Identification of an abdominal ganglion neuron antagonizing L7-driven gill contraction in Aplysia

Gill motor neuron L7-induced longitudinal shortening of the gill in Aplysia kurodai and A. juliana was suppressed when extracellular stimuli were applied to a restricted dorsal central region of the abdominal ganglion. We found a neuron there which antagonized the L7-driven contraction. Since the contraction was suppressed when the identified neuron was activated simultaneously with L7, we refer to the newly found neuron as “Anti-L7”. Anti-L7 did not change the L7 impulse generation in the abdominal ganglion. No direct synaptic connection from L7 to Anti-L7 was detected. A fluorescent dye injected into the soma of Anti-L7 revealed that the neuron sent axonal branches to the branchial nerve. These results may show that Anti-L7 antagonizes L7 at the periphery inside the gill, rather than in the abdominal ganglion. EJPs induced by L7 were unaffected by Anti-L7. Activation of Anti-L7 alone did not induce any change in tone or membrane potential of the gill musculature. The suppressive effect of Anti-L7 lasts many seconds after the cessation of a train of Anti-L7 impulses. The results may suggest that the suppression is mediated through an inhibitory neuromodulatory mechanism without inhibition of L7 itself. Accepted: 1 April 1999     

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.     

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.     

Modulation of the Aplysia gill withdrawal reflex by dopamine

The ability of dopamine to modulate gill contractions was tested in Aplysia. When dopamine was perfused through the gill vasculature, gill contractions caused by siphon stimulation (gill withdrawal reflex) and by depolarization of the gill motor neuron L7 were increased in amplitude, as compared with those evoked during seawater perfusion. Habituation of gill movements, brought about by repetitive stimulation of the siphon or of L7, was prevented by dopamine. Despite the absence of reflex habituation, the number of action potentials in central gill motor neurons, evoked by siphon stimulation, showed normal decrement. Dopamine's effects were blocked when the ctenidial nerve was cut or when L7 hyperpolarized. These data suggest that dopamine acts peripherally to increase the efficacy of L7's synaptic transmission onto gill muscle or elements of the gill neural plexus.     

Neurotransmitter receptors on gill muscle fibers and the gill peripheral nerve plexus in Aplysia.

Isolated pinnules of the gill of Aplysia contract when dopamine (DA) is perfused through the bath. The contraction is not blocked by high-Mg2+ seawater, and reflects excitatory receptors for DA on the smooth muscle cells of the gill. The pinnule often shows irregular, spontaneous contractions which are blocked by high-Mg2+ seawater and 30 mM CoCl2. These contractions reflect spontaneous activity of a peripheral nerve plexus. No other transmitter was found to be directly excitatory on the muscle fibers, although there are inhibitory receptors for serotonin (5-HT). Tactile stimulation of the pinnule evoked a two-component contractile reflex contraction due to activation of the peripheral nerve plexus. Acetylcholine, octopamine, and 5-HT but not several other transmitters depressed these responses, presumably due to inhibitory receptors on the neurons of the peripheral plexus.     

The development of central nervous system control of the gill withdrawal reflex evoked by siphon stimulation in Aplysia

In older Aplysia, the central nervous system (CNS) (abdominal ganglion) exerts suppressive and facilitatory control over the peripheral nervous system (PNS) which initially mediates the gill withdrawal reflex and its subsequent habituation evoked by tactile stimulation of the siphon. In young animals, both the suppressive and facilitatory CNS control were found to be absent. In older animals, removal of branchial nerve (Br) input to the gill resulted in a significantly reduced reflex latency and, with ctenidial (Ct) and siphon (Sn) nerves intact, a significantly increased reflex amplitude and an inability of the reflex to habituate with repeated siphon stimulation. In young animals, removal of Br had no effect on reflex latency and with Ct and Sn intact, the reflex amplitude latency was not increased and the reflex habituated. Older animals can easily discriminate between different intensity stimuli applied to the siphon as evidenced by differences in reflex amplitude, rates of habituation, and evoked neural activity. On the other hand, young animals cannot discriminate well between different stimulus intensities. The lack of CNS control in young animals was found to be due to incompletely developed neural processes within the abdominal ganglion and not the PNS. The lack of CNS control in young Aplysia results in gill reflex behaviours being less adaptive in light of changing stimulus conditions, but may be of positive survival value in that the young will not habituate as easily. The fact that CNS control is present in older animals strengthens the idea that in any analysis of the underlying neural mechanisms of habituation the entire integrated CNS-PNS must be taken into account.     

