L'innervation monoaminergique de l'Eminence Médiane — Etude radioautographique et pharmacologique chez le CanardAnas platyrhynchos

Résumé Dans l'Eminence Médiane du Canard, la Noradrénaline et la Dopamine tritiées sont captées et retenues auniveau de la zone interne et en périphérie des vaisseaux portes hypophysaires. Cette rétention, observée par radioautographie après incubation in-vitro ou injection intraventriculaire des médiateurs, est considérablement diminuée par la réserpine. Une ou deux injections intraventriculaires de 6-Hydroxy-Dopamine laissent subsister quelques fibres capables de capter la Noradrénaline-³H. Après trois injections de 6-Hydroxy-Dopamine, on n'observe plus aucune incorporation du médiateur.Ces données radioautographiques et pharmacologiques attestent que les fibres de la zone interne de l'Eminence Médiane, qui captent la Noradrénaline et ses précurseurs et qui sont détruites par la 6-Hydroxy-Dopamine, sont de nature catécholaminergique.En microscopie électronique, on constate que les fibres marquées contiennent des vésicules de types divers avec fréquemment un coeur dense excentré. Ces fibres présentent des contacts synaptiques avec des dendrites ou péricaryons neuronaux qui ne retiennent pas le traceur et qui reçoivent également d'autres afférences synaptiques non marquées. Ce fait permet d'établir que les neurones infundibulaires présents dans l'Eminence Médiane sont susceptibles d'une régulation mettant en jeu des catécholamines et d'autres types de neurotransmetteurs.

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Monoaminergic innervation of the median eminence—Radioautographic and pharmacological study in the duck,Anas platyrhynchos I. Catecholaminergic innervation

Summary When³H-norepinephrine or³H-dopamine are given to the median eminence of the Duck after an injection into the IIIrd ventricle or afterin vitro incubation, an intense and preferential accumulation of the tracer is observed in the internal zone and in the vicinity of the primary portal vessels.The amount of labeled catecholamines in the median eminence is greatly diminished by reserpine. After one or two injections of 6-hydroxy-dopamine into the third ventricle followed by application of³H-norepinephrine, only a few fibers are labeled; after three injections of this drug, nearly no uptake of³H-norepinephrine is seen.This radioautographic and pharmacological approach indicates that the axons of the internal zone of the median eminence, which are able to take up and store labeled norepinephrine and its precursors and which are destroyed by 6-hydroxy-dopamine, are catecholaminergic.The ultrastructural examination of the internal zone shows that the label is confined to axonal varicosities containing various types of vesicles possessing frequently an eccentric core. The labeled axonal varicosities were found in synaptic contact with unlabeled dendrites and also with unlabeled perikarya. The postsynaptic structures which receive labeled presynaptic axons display also synaptic contacts with other unlabeled axons. This fact suggests that neurons of the median eminence are probably modulated by catecholamines and other neurotransmitters either on their dendrites and even on their soma.

Avec la collaboration de Melle. S. Bosc.     

The association of crustacean cardioactive peptide with the spermatheca of the African migratory locust, Locusta migratoria

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons in the nerves that project to the spermatheca of 3-4 week old adult female locusts. In addition, lightly stained CCAP-like immunoreactive processes were localized over the spermathecae. The amount of CCAP in the spermathecal tissue was quantified using an enzyme-linked immunosorbent assay (ELISA) performed on extracts of the whole spermatheca, and on its constituent parts, namely the sperm sac, coiled duct and straight duct. The spermatheca contains 920+/-273 fmol (mean+/-SE) of CCAP equivalents, with the majority localized in the coiled duct. There are age-related differences in the amount of CCAP present in the spermathecae with less content in spermathecae from 1 to 5 day old and greater content in spermathecae from 3 to 4 week old adults. There was also no difference in CCAP content of spermathecae in mated and virgin 3 to 4 week old adults. Reversed phase-high performance liquid chromatography (RP-HPLC) followed by ELISA further confirmed the presence of CCAP-like material in extracts of locust spermathecae. Physiological assays demonstrated that CCAP increased the basal tonus and frequency of spontaneous contractions of the spermatheca, with thresholds between 10(-10) and 10(-9)M and maxima at 10(-7)M CCAP. CCAP also increases the amplitude of neurally evoked contractions with a threshold less than 10(-11)M and a maximum of 10(-7)M CCAP. The present study suggests that CCAP acts as a neuromodulator/neurotransmitter at the spermathecal visceral tissue of female Locusta migratoria.     

