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.     

Bradycardial response inAplysia exposed to air

Summary Heart rate was chronically monitored (Figs. 1, 3) in two species of the marine gastropodAplysia. The warm waterA. brasiliana have an average basal heart rate in water of 33 min^–1, whereas the cold waterA. californica's heart rate is 20.6 min^–1. The heart rate in both species shows a strong temperature dependence and the difference in basal heart rate is negligible when measured at the same temperature (Fig. 2). Both species show a consistent bradycardia when exposed to air (Fig. 4):A. brasiliana showed a 43% average decrease in air, whereasA. californica showed only a 16.5% decrease. Removal of the abdominal ganglion produced no significant decrease in heart rate in either species, nor did it reduce the bradycardial response to air exposure inA. californica (Fig. 8). However, it significantly reduced, but did not abolish, the bradycardia inA. brasiliana (Figs. 5, 6, 7). We conclude that the bradycardia has a significant central component inA. brasiliana, but is peripherally mediated inA. californica. The bradycardial response to air exposure may be analogous to the diving response in air breathing vertebrates.     

Temperature dependence of egg laying inAplysia brasiliana andA. californica

Summary The temperature dependence of egg laying was examined in winter-caughtAplysia. Cold-waterAplysia californica and warm-waterA. brasiliana were individually housed in the same large aquarium for 16 days at 15°C, and then for 16 days at 20°C. Initially, the majority of theA. californica were not reproductively mature (as determined by injections of atrial gland extracts) whereas all of theA. brasiliana were reproductively mature. When the temperature was increased from 15 to 20°C, both species showed a marked increase in the frequency of egg laying. At both temperatures,A. brasiliana laid eggs more frequently but produced smaller egg masses thanA. californica. We conclude that increased egg laying inA. californica was attibutable both to facilitation of oogenesis in previously reproductively immature animals and to increased activity of the bag cells which release an egg-laying hormone. Increased egg laying inA. brasiliana was attributable primarily to increased bag cell activity.     

Arginine vasotocin,an endogenous neuropeptide of Aplysia,suppresses the gill withdrawal reflex and reduces the evoked synaptic input to central gill motor neurons

The superfusion (15 min) of arginine vasotocin (AVT; 10^?9–10^?12M) over the abdominal ganglion of Aplysia californica suppressed the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon, increased the rate of gill reflex habituation, and decreased the evoked synaptic activity to central gill motor neurons. The suppressive effects of AVT on gill reflex behaviors were not due to toxic effects of the hormone since the effects were completely reversible following washout and 3 h rest. The results obtained with AVT were similar to those previously found using the mammalian neuropeptide arginine vasopressin. AVT may act by increasing the activity of central neurons which exert suppressive control over both gill reflex behaviors and evoked activity to central gill motor neurons.     

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.     

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.     

Neural control of the pyloric region in the foregut of the shrimp Penaeus (Decapoda: Penaeidae)

Pyloric pattern-generating neurons that control the pyloric region of the foregut were identified in the stomatogastric ganglion of the most primitive decapod genus Penaeus. Five types of motor neurons and one interneuron are involved in generation of pyloric motor pattern. One cell type of motor neurons innervates muscles of both the gastric mill and the pylorus like the gastric motor neurons in Cancer, but unlike those in Panulirus. These identified neurons are connected to each other either by electrical or inhibitory chemical synapses to construct the neural circuit. This pyloric circuit is similar to the homologous circuit of other crustacean species though some differences are seen in synaptic connections, supporting the hypothesis that the basic design of the neural circuit has been conserved during evolution of the Malacostraca, and that differences have occurred in the synaptic connectivity as the foregut structure has become complex. The motor neurons use either acetylcholine or glutamate as a neurotransmitter like in reptantians. The foregut structure, the number of the pyloric cells, muscle innervation, neurotransmitters, and circuitry are compared among malacostracan crustaceans to provide insight into how the neural circuits change and evolve to produce the motor patterns mediating behaviour. Accepted: 18 April 1997     

A single pair of interneurons controls motor neuron activity during pre-ecdysis compression behavior in larval Manduca sexta

