of N-[3H]-acetyl-D-galactosamine into glycolipid is not affected by anisomycin, an effective inhibitor of protein synthesis, the export into the axon of membranes containing the newly synthesized lipid is completely blocked by the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of an identified serotonergic neuron in free moving Aplysia correlates with behavioral arousal

Extracellular recordings of the metacerebral cell (MCC), a serotonergic neuron inAplysia, were obtained in free moving, undrugged animals. MCC activity was evoked by exposure to food. Arousal level was manipulated by satiating the animals or exposing them to a noxious stimulus. We found that the amount of evoked MCC activity correlated with the level of arousal of the animal.     

Synaptic actions of identified peptidergic neuron R15 in Aplysia. I. Activation of respiratory pumping

The purpose of the study described in this and the following two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 increases the frequency of bursting in the R25/L25 network that triggers respiratory pumping. This excitatory modulatory effect appears to be mediated by R15 alpha 1 peptide. R15 activates both strong and weak modes of respiratory pumping. In contrast, the two R20 cells, which are thought to use the neuropeptides SCPA and SCPB as transmitters, elicit only strong episodes of respiratory pumping. The synaptic actions of R15 also differ from those of the R20 cells in being longer lasting and in exhibiting profound desensitization. Chronic recording of R15 activity in vivo indicates that it does not burst spontaneously in the intact animal, so the synaptic actions of R15 are not chronically desensitized. The neuroendocrine bag cells, which initiate egg laying, had been shown by others to excite R15 and the R25/L25 network that triggers respiratory pumping. Our data indicate that the excitatory effects of the bag cells on the R25/L25 cells are mediated in part by R15.     

Synaptic actions of identified peptidergic neuron R15 in Aplysia. III. Activation of the large hermaphroditic duct

The purpose of the study described in this and the preceding two companion papers was to determine the synaptic actions of neuron R15, an endogenously bursting neurosecretory cell in Aplysia, as a step toward determining its physiological function. The results described in this paper demonstrate that activity in R15 activates anterograde peristaltic movements in the segment of the large hermaphroditic duct through which eggs move during egg-laying behavior. This action is mimicked by R15 alpha 1 peptide, a putative transmitter of R15. The neuroendocrine bag cells, which initiate egg laying when they fire in a population burst, have been shown by others to excite R15. Our data suggest that R15 mediates excitatory effects of the bag cells on the large hermaphroditic duct. Taken with the results of the two companion papers, these data support the hypothesis that R15 integrates various aspects of egg-laying behavior. The desensitization of R15's postsynaptic actions may complement the long-lasting refractoriness of the bag cells described by others, with both effects contributing to the episodic nature of egg laying.     

Movement of newly synthesized membrane by fast transport along the axon of an identified Aplysia neuron

In order to ascertain the form in which newly synthesized membrane is moved by fast axonal transport, we examined the distribution of label along an axon of the identified giant serotoninergic neuron (GCN) in the cerebral ganglion of Aplysia californica. Membrane glycoproteins were labeled by intrasomatic injection of 3H-fucose, and segments of GCN's axon in the posterior lip nerve containing the transported organelles were examined at 1, 5, 15, and 24 hours by quantitative electron-microscopic autoradiography. To show that membrane which is rapidly transported is contained only in discrete organelles rather than in continuous sheets of axoplasmic reticulum, we systematically varied conditions of fixation. We found that we could distinguish vesicles from axoplasmic reticulum most reliably in axons fixed with 2% formaldehyde and 2% glutaraldehyde in cacodylate buffer. At short times after intrasomatic injection of 3H-fucose, dense-cored vesicles and multivesicular tubules were the only axonal organelles labeled.     