The pregenital abdominal musculature in phasmids and its implications for the basal phylogeny of Phasmatodea (Insecta: Polyneoptera)

Recently several conflicting hypotheses concerning the basal phylogenetic relationships within the Phasmatodea (stick and leaf insects) have emerged. In previous studies, musculature of the abdomen proved to be quite informative for identifying basal taxa among Phasmatodea and led to conclusions regarding the basal splitting events within the group. However, this character complex was not studied thoroughly for a representative number of species, and usually muscle innervation was omitted. In the present study the musculature and nerve topography of mid-abdominal segments in both sexes of seven phasmid species are described and compared in detail for the first time including all putative basal taxa, e.g. members of Timema, Agathemera, Phylliinae, Aschiphasmatinae and Heteropteryginae. The ground pattern of the muscle and nerve arrangement of mid-abdominal segments, i.e. of those not modified due to association with the thorax or genitalia, is reconstructed. In Timema, the inner ventral longitudinal muscles are present, whereas they are lost in all remaining Phasmatodea (Euphasmatodea). The ventral longitudinal muscles in the abdomen of Agathemera, which span the whole length of each segment, do not represent the plesiomorphic condition as previously assumed, but might be a result of secondary elongation of the external ventral longitudinal muscles. Sexual dimorphism, common within the Phasmatodea, also applies to the muscle arrangement in the abdomen of some species. Only in the females of Haaniella dehaanii (Heteropteryginae) and Phyllium celebicum (Phylliinae) the ventral external longitudinal muscles are elongated and span the length of the whole segment, possibly as a result of convergent evolution.     

Regulation of cardiac rhythm in hibernating mammals

The dramatic fall in heart rate exhibited by mammals entering hibernation begins before there is any noticeable fall in body temperature. The initial, progressive decrease in heart rate is the result of a cyclic parasympathetic activation that induces skipped beats and regular asystoles as well as slows the even heart beat. As body temperature subsequently falls, the parasympathetic influence is progressively withdrawn and periods of parasympathetic and sympathetic dominance alternate and give rise to regular periods of arrhythmia (tachycardia followed by bradycardia), and occasional long asystoles or periods of highly irregular cardiac activity. Superimposed on this is a vagally-mediated, respiratory sinus arrhythmia that is accentuated in species that breathe episodically. These events give way to a uniform heart rate in deep hibernation at low temperatures where both parasympathetic and sympathetic tone appear absent. The complete absence of tone is not a function of reduced temperature but is reflective of the state of deep, steady state hibernation. The elevation in heart rate that accompanies the onset of arousal is the result of dramatic increases in sympathetic activation that precede any increases in body temperature. As body temperature then rises, sympathetic influence is slowly withdrawn. Arrhythmias are also common during natural arousals or shifts from lower to warmer hibernation temperatures as periods of parasympathetic and sympathetic dominance again alternate en route to re-establishing a steady state in euthermia. The mechanism behind, and the biological significance of, cardiac changes mediated through orchestrated arrhythmias remain unknown.     

Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster

Octopamine is an important neuroactive substance that modulates several physiological functions and behaviors of invertebrate species. Its biosynthesis involves two steps, one of which is catalyzed by Tyramine beta-hydroxylase enzyme (TBH). The Tbetah gene has been previously cloned from Drosophila melanogaster, and null mutations have been generated resulting in octopamine-less flies that show profound female sterility. Here, I show that ovulation process is defective in the mutant females resulting in blockage of mature oocytes within the ovaries. The phenotype is conditionally rescued by expressing a Tbetah cDNA under the control of a hsp70 promoter in adult females. Fertility of the mutant females is also restored when TBH is expressed, via the GAL4-UAS system, in cells of the CNS abdominal ganglion that express TBH and produce octopamine. This neuronal population differs from the dopamine- and serotonin-expressing cells indicating distinct patterns of expression and function of the three substances in the region. Finally, I demonstrate that these TBH-expressing cells project to the periphery where they innervate the ovaries and the oviducts of the reproductive system. The above results point to a neuronal focus that can synthesize and release octopamine in specific sites of the female reproductive system where the amine is required to trigger ovulation.     

Close apposition of dynorphin-positive nerve fibres to lymphocytes in the liver suggests opioidergic neuroimmunomodulation

The liver is innervated by sympathetic efferent, spinal afferent, vagal afferent and probably also vagal efferent fibres. To assess potential functional roles of the various neuronal subsets, data on transmitter systems are of crucial importance. This study was aimed at elucidating a possible opioidergic system in the mouse and rat liver. In particular relationships of opioidergic neurons to immune cells were emphasised. Material from perfusion-fixed mice (n=29) of different strains (BALB/c, NMRI, C57Bl6, SV 129 inbred) and Wistar rats (n=7) was cryosectioned at 12–14 m and incubated for single or double immunofluorescence. Antibodies directed against dynorphin A, met-enkephalin, endomorphin 1 and 2, -, - and -opioid receptors (MOR, KOR, DOR), tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), CD4, CD8 and macrophages were used. Binding sites were detected using Cy3-, FITC-, DTAF-, Cy2-, Alexa 555- and Texas red-tagged secondary antibodies. Specimens were analysed using confocal laser scanning microscopy (CLSM). Numerous nerve fibres staining for dynorphin were found in periportal areas of both mouse and rat livers. Neither met-enkephalin nor endomorphin could be detected in axons. No immunopositive neuronal cell bodies or other cellular elements were seen. All dynorphin positive fibres costained for TH while not every TH-positive fibre costained for dynorphin. Thus, most if not all dynorphin-positive nerve fibres may be of sympathetic origin. KOR immunostaining could be localised to round mononuclear cells which often costained for CD4, less frequently for CD8 and rarely for the pan-macrophage marker BM8. Altogether, about 45% of KOR-positive cells were identified as T-lymphocytes. In some instances, close appositions of dynorphin-positive axons to KOR-positive cells were revealed by CLSM. No KOR immunoreactivity was detected in nerve fibres. Hence, sympathetic neurons innervating the liver may interfere with inflammatory processes, in addition to their well-established ₂-adrenergic effect, via an opioidergic action on immune cells.     