Manduca sexta molts several times as a larva (caterpillar) before becoming a pupa and then an adult moth. Each molt culminates in ecdysis behavior, during which the old cuticle is shed. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which includes rhythmic, synchronous compressions of the abdomen. A previous study indicated that motor neuron activity during pre-ecdysis compression behavior is driven by an ascending neural pathway from the terminal abdominal ganglion. The present study describes a pair of interneurons, designated IN-402, that are located in the terminal ganglion and belong to the ascending pathway. Each IN-402 is synchronously active with pre-ecdysis compression motor bursts, and bilaterally excites compression motor neurons throughout the abdominal nerve cord via apparently monosynaptic connections. The pair of IN-402s appears to be the sole source of rhythmic synaptic drive to the motor neurons during the pre-ecdysis compression motor pattern. These interneurons play a key role in the production of larval pre-ecdysis behavior, and are candidates for contributing to the developmental weakening of pre-ecdysis behavior at pupation.Abbreviations A3, A4... abdominal ganglion 3, abdominal ganglion 4... - AT terminal abdominal ganglion - DN _A anterior branch of the dorsal nerve - EH eclosion hormone - EPSP excitatory postsynaptic potential     

Differing afferent connections of spiking and nonspiking wind-sensitive local interneurons in the terminal abdominal ganglion of the cricket Gryllus bimaculatus

Fifteen local spiking interneurons (LSIs) and twentyone local non-spiking interneurons (LNIs) were identified in the terminal abdominal ganglion (TAG) of the cricket Gryllus bimaculatus on the basis of intracellular recording and staining (Figs. 1, 5, 6). Although the majority of LNIs showed sharp directionalities (Fig. 7) the LSIs did not (Fig. 3). The directionality of LNIs varied with the recording sites within a single cell (Fig. 8). Electrical stimulations of the cereal sensory nerve suggested that the LNIs are connected monosynaptically with the sensory afferents of both the cerci, and that LSIs may possess a variety of bilateral combinations of polysynaptic connections with the sensory afferents. We found that the spiking and the non-spiking local interneurons in the cereal sensory system differ not only in their membrane properties, but also in their afferent connections, and concluded that their differing connectivity to the sensory afferents will associate them with different roles in signal processing.Abbreviations TAG terminal abdominal ganglion - LSI local spiking interneuron - LNI local non-spiking interneurons - CNS central nervous system - PSP post synaptic potential - GI giant interneuron     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

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.     

Motor pattern generation of the posterior cardiac plate-pyloric system in the stomatogastric ganglion of the mantis shrimp Squilla oratoria

Activity patterns of the constituent neurons of the posterior cardiac plate-pyloric system in the stomatogastric ganglion of the mantis shrimp Squilla oratoria were studied by recording spontaneous burst discharges intracellularly from neuronal somata. These neurons were identified electrophysiologically, and synaptic connections among them were qualitatively analysed. The posterior cardiac plate constrictor, pyloric constrictor, pyloric dilator and ventricular dilator motoneurons, and the pyloric interneuron were involved in the posterior cardiac plate-pyloric system. All the cell types could produce slow burst-forming potentials which led to repetitive spike discharges. These neurons generated sequentially patterned outputs. Most commonly, the posterior cardiac plate neuron activity was followed by the activity of pyloric constrictor neurons, and then by the activity of pyloric dilator/pyloric interneuron, and ventricular dilator neurons. The motoneurons and interneuron in the posterior cardiac plate-pyloric system were connected to each other either by electrical or by inhibitory chemical synapses, and thus constructed the neural circuit characterized by a wiring diagram which was structurally similar to the pyloric circuit of decapods. The circuitry in the stomatogastric ganglion was strongly conserved during evolution between stomatopods and decapods, despite significant changes in the peripheral structure of the foregut. There were more electrical synapses in stomatopods, and more reciprocal inhibitory synapses in decapods.Abbreviations EJP excitatory junctional potential - IPSP inhibitory postsynaptic potential - CoG commissural ganglion - CPG central pattern generator - ion inferior oesophageal nerve - OG oesophageal ganglion - pcp posterior cardiac plate - son superior oesophageal nerve - STG stomatogastric ganglion - stn stomatogastric nerve - PY pyloric constrictor - PD pyloric dilator - VD ventricular dilator - AB pyloric interneuron - lvn lateral ventricular nerves - tcpm transverse cardiac plate muscle     

Target neuron specification of short-term synaptic facilitation and depression in the cricket CNS