Modulatory synaptic actions of an identified histaminergic neuron on the serotonergic metacerebral cell of Aplysia

Possible sources of excitatory synaptic input to the serotonergic metacerebral cell (MCC) were determined by stimulating various neurons in the cerebral ganglion. Firing of the previously identified histaminergic neuron C2 was found to produce synaptic input to the MCC. The synaptic input consists of fast excitatory-inhibitory synaptic potentials on a background of a slow EPSP. The slow EPSP appears to be monosynaptic and chemically mediated since it persists in a solution of high divalent cations; broadening of the presynaptic spike enhances the EPSP; the size of the EPSP is a function of the Mg2+ and Ca2+ concentrations of the bathing solution; and the EPSP can be mimicked by application of histamine to the MCC. The slow EPSP, in addition to firing the MCC, can increase the excitability of the cell, even under conditions in which C2 is fired at a rate too slow to produce a measurable EPSP when the MCC is at rest potential. This property appears to be due to the fact that the slow EPSP results from an apparent decrease of membrane conductance so that the size of the EPSP increases markedly as the cell is depolarized, and the EPSP appears to be highly voltage-dependent so that it is small or absent close to the rest potential of the MCC. When the MCC is voltage-clamped, application of histamine to the bath results in an inward current that disappears when the MCC is hyperpolarized. The potential at which the histamine-induced current reverses or disappears is dependent on the concentration of external potassium, suggesting that, at least in part, the slow EPSP is due to a decrease of potassium conductance. The data on C2 are consistent with its being an element of the neuronal system that mediates a state of food arousal in Aplysia.     

Isolation of a peptide initiating burst activity in an identified neuron of Helix pomatia

A peptide was isolated from the water-soluble fraction of brain extract of the snail (Helix pomatia). The peptide initiated or enhanced the bursting activity of the identified neuron RPa1. The effect of the peptide application was similar to the effect of the total water-soluble fraction. The action of the peptide on neuronal bursting activity was not identical to the action of other known peptides. It is suggested that under normal conditions the peptide is released from the presynaptic terminals of a nonidentified interneuron on the soma of RPa1 neuron initiating its bursting activity.     

Synaptic connections of buccal mechanosensory neurons in the opisthobranch mollusc, Navanax inermis

Mechanical stimulation of various areas of the pharyngeal wall and lips can produce EPSPs and IPSPs, as well as abruptly rising impulses, in primary sensory cells. IPSP fields are generally larger than EPSP fields and these fields are distributed without obvious order around fields from which afferent spikes are evoked. Apparently monosynaptic excitatory and inhibitory contacts are formed between primary sensory neurons. These synapses are blocked by high Mg2+ indicating chemical transmission. IPSPs are inverted by Cl- injection. Excitatory inputs can be electrically far from the soma. Sensory cells form apparently monosynsptic excitatory or inhibitory contacts on motoneurons mediating pharyngeal expansion. Brief sensory excitation can initiate sustained firing within this neuronal population and sustained synaptic activity in motoneurons. Interactions of sensory neurons may be important in information processing and in generating motor paterns. These neurons serve both primary sensory and interneuronal functions.     

Proteins rapidly transported to the synapses of a single identified neuron of Aplysia californica

The cell body of R2, a giant cholinergic neuron of Aplysia californica, resides in the abdominal ganglion, whereas its synapses are on thousands of unicellular mucus glands located in the skin. Due to the great spatial separation between the site of macromolecular synthesis and the presynaptic terminals, rapid axonal transport can be used to segregate synaptic proteins from those to be used elsewhere in the cell. The proteins of R2 were labeled by incubating the abdominal ganglion in [35S]methionine for 5 hr in a chamber separated from the rest of the isolated central nervous system. After 50 hr, 28 radiolabeled proteins were reproducibly found by one- and two-dimensional polyacrylamide gel electrophoresis to be transported to the distal regions of peripheral nerves P6, P7, and P8 that innervate the parapodia and middle body wall. We are sure that R2 is the source of these proteins since radioautography of sections taken throughout the nervous system, complemented by cobalt tracings, showed that R2 is the only neuron in the abdominal ganglion with axons in these nerves. Nine of the 28 transported proteins are glycoproteins since they were also labeled after injecting R2's cell body with [3H]-L-fucose. There is evidence that the proteins and glycoproteins are destined for R2's presynaptic terminals. For example, in experiments in which the body wall and parapodium remained attached to the nerves, the proteins were transported to the skin region that contains the glands. Moreover, analyses of the distribution of the rapidly transported proteins by qualitative radioautography and by extrusion of axoplasm indicated that none are constituents of the axolemma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of a voltage-dependent sodium current by GABA (identified neurons, buccal ganglia, Helix pomatia