Vascular resistance and vasoactivity of gills and pulmonary artery of the salamander,Ambystoma tigrinum

Summary In order to understand the blood flow patterns and their regulation in the gills and pulmonary artery ofAmbystoma tigrinum, the vascular resistance and vasoactivity of the two major branchial perfusion pathways and a vascular plexus in the pulmonary artery were investigated using an isolated-tissue perfusion method. Acetylcholine and epinephrine were both pressor agents in all three vascular segments. Angiotensin II also constricted the branchial respiratory vasculature. Norephinephrine was primarily a vasodilator in the branchial respiratory vasculature, however, it had no effect on the shunt vessels of the gill or the pulmonary arterial plexus. Both gill circulations were insensitive to alterations in CO₂ and pH. Anoxia produced a slight vasodilation of the branchial respiratory vessels but had no effect on the shunt vasculature. Mild hypoxia had no effect on either branchial circulations. The results suggest that: (1) blood flow through the respiratory section of the gill may vary between 8 and 47% of total gill flow, (2) the major perfusion pathway to the lung is probably from the efferent artery of the third gill through the ductus arteriosus and then into the pulmonary artery, (3) O₂, CO₂ and pH exert no local control of branchial perfusion, (4) both cholinergic and adrenergic regulation of branchial and proximal pulmonary arterial vascular resistance is possible, (5) a rise in circulating norepinephrine should increase blood flow to the respiratory section of the gill.Abbreviations AII angiotensin II - ACh acetylcholine - EPi epinephrine - NE norepinephrine     

Nitric oxide synthase in the glossopharyngeal and vagal afferent pathway of a teleost, Takifugu niphobles

To examine the presence of nitric oxide synthase (NOS) in the sensory system of the glossopharyngeal and vagus nerves of teleosts, nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity and immunoreactivity for NOS were examined in the puffer fish Takifugu niphobles. The nitrergic sensory neurons were located in the ganglia of both the glossopharyngeal and the vagal nerves. In the vagal ganglion, positive neurons were found in the subpopulations for the branchial rami and the coelomic visceral ramus, but not for the posterior ramus or the lateral line ramus. In the medulla, nitrergic afferent terminals were found in the glossopharyngeal lobe, the vagal lobe, and the commissural nucleus. In the gill structure, the nitrergic nerve fibers were seen in the nerve bundles running along the efferent branchial artery of all three gill arches. These fibers appeared to terminate in the proximal portion of the efferent filament arteries of three gill arches. On the other hand, autonomic neurons innervating the gill arches were unstained. These results suggest that nitrergic sensory neurons in the glossopharyngeal and vagal ganglia project their peripheral processes through the branchial rami to a specific portion of the branchial arteries, and they might play a role in baroreception of this fish. A possible role for nitric oxide (NO) in baroreception is also discussed.     