We investigated the role of retrograde signals in the regulation of short-term synaptic depression and facilitation by characterizing the form of plasticity expressed at novel synapses on four giant interneurons in the cricket cercal sensory system. We induced the formation of novel synapses by transplanting a mesothoracic leg and its associated sensory neurons to the cricket terminal abdominal segment. Axons of ectopic leg sensory neurons regenerated and innervated the host terminal abdominal ganglion forming monosynaptic connections with the medial giant interneuron (MGI), lateral giant interneuron (LGI), and interneurons 7-1a and 9-2a. The plasticity expressed by these synapses was characterized by stimulating a sensory neuron with pairs of stimuli at various frequencies or with trains of 10 stimuli delivered at 100 Hz and measuring the change in excitatory postsynaptic potential amplitude recorded in the postsynaptic neuron. Novel synapses of a leg tactile hair on 7-1a depressed, as did control synapses of cercal sensory neurons on this interneuron. Novel synapses of leg campaniform sensilla (CS) sensory neurons on MGI, like MGI's control synapses, always facilitated. The form of plasticity expressed by novel synapses is thus consistent with that observed at control synapses. Leg CS synapses with 9-2a also facilitated; however, the plasticity expressed by these sensory neurons is dependent on the identity of the postsynaptic cell since the synapses these same sensory neurons formed with LGI always depressed. We conclude that the form of plasticity expressed at these synaptic connections is determined retrogradely by the postsynaptic cell. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 700–714, 1998     

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.     

Distribution of synapses on two types of ascending interneurons in the crayfish,Procambarus clarkii

Summary About 60 pairs of ascending interneurons are present in the terminal ganglion of the crayfish Procambarus clarkii (Girard). Some of these interneurons have been impaled intracellularly, characterized physiologically, and then labeled with horseradish peroxidase (HRP) to examine the distribution and ultrastructure of synapses. A close relationship between ultrastructure and physiological properties has been found between two types of interneurons, which either have a pre-motor effect upon motor neurons or have no such effect. In one interneuron with a pre-motor effect (6D2), input and output synapses are intermingled on thicker branches, whereas only input synapses are found on small diameter branches. Only input synapses have been observed on the branches in another interneuron with-out a pre-motor effect (6B1). No differences in branch morphology are found in these two interneurons. Interneuron 6D2 contains large numbers of small round agranular vesicles, but the same type of synaptic vesicles is rarely seen in interneuron 6B1, which has no output synapses. Our results indicate a good correlation between the synaptic distribution and pre-motor effects of interneurons in the terminal ganglion.Abbreviations A6, 7 Sixth and seventh abdominal segment of the terminal ganglion - AVC anterior ventral commissure - DC I dorsal commissure I - DIT dorsal intermediate tract - DMT dorsal medial tract - eLG extra lateral giant interneuron - LVT lateral ventral tract - LG lateral giant interneuron - LVT lateral ventral tract - MDT median dorsal tract - MG medial giant interneuron - MoG motor giant neuron - MVT median ventral tract - PVC posterior ventral commissure - R1s sensory fiber tract of nerve root 1 - R3m motor fiber tract of nerve root 3 - R4–7 nerve roots 4–7 - SC I,II sensory commissure I,II - VC I,III ventral commissure I, III - VIT ventral intermediate tract - VLT ventral lateral tract - VMT ventral medial tract     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

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.     

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.     

Dopamine-mediated calcium channel regulation in synaptic suppression in L. stagnalis interneurons

D2 dopamine receptor-mediated suppression of synaptic transmission from interneurons plays a key role in neurobiological functions across species, ranging from respiration to memory formation. In this study, we investigated the mechanisms of D2 receptor-dependent suppression using soma-soma synapse between respiratory interneuron VD4 and LPeD1 in the mollusk Lymnaea stagnalis (L. stagnalis). We studied the effects of dopamine on voltage-dependent Ca²⁺ current and synaptic vesicle release from the VD4. We report that dopamine inhibits voltage-dependent Ca²⁺ current in the VD4 by both voltage-dependent and -independent mechanisms. Dopamine also suppresses synaptic vesicle release downstream of activity-dependent Ca²⁺ influx. Our study demonstrated that dopamine acts through D2 receptors to inhibit interneuron synaptic transmission through both voltage-dependent Ca²⁺ channel-dependent and -independent pathways. Taken together, these findings expand our understanding of dopamine function and fundamental mechanisms that shape the dynamics of neural circuit.