The action of systemically applied GABA on voltage-dependent currents of the identified neurons B1–B4 in the buccal ganglia of Helix pomatia were investigated by conventional voltage clamp techniques. In the B4 neuron, superfusion with sodium-free solution or addition of tetrodotoxin to the bath medium abolished the voltage dependent inward current. This voltage dependent sodium current was reduced with GABA application. Muscimol exerted the same effect as GABA whereas administration of baclofen had no effect. Voltage-dependent sodium and calcium currents of the neurons B1–B3 remained unchanged with GABA application. It is concluded that GABA is capable of reducing voltage-dependent sodium currents of distinct neuronal individuals via a GABA_A receptor-like structure.     

Synaptic connections of a low-threshold mechanoreceptive primary neuron within the trigeminal subnucleus oralis.

The central axon of a primary afferent neuron that responded to indentation of the glabrous skin of the lower lip in a slowly adapting fashion was intra-axonally injected with horseradish peroxidase. The labeled terminal within the subnucleus oralis was examined electron microscopically. The labeled ending had a pale axoplasm and contained clear spherical synaptic vesicles. The labeled ending formed a synaptic triad with a dendrite and an unlabeled axonal ending with pleomorphic vesicles (a mixture of oval, flattened and dense core vesicles). The labeled primary ending was presynaptic only to the dendrite, while the unlabeled ending was presynaptic to both the dendrite and the labeled primary ending.     

Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15

Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rate of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.     

Ionic mechanism of a hyperpolarizing glutamate effect on two identified neurons in the buccal ganglion of Aplysia

The ionic mechanism of a membrane effect of L-glutamate on two identified neurons in the buccal ganglion of Aplysia kurodai was investigated with conventional microelectrode techniques and glutamate iontophoresis. Bath-applied and iontophoresed glutamate hyperpolarized the membrane and increased the membrane conductance. The hyperpolarizing glutamate response decreased in amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of the hyperpolarizing glutamate response was close to the ECl (-60 mV). The reversal potential changed by 22.4 mV when the external chloride concentration was altered by a factor of 5. The relationship between the iontophoretically applied current and the membrane conductance changes was suggestive of two glutamate molecules reacting with a single receptor site. The hyperpolarizing glutamate response was essentially unaffected by 2-amino-4-phosphonobutyric acid (2-APB), L-proline, and quinuclidinyl benzilate (QNB). It was concluded that the hyperpolarizing glutamate response was generated by an activation of Cl- conductance.     

Synaptic connections of a periodontal primary afferent neuron within the subnucleus oralis of the cat

The central axon of a primary afferent neuron that responded to light mechanical stimulation of the lower premolar teeth in a fast adapting fashion was intra-axonally injected with horseradish peroxidase in the cat. The labeled terminals within the rostrodorsomedial (Vo.r) and dorsomedial (Vo.dm) parts of subnucleus oralis were examined electron microscopically. The labeled ending had pale axoplasm, contained clear spherical synaptic vesicles, and formed multiple synapses with dendrites and/or unlabeled axonal endings with pleomorphic vesicles (P-endings). In these synaptic contacts, the labeled primary ending was presynaptic to dendrites and postsynaptic to P-endings. Labeled endings simultaneously synapsing with both dendrites and P-endings were more frequent in Vo.dm (28%) than in Vo.r (8.3%).     