Different effects of the biogenic amines dopamine,serotonin and octopamine on the thoracic and abdominal portions of the escape circuit in the cockroach

1. The escape behavior of the cockroach, Periplaneta americana, is known to be modulated under various behavioral conditions (Camhi and Volman 1978; Camhi and Nolen 1981; Camhi 1988). Some of these modulatory effects occur in the last abdominal ganglion (Daley and Delcomyn 1981a, b; Libersat et al. 1989) and others in the thoracic ganglia (Camhi 1988). Neuromodulator substances are known to underlie behavioral modulation in various animals. Therefore, we have sought to determine whether topical application of putative neuromodulators of the escape circuit enhance or depress this circuit, and whether these effects differ in the last abdominal vs. the thoracic ganglia. 2. Topical application of the biogenic amines serotonin and dopamine to the metathoracic ganglion modulates the escape circuitry within this ganglion; serotonin decreases and dopamine enhances the response of leg motoneurons to activation of interneurons in the abdominal nerve cord by electrical or wind stimulation. 3. The neuropil of the thoracic ganglia contains many catecholamine-histofluorescent processes bearing varicosities, providing a possible anatomical substrate for dopamine release sites. 4. Topical application of octopamine to the terminal abdominal ganglion enhances the response of abdominal interneurons to wind stimulation of the cerci. In contrast, serotonin and dopamine have no effect at this site. 5. It is proposed that release of these biogenic amines may contribute to the known modulation of the cockroach escape response.     

The aminergic control of cockroach salivary glands

The acinar salivary glands of cockroaches receive a dual innervation from the subesophageal ganglion and the stomatogastric nervous system. Acinar cells are surrounded by a plexus of dopaminergic and serotonergic varicose fibers. In addition, serotonergic terminals lie deep in the extracellular spaces between acinar cells. Excitation-secretion coupling in cockroach salivary glands is stimulated by both dopamine and serotonin. These monoamines cause increases in the intracellular concentrations of cAMP and Ca(2+). Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, two elementary secretory processes, namely electrolyte/water secretion and protein secretion, are triggered by different aminergic transmitters. Because of its simplicity and experimental accessibility, cockroach salivary glands have been used extensively as a model system to study the cellular actions of biogenic amines and to examine the pharmacological properties of biogenic amine receptors. In this review, we summarize current knowledge concerning the aminergic control of cockroach salivary glands and discuss our efforts to characterize Periplaneta biogenic amine receptors molecularly.     

Effects of serotonin on the cardio-circulatory system of the European eel (Anguilla anguilla) in vivo

The effects of serotonin on continuously recorded cardiac parameters (heart rate, cardiac output, cardiac stroke volume), ventral and dorsal aortic blood pressures, branchial and systemic vascular resistances were investigated in the European eel in vivo. Intravenous administration of serotonin (30 g · kg^–1) caused a marked bradycardia (45%) and a simultaneous decrease in cardiac output (50%), ventral (35%) and dorsal (50%) aortic blood pressures. Branchial resistance was markedly increased (60%) and systemic resistance decreased (30%). Cardiac stroke volume remained unchanged. The effects of serotonin on cardiac mained unchanged. The effects of serotonin on cardiac parameters were suppressed either by methysergide or a bilateral section of the cardiac vagus. Bradycardia could then be regarded as the consequence of a vagal mechanism triggered by serotonin action on central methysergide-sensitive serotonergic receptors. No inotropic effect of serotonin was observed. This lack of myocardiac contractility modification is discussed. The serotonin-mediated branchial vasoconstriction was attenuated by vagotomy, whereas the residual increase in branchial resistance (40%) was suppressed by methysergide. The serotonin-mediated branchial vasoconstriction could be the consequence of both a passive mechanism (compliance) caused by the decrease in cardiac output and an active mechanism involving methysergide-sensitive serotonergic receptors of the branchial vasculature. A possible involvement of this vasomotor effect in gill oxygen uptake is discussed. The serotonin-induced systemic vasodilation was insensitive either to cardiac vagotomy or to 5-HT_1/2, 5-HT₃ and 5-HT₄ receptor antagonists, suggesting the involvement of a local mechanism which remains to be assessed.Abbreviations CSV cardiac stroke volume - DAP dorsal aortic pressure - HR heart rate - QC cardiac output - VAP ventral aortic pressure - VR _b branchial vascular resistance - VR _s systemic vascular resistance - VR _t total vascular resistance - 5-HT 5-Hydroxytryptamine serotonin - RBI Research Biochemical Incorporated, metoclopramide HCl     

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.     

