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.     

Serotonin-containing Cells in the Nervous System and Other Tissues During Ontogeny of a Lancelet,Branchiostoma floridae

Abstract Serotonin-containing cells are described by immunohistochemistry throughout lancelet ontogeny. Such cells are first detected in the 2-day larva: these are (1) enterochromaffin cells in the inner epithelium of the gut and (2) anterior serotonergic neurons at the rostral end of the nerve cord. In the 6-day larva, relatively low levels of serotonin appear in ventro-lateral perikarya and cell processes of intraspinal serotonergic neurons scattered along the nerve cord. In the 18-day (early metamorphic) larva, antero-lateral serotonergic neurons are detected near the rostral end of the nerve cord as two small, bilateral clusters of perikarya with axons that descend the nerve cord; at later developmental stages, these axons extend almost to the posterior end of the body. In the 21-day (mid-metamorphic) larva, serotonin can no longer be detected in the anterior serotonergic neurons, but serotonin-containing cells are found subjacent to the inner epithelium of the digestive caecum and in the peribranchial epithelium covering the primary gill bars. In the discussion, we suggest that the anterior serotonergic neurons may play a role in larval photoreception and that the antero-lateral serotonergic neurons may be homologous to vertebrate hindbrain neurons with axons descending the spinal cord to modulate undulation (if this homology is valid, the anterior limit of the lancelet hindbrain would be roughly 100 μm behind the rostral tip of the nerve cord).     

Neurons controlling the gill vasculature in five species of teleosts

Summary A plexus of nerve fibers encompassing neuronal perikarya is present within the gill filament; it surrounds the proximal portion of the efferent filament artery and the efferent lamellar arterioles. This innervation resembles the pattern described for the area around the sphincter of the efferent filament artery: acetylcholinesterase-positive neurons and fibers, fast-fading yellow-fluorescent neurons and fibers, long-lasting green-fluorescent fibers. In addition, synaptic contacts between the different components suggest functional interrelationships. Nerves evidently control the efferent limb of the filament circulation including the sphincter of the efferent filament arteries, the proximal portion of the efferent filament arteries proper, and their corresponding efferent lamellar arterioles. However, the distal portion of this system is poorly innervated.     

The sphincter of the efferent filament artery in teleost gills: I. Structure and parasympathetic innervation

Previous studies have shown the existence of a sphincter in the efferent filament artery of the teleost gill and its constrictory response to acetylcholine (ACH) and vagal stimulation. This study deals with the muscular organization of this sphincter and the distribution of its innervation as elucidated by degeneration methods and cytochemistry. The sphincter innervation is supplied by the protrematic vagus nerves. Nerve endings filled with cholinergic-type vesicles are located in close association with the adventitial smooth muscle cells and display a strong acetylcholinesterase (ACHE) activity. Section of the protrematic vagus nerve induces a nearly complete degeneration of the sphincter innervation. ACHE-positive nerve cell bodies are present both in the sphincter area and in the protrematic vagus nerve. These results suggest that innervation of the sphincter in the efferent filament artery is cholinergic through the activity of postganglionic axons of the parasympathetic system.     

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.     

Effects ofp-chloroamphetamine on brain serotonin neurons

p-Chloroamphetamine (PCA) is a useful pharmacologic tool for selectively increasing brain serotonin function acutely by release of serotonin into the synaptic cleft. PCA produces behavioral, neurochemical and neuroendocrine effects believed due to serotonin release after doses in the range of 0.5–5 mg/kg. At higher doses and at longer times, PCA causes depletion of brain serotonin. The mechanisms of this depletion are not well understood but require the serotonin uptake carrier. Antagonism of PCA-induced depletion of brain serotonin is a useful means of assessing the ability of a compound to block the serotonin uptake carrier on brain serotonin neurons. PCA can also be used as a neurotoxic agent to deplete brain serotonin in functional studies, apparently by destroying some serotonergic nerve terminals. Used in this way, PCA has an advantage over 5,6- and 5,7-dihydroxytryptamines in being effective by systemic injection, and it affects brain serotonergic projections with a different neuroanatomic specificity than the dihydroxytryptamines.Special issue dedicated to Dr. Morris H. Aprison.     

