Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 on the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.     

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.     

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.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

In vivo responses of paired giant mechanoreceptor neurons in Aplysia abdominal ganglion.

Two neurons with cell bodies symmetrically located in the abdominal ganglion and giant axons in the left (L1) and right (R1) pleurovisceral connectives of Aplysia californica were examined in vivo and in vitro. Direct stimulation of R1 and L1 in the intact animal does not elicit any observable behavior, suggesting that they are neither motoneurons nor command neurons. These cells respond in vivo to sudden onset mechanical stimulation of widespread regions of the body. R1 and L1 spikes are initiated in at least three different loci: (1) the peripheral axon in the foot, (2) the neuropil of the pleural and/or pedal ganglion, and (3) the neuropil of the abdominal ganglion. Furthermore, R1 and L1 probably have two different mechanisms for spike initiation: (1) sensory (foot), and (2) synaptic (abdominal and/or head ganglia). The different loci for spike initiation account for the bidirectional conduction of R1 and L1 spikes. As sensory (mechanoreceptor) neurons, R1 and L1 have peripheral axons in the ipsilateral posterior pedal nerve, show low threshold responses to stimulation of the ipsilateral posterior foot, they are rapidly adapting their responses do not decrease with repetition, and they are not blocked by high Mg++/low Ca++ solutions. As synaptically-driven neurons, R1 and L1 have widespread bilateral responsiveness, their responses decrease with repetition and their inputs are blocked with high Mg++/low Ca++ solutions. These neurons integrate sensory and synaptic inputs and conduct bidirectionally, however, their output connections must be specified before their behavioral function can be understood.     

Ganglionic circulation and its effects on neurons controlling cardiovascular functions in Aplysia californica

The abdominal ganglion of the mollusk Aplysia californica receives most of its blood supply through a small caudal artery that branches off the anterior aorta near its junction with the heart. Injection of an ink/gelatin mixture into the caudal artery revealed a consistent pattern of arterial branching within the ganglion and a general proximity of larger vessels to identified neurons controlling circulation in this animal. This morphological arrangement was particularly evident for the heart excitor interneuron, cell L10, which lies next to the caudal artery near its entry into the ganglion. In electrophysiological experiments, L10 was excited when blood flow or oxygen tension within the ganglion was reduced. This effect was expressed as a gradual increase in impulse frequency of L10 and conversion from tonic to bursting mode of spike discharge. L10 follower cells in the RB and LD neuron clusters were affected synaptically by the changes in L10 activity, while other follower cells (L3 and RD neurons) responded independently of L10's synaptic influence. The neurosecretory white cells (R3 to R14) that innervate the major arteries and pericardial tissues were also excited when ganglionic circulation was interrupted. In innervated preparations of the heart and respiratory organs, decreased circulation through the abdominal ganglion stimulated a transient increase in the rate and amplitude of respiratory (gill) pumping and pericardial contractions and caused a sustained increase in activity of the heart. Both responses increase cardiac output and both appear to involve a direct influence of ganglionic circulation on interneurons controlling the gill and heart. These results indicate that the cell-specific patterns of excitation and inhibition caused by fluctuations in ganglionic circulation may be important factors for maintaining circulatory homeostasis in this animal.     

The abdominal ganglion of Aplysia brasiliana: a comparative morphological and electrophysiological study, with notes on A. dactylomela.

The ultrastructure and electrophysiological properties of neurons in the abdominal (visceral) ganglion of the marine opisthobranch gastropod Aplysia brasiliana have been investigated to determine whether this preparation compares favorably with the well studied A. californica for neurobiological research. In general, the topography, morphology and physiological characteristics, including synaptic connections, of neurons in this ganglion are quite similar to those of A. californica. There is close correspondence between the two animals in terms of each of the identified cells or neuronal clusters in the ganglion, including the presence of the cell L10 (interneuron I) in A. brasiliana which makes synaptic connections comparable with those in A. californica. New follower cells of this interneuron have been found in A. brasiliana. This species offers some advantages in that the connective tissue surrounding the ganglion is thinner and more transparent, making cell identification and penetration easier. A. brasiliana appears to exhibit the behaviors of A. californica that have been used in previous functional analyses of neural circuits. In addition, this species swims and exhibits a "burrowing" activity less commonly seen in A. californica. The rich repertoire of behaviors and accessibility of large identifiable and functionally interconnected neurons makes this species of Aplysia an excellent model preparation for future neurobiological studies. Similar, less thorough, investigations of the abdominal ganglion of A. dactylomela indicate that this species is also very similar to A. californica in terms of the identified cells in the abdominal ganglion.     

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.     

Circuits constructed from identified Aplysia neurons exhibit multiple patterns of persistent activity.

We have used identified neurons from the abdominal ganglion of the mollusc Aplysia to construct and analyze two circuits in vitro. Each of these circuits was capable of producing two patterns of persistent activity; that is, they had bistable output states. The output could be switched between the stable states by a brief, external input. One circuit consisted of cocultured L10 and left upper quadrant (LUQ) neurons that formed reciprocal, inhibitory connections. In one stable state L10 was active and the LUQ was quiescent, whereas in the other stable state L10 was quiescent and the LUQ was active. A second circuit consisted of co-cultured L7 and L12 neurons that formed reciprocal, excitatory connections. In this circuit, both cells were quiescent in one stable state and both cells fired continuously in the other state. Bistable output in both circuits resulted from the nonlinear firing characteristics of each neuron and the feedback between the two neurons. We explored how the stability of the neuronal output could be controlled by the background currents injected into each neuron. We observed a relatively well-defined range of currents for which bistability occurred, consistent with the values expected from the measured strengths of the connections and a simple model. Outside of the range, the output was stable in only a single state. These results suggest how stable patterns of output are produced by some in vivo circuits and how command neurons from higher neural centers may control the activity of these circuits. The criteria that guided us in forming our circuits in culture were derived from theoretical studies on the properties of certain neuronal network models (e.g., Hopfield, J. J. 1984. Proc. Natl. Acad. Sci. USA. 81:3088-3092). Our results show that circuits consisting of only two co-cultured neurons can exhibit bistable output states of the form hypothesized to occur in populations of neurons.     