Characterization of neuronal regeneration in the abdominal ganglion of Aplysia californica

The ability of neurons in the abdominal ganglion of Aplysia to regenerate their axons following branchial nerve crush was studied using retrograde staining and intracellular dye injection. The duration of the gill withdrawal reflex (GWR) was measured prior to and following nerve crush. Three days after crushing the nerve, the duration of the gill withdrawal reflex was reduced to 20% of control levels. There was rapid recovery 19 days after crushing the branchial nerve. The GWR duration returned to control levels by postlesion days 25–27. Some of the behavioral recovery can be attributed to axonal regeneration. Regeneration, as evidenced by retrograde staining, was first observed by postlesion day 15. The number of stained neurons in ganglia with crushes increased until postlesion day 33. The number of stained neurons in experimental animals was always less than that of controls (67 ± 9% at postlesion day 56). More axonal regeneration was seen in the hemiganglion ipsilateral to the branchial nerve. Regeneration after 32 days postlesion was 60 ± 5% of controls in the ipsilateral hemiganglion, as opposed to 29 ± 6% in the contralateral hemiganglion. Regeneration of individual neurons was also demonstrated. Identified neuron R2 was shown by intracellular dye injection and electrical stimulation of antidromic action potentials to have an axon in the branchial nerve in all ganglia allowed to regenerate for longer than 32 days. These results indicate that in Aplysia, despite behavioral recovery, complete axonal regeneration does not occur in a large segment of the neurons in the adult central nervous system. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 160–172, 1998     

Parallel processing of mechanosensory inputs from the locust ovipositor by intersegmental and local interneurones

The neural pathways underlying the processing of signals from locust (Schistocerca gregaria) ovipositor hairs by different classes of interneurones are investigated.Spikes in the sensory neurones from these hairs evoke chemically-mediated, unitary EPSPs with a short and constant latency in six identified non-giant projection interneurones with cell bodies in the terminal abdominal ganglion. Five of these interneurones receive direct inputs from the valves ipsilateral to their neuropilar branches, whereas the other receives direct inputs from valves on both sides. The sensory neurone from a single hair makes divergent connections with several interneurones and those from different hairs make convergent connections with a given interneurone. The amplitude of the EPSPs evoked depends on the position of a hair along the proximal-distal axis of the valve, with sensory neurones from more distal hairs generating larger amplitude EPSPs.Deflection of hairs also excites three of the four giant projection interneurones through polysynaptic pathways and some local interneurones in the terminal abdominal ganglion through monosynaptic connections. Branches of non-giant projection interneurones, local interneurones, but not those of the giant interneurones, overlap the axon terminals of the ovipositor hair afferents in the terminal abdominal ganglion.     

Early development of the serotonergic and dopaminergic nervous system in Spisula solidissima (surf clam) larvae.

We have defined the development of the serotonergic and dopaminergic components of the central nervous system in the early Spisula solidissima (surf clam) embryo using HPLC and immunocytochemistry. HPLC analysis reveals norepinephrine, dopamine, and serotonin are present at 24 h post-fertilization. Immunocytochemistry shows that the serotonergic nervous system emerges during the late trochophore stage with the development of a single serotonergic cell, C/A1, in the cerebral/apical ganglion. After 48 h, a second serotonergic cell forms, C/A2, which is connected to C/A1 by two serotonergic processes, and a single serotonergic cell emerges in the visceral ganglion, V1. At 72 h, a new serotonergic cell body develops in the cerebral/apical ganglion, C/A3. After 96 h, the cerebral/apical ganglion and visceral ganglion are connected by a serotonergic process. Expression of the dopamine receptor, D2, begins by 24 h with a generalized expression in the region of the developing gut. D2 expression in the gut ceases by 48 h. At 48 h, a network of fibers forms dorsolateral to the mouth. By 72 h, D2 expressing projections emerge from this network.     