Identification and characterization of neurons initiating patterned neural activity in the buccal ganglia of Aplysia

Two patterns of neural activity were identified in excised buccal ganglia of Aplysia californica. Both are expressed in many cells, and each can be expressed independently. Using cells B4 and B5 as monitors of the activity patterns, we searched the buccal ganglia for cells initiating the patterns. Two electrically coupled cells, B31 and B32, can initiate what we termed pattern 2. The cells are active before pattern 2 is expressed. Stimuli initiating pattern 2 excite B31/B32. Depolarizing B31/B32 induces the pattern, while hyperpolarizing them can prevent its expression. The cells have unusual features. Their somata do not sustain conventional action potentials, and depolarization causes a regenerative response. B33 differs from B31/B32 in that its soma sustains conventional action potentials but otherwise has similar features. B34 also seems to be inexcitable but has weaker synaptic input than B31/B32 and appears unable to induce pattern 2. B35 and B36 have prominent regenerative capabilities. B35 is also able to initiate pattern 2. B37 is presynaptic to B31/B32 and can initiate pattern 2 via its effects on them. The newly identified cells provide a starting point for investigating factors that initiate and control different patterns of neural activity in the buccal ganglia. Since the buccal ganglia are involved in generating feeding behavior, further studies on the newly identified cells may provide insights into the neural control of feeding behavior, and provide a neural substrate for studying modulation of the feeding patterns by associative learning.     

Enhanced synaptic transmission at identified synaptic connections in the cerebral ganglion of Aplysia.

The identified A-B neuron synaptic connections in the cerebral ganglion of Aplysia exhibited a novel form of enhanced synaptic transmission. A brief high-frequency train of action potentials (2 s, 10-30 Hz) in the presynaptic A neurons produced a long-lasting increase in the amplitude of excitatory postsynaptic potentials (EPSPs) in B neurons. The increase in synaptic efficacy was termed slow developing potentiation (SDP) since the EPSP amplitude increased slowly with the peak occurring 5 min after the tetanizing train. Peak EPSP amplitudes increased relative to the initial EPSP by an average of greater than 250%. SDP decayed as a single exponential with a time constant of tau = 24 min. The enhanced transmission was neuron specific. Only the connections made by the tetanized A neuron were potentiated. However, potentiation apparently occurred at all the synapses made by the tetanized A neuron. Tetanizing the postsynaptic B neurons neither induced, nor when paired with A neuron tetanization, increased SDP. SDP appears to be primarily due to increased transmitter release by the presynaptic neuron.     

Biochemical and morphological correlates of transmitter type in C2, an identified histaminergic neuron in Aplysia

There is compelling evidence that histamine serves as a neurotransmitter in C2, a pair of symmetrical neurons in the cerebral ganglion of Aplysia californica. These cells had previously been shown to contain high concentrations both of histamine and of its biosynthetic enzyme, histidine decarboxylase; in addition, 3H-histamine injected intrasomatically was found to move along C2's axons by fast transport. Furthermore, several actions of C2 on identified follower cells were simulated by the application of histamine. We have now characterized this identified neuron further. C2 converts 3H-histidine to histamine: 16% of the labeled precursor was converted to histamine 1 hour after intrasomatic injection. Synthesis of 3H-histamine is specific, since no conversion occurred after injection of other identified Aplysia neurons that are known to use other neurotransmitter substances. We also examined the fine structure of C2's cell body, axons, and axon terminals within the cerebral ganglion and in the nerves that carry its three peripheral branches, identified after injection of Lucifer Yellow, 3H-histamine, or horseradish peroxidase. Characteristic dense-core vesicles are present in all regions of the neuron, and are labeled after intrasomatic injection of 3H-histamine. These 100-nm vesicles together with 60-nm electron-lucent vesicles fill the varicose extensions of C2's neurites that are widely distributed within the ganglion, but only the smaller vesicles cluster at the membrane specializations presumed to be active zones that make contact with many neurons. The widespread distribution of axon terminals and varicosities is consistent with the idea that C2 is modulatory in function; 3H-histamine is taken up selectively by the cell body and axons of C2 and of several other putative histaminergic neurons in a Na+ -dependent manner. Characterization of these biochemical and morphological features of C2 adds to the large amount of information already available to make this identified cell a standard for identifying other neurons that use histamine as a transmitter.