Terminal processes of serotonin neurons in the caudal spinal cord of the molly,Poecilia latipinna,project to the leptomeninges and urophysis

Summary The caudal neurosecretory complex of poeciliids has previously been shown to be innervated by extranuclear and intrinsic serotonergic projections. In the present study, immunohistochemical techniques were used to characterize fibers originating from serotonin neurons intrinsic to the caudal spinal cord. Bipolar and multipolar neurons were oriented ventromedially, and contained numerous large granular vesicles. Three types of serotonergic fibers were distinguished based on their distribution and morphology. Intrinsic Type-A fibers branched into varicose segments near the ventrolateral surface of the spinal cord and contacted the basal lamina beneath the leptomeninges. Type-B fibers coursed longitudinally to enter the urophysis, where they diverged and terminated around fenestrated capillaries. Labelled vesicles in Type-A and Type-B terminals were the same size as those in labelled cells and in unlabelled neurosecretory terminals in the urophysis. Type-C small varicose fibers branched within the neuropil of the caudal neurosecretory complex. Serotonin may be secreted into the submeningeal cerebrospinal fluid, the urophysis, and the caudal vein by Type-A and Type-B fibers, whereas, Type-C fibers may be processes of serotonergic interneurons in the neuroendocrine nucleus. The possibility that urotensins I and II or arginine vasotocin were colocalized in the processes of the intrinsic serotonin neurons was investigated immunohistochemically. The negative results of these experiments suggest that serotonin-containing neurons may represent a neurochemically distinct subpopulation in the caudal neurosecretory complex.     

Smooth muscles in relation to the gill skeleton of Perca fluviatilis: organization and innervation

Summary Two laminae composed of smooth muscles, elastic tissue and collagen have been described in relation with the gill skeleton in Perca fluviatilis. A transverse smoothmuscle lamina joins the base of the cartilage rods of the two opposite hemibranchs. A longitudinal smooth-muscle lamina runs parallel to the afferent branchial artery and joins the cartilage rods from one filament to the other. In both laminae, the formaldehyde-induced fluorescence technique (Falck-Hillarp) reveals a network of nerve fibers displaying a green fluorescence characteristic of catecholamines. At the ultrastructural level, the presence of nerve endings containing clear and granular vesicles, and the degeneration of these endings after 6-hydroxydopamine treatment confirm the aminergic nature and the sympathetic origin of this innervation. Surgical denervation brings evidence that the innervation of both laminae is supplied by the metatrematic branches of the branchial nerves. The role of these smooth-muscle laminae remains speculative.     

Variation in serotonergic and dopaminergic neuronal survival in the central nervous system of adult <Emphasis Type="Italic">Drosophila</Emphasis>

Loss of serotonergic and dopaminergic neurons may have serious implications for normal brain function. Drosophila models of neurodegenerative diseases utilize the short life-span and simple anatomy of the fly to characterize the molecular and genetic processes characteristic of each dysfunctional state. In fly embryonic and larval ventral nerve cords, serotonergic and dopaminergic neurons are positioned in a stereotypic pattern that is reorganized during metamorphosis. In this study, we examine the adult pattern of serotonergic and dopaminergic neurons within the adult fly ventral nerve cord. We find that the number of cells lost following metamorphosis is highly variable. Changes in cell number attributable to age are therefore likely to be highly masked by developmental variation. The source of this variation is probably apoptosis-based cell loss during pupal development.This work was supported by a Keck Scholars Award and NINDS R29 37322 to BGC and by the University of Virginia Medical Scientist Training Program to PAS.     

4种弹涂鱼鳃的形态度量学比较及其生态学意义

通过对背眼虎鱼亚科中薄氏大弹涂鱼(Boleophthalmus boddarti)、青弹涂鱼(Scartelaos histophorus)、新几内亚弹涂鱼(Periophthalmus novaeguineaensis)和点弹涂鱼(P.spilotus)3属4种弹涂鱼鳃参数的测定,比较了各种之间鳃的形态度量学差异。结果表明,4种弹涂鱼的鳃参数(Y)与其体重(W)均符合方程logY=log a+b logW,且各鳃参数与体重的相关性显著(R2=0.50～0.98,P0.05)。等体重的弹涂鱼相比较,青弹涂鱼的总鳃丝数、总鳃丝长(mm)、鳃丝一侧鳃小片数(/mm)、总鳃面积(mm2)和相对鳃面积(mm2/g)均最大,薄氏大弹涂鱼相应鳃参数次之,新几内亚弹涂鱼和点弹涂鱼相应鳃参数较小。弹涂鱼鳃结构的这种梯度退化,表明青弹涂鱼和薄氏大弹涂鱼水生性较强,而新几内亚弹涂鱼和点弹涂鱼陆生性较强。4种弹涂鱼的总鳃丝长和总鳃面积明显小于其他等体重水生鱼类,这与弹涂鱼的两栖生活特征相符。     