Neuronal background of activation of estivated snails,with special attention to the monoaminergic system: a biochemical,physiological, and neuroanatomical study

Osmotic stimulation activates both estivated and inactivated specimens of Helix pomatia and increases their central arousal. High-pressure liquid chromatography has shown that, during activation, the level of both serotonin and dopamine decreases in the central nervous system (CNS) but increases in the foot and heart, organs that are involved in the eversion of the body. In isolated CNS from activated animals, the firing frequency of the heart-modulator serotonergic (RPas) neurons is significantly higher than that in the CNS of estivated or inactivated animals. These neurons innervate both the heart and the anterior aorta. In semi-intact preparations, distilled water (an osmotic stimulus) applied to the mantle collar increases their firing frequency, whereas tactile stimulation evokes their inhibition. Extracellularly applied monoamines mimic the effect of peripheral stimuli: serotonin (0.1–10 μM) increases the activity of the RPas neurons, whereas dopamine (0.1–10 μM) inhibits their activity. Tyrosine-hydroxylase immunocytochemistry and retrograde neurobiotin tracing have revealed similar bipolar receptor cells in the mantle collar and tail, organs that are exposed to environmental stimuli in estivated animals. Serotonin immunocytochemistry carried out on the same tissues does not visualize receptor cells but labels a dense network of fibers that appear to innervate neurobiotin-labeled receptor cells. The combination of neurobiotin-labeling of RPas neurons and immunolabeling suggests that RPas neurons receive direct dopaminergic inputs from receptor cells and serotonergic inputs from central serotonergic neurons, indicating that central serotonergic neurons are interconnected. Thus, the RPas neurons may belong to neuronal elements of the arousal system. This work was supported by Hungarian OTKA grants T037389, T046580, T037505, and K63451.     

Development and sensitivity to serotonin of Drosophila serotonergic varicosities in the central nervous system

Serotonin is a classical small-molecule neurotransmitter with known effects on developmental processes. Previous studies have shown a developmental role for serotonin in the fly peripheral nervous system. In this study, we show that serotonin can modulate the development of serotonergic varicosities within the fly central nervous system. We have developed a system to examine the development of serotonergic varicosities in the larval CNS. We use this method to describe the normal serotonergic development in the A7 abdominal ganglion. From first to third instar larvae, the volume of the neuropil and number of serotonergic varicosities increase substantially while the varicosity density remains relatively constant. We hypothesize that serotonin is an autoregulator for serotonergic varicosity density. We tested the sensitivity of serotonergic varicosities to serotonin by adding neurotransmitter at various stages to isolated larval ventral nerve cords. Addition of excess exogenous serotonin decreases native varicosity density in older larvae, and these acute effects are reversible. The effects of serotonin appear to be selective for serotonergic varicosities, as dopaminergic and corazonergic varicosities remain qualitatively intact following serotonin application.     

Demonstration of denervation supersensitivity: a pharmacological approach

1. Although the phenomenon of denervation supersensitivity has been extensively studied in peripheral cholinergically and adrenergically innervated organs, the consequences of denervation have not been studied in as much detail in serotonergically innervated s stems. This may be due in part to the difficulties associated with producing specific lesions of serotonergic systems and with quantifying physiological responses of the CNS due to serotonin. 2. Because the lateral ciliated cells of the gill of the bivalve mollusc, Mytilus edulis, are innervated by serotonergic motor neurons which are responsible for accelerating the beating rate of the cilia we used this as a model system with which to study the effects of denervation by physical transections of the branchial nerve and by pharmacological treatments with 5,6 dihydroxytryptamine. 3. Both treatments produced a supersensitive response of the ciliated cells to the superfusion of serotonin. Two mechanisms with different time courses may be responsible. 4. The initial faster acting component may be due to changes in re-uptake mechanisms for serotonin, while the later slowly developing component may be due to other causes, including a hyperplasia of postjunctional serotonin receptors.