Effect of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B_26–34), and the 8-amino acid alpha-bag cell peptide (α-BCP_1–8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both α-BCP_1–8 and B_26–34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells in mimicked by α-BCP_1–8. The excitatory effect of the atrial gland extract cannot be duplicated with B_26–34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B_26–34 seems to mimick α-BCP_1–8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

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.     

Abundant expression of ras proteins in Aplysia neurons

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.     

Electrophysiological studies of the gill ganglion inAplysia californica

1. An electrophysiological analysis was made of gill ganglion neurons in Aplysia californica. 2. Gill ganglion neurons behave similarly to neurons in the abdominal ganglion (the central nervous systems; CNS) that are involved with gill withdrawal behaviors. 3. Some gill ganglion neurons are motor neurons much like those in the CNS. 4. Neurons in the gill ganglion are electronically and dye-coupled. In addition, they receive common chemical synaptic inputs from the Int-II network in the CNS. 5. Tactile stimulation of the gill or siphon evokes synaptic activity in gill ganglion neurons whether or not the CNS is present. 6. Pedal nerve stimulation results in synaptic activity in gill ganglion neurons and facilitates synaptic input evoked by tactile stimulation of the gill or siphon. 7. Antibody staining reveals serotonin-like fibers in the branchial nerve close to the gill ganglion but no cell bodies in the ganglion. 8. The gill ganglion may play a role in the mediation of adaptive gill reflex behaviors. It may be one of the loci where the CNS and peripheral nervous system (PNS) interact and form an integrated circuit to mediate gill withdrawal reflex (GWR) behaviors.     

In vivo responses of paired giant mechanoreceptor neurons in Aplysia abdominal ganglion

Two neurons with cell bodies symmetrically located in the abdominal ganglion and giant axons in the left (L1) and right (R1) pleurovisceral connectives of Aplysia californica were examined in vivo and in vitro. Direct stimulation of R1 and L1 in the intact animal does not elicit any observable behavior, suggesting that they are neither motoneurons nor command neurons. These cells respond in vivo to sudden onset mechanical stimulation of widespread regions of the body. R1 and L1 spikes are initiated in at least three different loci: (1) the peripheral axon in the foot, (2) the neuropil of the pleural and/or pedal ganglion, and (3) the neuropil of the abdominal ganglion. Furthermore, R1 and L1 probably have two different mechanisms for spike initiation: (1) sensory (foot), and (2) synaptic (abominal and/or head ganglia). The different loci for spike initiation account for the bidirectional conduction of R1 and L1 spikes. As sensory (mechanoreceptor) neurons, R1 and L1 have peripheral axons in the ipsilateral posterior pedal nerve, show low threshold responses to stimulation of the ipsilateral posterior foot, they are rapidly adapting their responses do not decrease with repetion, and they are not blocked by high Mg⁺⁺/low Ca⁺⁺ solutions. As synaptically-driven neurons, R1 and L1 have widespread bilateral responsiveness, their responses decrease with repetition and their inputs are blocked with high Mg⁺⁺/low Ca⁺⁺ solutions. These neurons integrate sensory and synaptic inputs and conduct bidirectionally, however, their output connections must be specified before their behavioral function can be understood.     

Serotonin-like immunoreactivity in the ventral nerve cord of the Colorado potato beetle,Leptinotarsa decemlineata: Identification of five different neuron classes

Summary In an immunohistochemical study of the ventral nerve cord of L. decemlineata, five distinct neuron categories were distinguished: 1) Two paired segmental twin interneurons occur in each ganglion or neuromere; their axons distribute processes over almost the entire nerve cord and run to the cerebral ganglion complex. In contrast, other axons are distributed locally. 2) Four large frontal neurosecretory neurons occur in the suboesophageal ganglion (SOG), two of which have axons that run into the mandibular nerves to form a neurohemal plexus on the surface of cerebral nerves. 3) A pair of large caudal neurons occur in the terminal ganglion and innervate the hindgut. 4) Local miniature interneurons occur in the SOG. 5) Terminal neurons are present in the last abdominal ganglion. Segmental twin interneurons appear to be grouped into 3 functional units spanning several ganglia. Their axons run to specific projection areas, which separate the functional units, and which mark the externally visible separation of condensed ganglion complexes. A possible role of the most caudal functional unit might be the synaptic control of caudal neurons innervating the hindgut.     

Endogenous peptides work at multiple sites in the nervous system in the control of gill behaviors in Aplysia

The suprafusion of two endogenous neuropeptides, arginine vasotocin (AVT) and small cardioactive peptide B (SCPB), over the abdominal ganglion of Aplysia californica significantly affects the ability of a central gill motor neuron to elicit a gill withdrawal response. Gill motor neurons L7 or LDG1 were depolarized to produce the same number of action potentials (APs) on each trial. When AVT (10(-6)M) was suprafused, the motor neurons' ability to elicit a gill movement was suppressed; while SCPB (10(-6)M) superfusion facilitated the response. Neither peptide altered the passive membrane properties of the motor neurons nor did they affect the duration of their APs. These results are consistent with the hypothesis that the peptides act via central control neurons which exert both suppressive and facilitatory control over gill reflex behaviors and associated neural activity.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target.

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transverse nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell-specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cell bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at approximately 42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Within this region of the nerve, the growth cones of both cell types-exhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.