Aminergic cellular organization in the gills of Aplysia species

The constituent elements of the gills of Aplysia kurodai and A. juliana were examined for the presence of biogenic amines using histochemical, immunocytochemical, and HPLC techniques. Aminergic elements were revealed by glyoxylic acid-induced fluorescence in the branchial nerve, branchial ganglion, branchial vessels, and pinnules in both species. Three types of fluorescent cells were found in the neural plexus of the gill in each species. Two of them might be sensory neurons. Although HPLC analysis showed the presence of serotonin and dopamine in all gill structures including fluorescent neural elements, there were regional differences in concentrations of the monoamines. It was noted in the pinnules that there was a much higher concentration of dopamine than serotonin. Serotonin immunocytochemistry revealed neural processes which were immunoreactive to antiserotonin antibody, but serotonin immunoreactivity could not be found in a population of branchioganglionic neuron (BGN) somata. Serotonergic elements in the ganglion may be processes of the central ganglion, while dopaminergic elements may be processes of neurons in the neural plexus, located beyond the branchial ganglion. BGNs were activated by bath-applied dopamine and serotonin. These results suggest that dopaminergic sensory inputs from the neural plexus and serotonergic descending inputs from the abdominal ganglion may be among the inputs received by BGNs. It was found that serotonin depressed excitatory junctional potentials in muscle cells of the efferent branchial vessel, which were induced by an identified neuron of the abdominal ganglion. The aminergic cellular organization of the gill may involve serotonergic presynaptic-inhibitory fibers arising from the abdominal ganglion.     

Morphology and vascular anatomy of the gills of a primitive air-breathing fish,the bowfin (Amia calva)

Summary The morphology of the gills of a primitive air breather (Amia calva) was examined by light microscopy of semithin sections of gill filaments, and gill perfusion pathways were identified by scanning-electron microscopic analysis of corrosion replicas prepared by intravascular injection of methyl methacrylate. The arrangement of gill filaments and respiratory lamellae is similar to that of teleosts with the exception of an interfilamental support bar that is fused to the outer margins of lamellae on adjacent filaments. The prebranchial vasculature is also similar to that of teleosts, whereas the postbranchial circulation of arches III and IV is modified to permit selective perfusion of the air bladder. Gill filaments contain three distinct vascular systems: (1) the respiratory circulation which receives the entire cardiac output and perfuses the secondary lamellae; (2) a nutrient system that arises from the postlamellar circulation and perfuses filamental tissues; (3) a network of unknown function consisting of subepithelial sinusoids surrounding afferent and efferent margins of the filament and traversing the filament beneath the interlamellar epithelium. Prelamellar arteriovenous anastomoses (AVAs) are rare, postlamellar AVAs are common especially at the base of the filament where they form a dense network of small tortuous vessels before coalescing into a large filamental nutrient artery. Unlike in most teleosts, the outer vascular margins of the lamellae are embedded in the interfilamental support bar and become the sole vasculature of this tissue. Arterial-arterial lamellar bypass vessels were not observed. Previously observed decreases in oxygen transfer across the gills during air breathing can be explained only by redistribution of blood flow between or within the respiratory lamellae.Supported by NSF Grant No. PCM 79-23073The author wishes to thank Miss K. Drajus and D. Kullman for their excellent technical assistance and Dr. W. Gingerich, Mr. J. Crowther and D. Zurn for help in obtaining bowfin     

Ultrastructure of the presumed ion-transporting cells in the gills of ammocoete lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (Bloch)

Summary Mitochondria-rich cells were located in the interplatelet area of gill filaments from ammocoete Lampetra fluviatilis and L. planeri. The ultrastructure of this cell type differs from typical teleost chloride cells by the absence of a tubular, smooth endoplasmic reticulum (SER). This difference is discussed in relation to the presumed functions of the cell and to the evolutionary histories of lampreys and teleosts. It is concluded that the mitochondria-rich cell is responsible for the active uptake of ions by the ammocoete gill.     

Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity

Summary 1. During early ontogeny, the serotonergic neurons in the brain stem of the three-spined stickleback shows a temporal and spatial developmental pattern that closely resembles that of amniotes.2. However, in the adult fish, only the midline nuclei of the rostral group (dorsal and median raphe nuclei) and the dorsal lateral tegmental nucleus are consistently serotonin-immunoreactive (5-HTir), whereas the groups of the upper and lower rhombencephalon (raphe pontis, raphe magnus, and raphe pallidus/obscurus nuclei) are variable and, when present, contain relatively small numbers of 5-HTir neurons.3. Using specific antisera against tryptophan 5-hydroxylase and aromaticl-amino acid decarboxylase, we have shown that the lateral B9 group and the groups of the upper and lower rhombencephalon are consistently present in adult sticklebacks. The results are discussed in relation to other known instances of neurotransmitter plasticity or transient neurotransmitter expression in teleost fish.4. While there are several instances of transient expression of neurotransmitter markers by discrete neuronal populations, there is so far no evidence of changes from one neurotransmitter phenotype to another in the brain of teleost fish. However, there are indications of plasticity of expression of catecholamines and indoleamines, and their respective synthesizing enzymes, as reflected in age-dependent changes and variation between individuals of different physiological status.5. As the brain grows continuously in teleost fish, and new neurons are added from proliferative regions, synaptic connections may be expected to undergo remodeling in all brain regions throughout life. Thus, the teleostean brain may be considered a suitable model for experimental studies of different aspects of neural plasticity.     

In vitro capsaicin-induced cytological changes and alteration in calcium distribution in giant serotonergic neurons of the snail Helix pomatia: a light- and electron-microscopic study

Morphological changes induced by capsaicin were studied in the serotonergic metacerebral giant neurons of the cerebral ganglia of Helix pomatia under in vitro conditions. Capsaicin at a concentration of 10^-4 M caused characteristic structural alterations in the giant serotonergic neurons but did not significantly influence serotonin immunoreactivity in the neurons. At the lightmicroscopic level, the most conspiciuous structural alterations were swelling of the cell bodies, which contained a swollen pale nucleus. Under the electron microscope, the nuclei,mitochondria and the cisternae of the endoplasmic reticulum were swollen in the capsaicin-affected metacerebral giant neurons. Electron-microscopic cytochemical techniques for calcium demonstration revealed electron-dense deposits in the swollen mitochondria and in the cisternae of the endoplasmic reticulum, suggesting an increased Ca²⁺ influx. The serotonergic metacerebral giant neurons could be labelled by cobalt (1 mM) in the presence of capsaicin (10^-4 M) suggesting that capsaicin opens the cation chanels of the capsaicin-sensitive neuronal membrane. The morphological and cytochemical alterations induced by capsaicin in the serotonergic metacerebral giant neurons of Helix pomatia closely resemble those induced in sensory neurons of mammalian dorsal root ganglion.This work was supported by OTKA grants No.: 2477, T016861, T017127 and ETT 587/93     

Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the presence of enterochromaffin cells

Summary The formaldehyde-induced fluorescence technique had shown 5-hydroxytryptamine-containing enteric neurons in the intestine of the teleost Platycephalus bassensis, but did not reveal such neurons in the intestine of Tetractenos glaber or Anguilla australis. Re-examination of these animals with 5-hydroxytryptamine immunohistochemistry showed immunoreactive enteric neurons in the intestine of all three teleost species. The 5-hydroxytryptamine-containing enteric neurons showed essentially the same morphology in all species examined: the somata were situated in the myenteric plexus, extending down into the circular muscle layer, but none were found in the submucosa; processes were found in the myenteric plexus, the circular muscle layer and the lamina propria. It was concluded that the neurons may innervate the muscle layers or the mucosal epithelium, but were unlikely to be interneurons. In a range of teleosts, enterochromaffin cells were found in the intestine of only those species in which the formaldehyde technique did not visualize neuronal 5-hydroxytryptamine. Available evidence suggests that, in vertebrates, 5-HT-containing enterochromaffin cells are lacking only where there is an innervation of the gut mucosa by nerve fibres containing high concentrations of 5-HT.     

Diffuse serotoninergic neurohemal systems associated with cerebral and suboesophageal nerves in the head of the Colorado potato beetle Leptinotarsa decemlineata

We analyzed the anatomy of two diffuse neurohemal systems for serotonin in the head of the Colorado potato beetle Leptinotarsa decemlineata by means of immunohistochemistry. One system is formed by axons from two bilateral pairs of neurons in the frontal margin of the suboesophageal ganglion that enter the ipsilateral mandibular nerve, emerge from this nerve at some distance from the suboesophageal ganglion, and cover all branches of the mandibular nerve with a dense plexus of immunoreactive axon swellings. The other system is formed by axons from two large neurons in the frontal ganglion that enter the ipsilateral frontal connectives, emerge from these connectives, and form a network of axon swellings on the labroforntal, pharyngeal, and antennal nerves and on the surface of the frontal ganglion. Immunohistochemical electron microscopy demonstrated that the axon swellings are located outside the neural sheaths of the nerves and hence in close contact with the hemolymph. We therefore suggest that these plexuses represent extensive neurohemal systems for serotonin. Most immunoreactive terminals are in direct contact with the hemolymph, and other terminals are closely associated with the muscles of the mandibles, labrum, and anterior pharynx, as well as with the salivary glands, indicating that these organs are under serotoninergic control.     

The linear cable theory as a model of gill blood flow

The vascular organization of the teleost gill suggests that blood flow distribution from the filamental artery to the respiratory lamellae is governed by relationships analogous to the cable conduction properties of a nerve axon. The space constant (λ) by definition is the distance along the gill filament at which the in-series resistance of the afferent filament artery equals the in-parallel resistance of the afferent lamellar arteriolar, lamellar, efferent lamellar arteriolar (ALA-L-ELA) segments. Constriction of the afferent filamental artery or uniform dilation of the ALA-L-ELA will decrease λ. As λ decreases, flow through the proximal (basal) lamellae greatly increases at the expense of distal lamellar perfusion. When λ increases in a system of finite length the flow profile must account for reflected pressures within the main vessel. The λ calculated from corrosion casts of gill vasculature is $\text{1}\text{4}$ to $\text{1}\text{2}$ the filament length. This favors blood flow through the proximal lamellae and when cardiac output increases, the proportion of cardiac output perfusing the proximal areas increases at the expense of distal lamellar blood flow. To offset these changes it is proposed that increased distal lamellar perfusion is achieved by simultaneous vasodilatation of distal and constriction of proximal ALA-L-ELA segments and dilation of the afferent filamental artery.     

The distribution of corticotropin-releasing factor-immunoreactive neurons and nerve fibers in the brain of the snake,Natrix maura

Summary The anatomical distribution of neurons and nerve fibers containing corticotropin-releasing factor (CRF) has been studied in the brain of the snake, Natrix maura, by means of immunocytochemistry using an antiserum against rat CRF. To test the possible coexistence of CRF with the neurohypophysial peptides arginine vasotocin (AVT) and mesotocin (MST) adjacent sections were stained with antisera against the two latter peptides. CRF-immunoreactive (CRF-IR) neurons exist in the paraventricular nucleus (PVN). In some neurons of the PVN, coexistence of CRF with MST or of CRF with AVT has been shown. Numerous CRF-IR fibers run along the hypothalamo-hypophysial tract and end in the outer layer of the median eminence. In addition, some fibers reach the neural lobe of the hypophysis. CRF-IR perikarya have also been identified in the following locations: dorsal cortex, nucleus accumbens, amygdala, subfornical organ, lamina terminalis, nucleus of the paraventricular organ, nucleus of the oculomotor nerve, nucleus of the trigeminal nerve, and reticular formation. In addition to all these locations CRF-IR fibers were also observed in the lateral septum, supraoptic nucleus, habenula, lateral forebrain bundle, paraventricular organ, hypothalamic ventromedial nucleus, raphe and interpeduncular nuclei.