Circadian variation in cardiac autonomic activity: reactivity measurements to different types of stressors

The role of endogenous circadian rhythmicity in autonomic cardiac reactivity to different stressors was investigated. A constant routine protocol was used with repeated exposure to a dual task and a cold pressor test. The 29 subjects were randomly divided into two groups in order to manipulate prior wakefulness. Group 1 started at 09:00 h immediately after a monitored sleep period, whereas group 2 started 12 h later. Measures of interbeat intervals (IBI), respiratory sinus arrythmia (RSA, a measure of parasympathetic activity), pre-ejection period (PEP, a measure of sympathetic activity), as well as core body temperature (CBT) were recorded continuously. Multilevel regression analyses (across-subjects) revealed significant (mainly 24 h) sinusoidal circadian variation in the response to both stressors for IBI and RSA, but not for PEP. Individual 24 + 12 h cosine fits demonstrated a relatively large interindividual variation of the phases of the IBI and RSA rhythms, as compared to that of the CBT rhythm. Sinusoidal by group interactions were found for IBI and PEP, but not for RSA. These findings were interpreted as an indication for endogenous circadian and exogenous parasympathetic (vagal) modulation of cardiac reactivity, while sympathetic reactivity is relatively unaffected by the endogenous circadian drive and mainly influenced by exogenous factors.     

Aldosterone modulates neural vasomotor response in hypertension: role of calcitonin gene-related peptide

Objective: We analyse the effect of aldosterone on vasomotor response induced by electrical field stimulation (EFS) in mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Results: Aldosterone (0.001–1 μM) reduced vasoconstrictor response to EFS in a dose- and time-dependent manner only in SHR. Thus, the rest of experiments were performed only in SHR. Aldosterone did not affect either noradrenaline response or release. Effect of aldosterone (1 μM) on EFS response was not affected by N^G-nitro-arginine-methyl esther (100 μM), and was abolished by capsaicin (0.5 μM) and the calcitonin gene-related peptide antagonist (CGRP 8–37, 0.5 μM). Calcitonin gene-related peptide (0.1 nM–0.1 μM) induced a concentration-dependent relaxation, which was enhanced by aldosterone (1 μM). Incubation with either spironolactone (1 μM), glibenclamide (10 μM), RU 486 10 μM, ODQ (10 μM) or cycloheximide (10 μM) significantly reduced the enhancement of CGRP-relaxation produced by aldosterone, while remained unmodified by SQ 22,536. Conclusions: Aldosterone decreases the vasoconstrictor response to EFS in mesenteric arteries from SHR but not from WKY. This effect is mediated by an increased response to the sensory neurotransmitter CGRP, substantially, through glucocorticoid receptors activation. Furthermore, this effect is mediated by an increase of cGMP synthesis and ATP-dependent potassium channel activation.     

Neuromast formation in the prehatching embryos of the Japanese flounder (Paralichthys olivaceus)

The present paper clarifies the initial development of the lateral line organs in the embryonic Japanese flounder, Paralichthys olivaceus. The first appearances of lateral line primordia, and the proliferation, distribution and morphological development of the free neuromasts, including nerve ending formation: establishment of hair cell innervations via the formation of synapses, were examined by light microscopy, scanning and transmission electron microscopy. The first pair of neuromast primordia appeared in the otic region ≈ 30 h prior to hatching and subsequently differentiated into free neuromasts, otic neuromasts, after ≈ 8 h. At hatching, a pair of free neuromasts and three pairs of neuromast primordia were present on the head, and three pairs of neuromast primordia were present on the trunk. The hair cell polarity of the otic neuromast until just prior to hatching was radial, but not bi‐directional. The typical afferent and efferent nerve endings in the otic neuromasts had formed by the time of hatching, suggesting that the otic neuromasts are functional prior to hatching. The three neuromast primordia located on each side of the trunk were derived from a long, narrow ectodermal cell cluster and erupted through the epidermis after hatching.     

A study of the peripheral innervation and muscle fibre types of Ictalurus nebulosus (Lesueur) and Ictalurus punctatus (Rafinesque)

The innervation pattern and fibre types of the axial musculature of two closely related catfish species with differing lifestyles, Ictalurus nebulosus (Lesueur) and I. punctatus (Rafinesque) were investigated. Both fish displayed the multiple innervation pattern in the red muscle. However, the white muscle of I. nebulosus demonstrated terminal innervation while I. puncrurus displayed multiply innervated white muscle fibres. Fibre typing utilizing histochemical techniques for glyco-gen, lipid, succinic dehydrogenase and glucose-6-phosphate dehydrogenase revealed the typical teleostean distribution of red, intermediate and white muscle fibres in both fish. Staining was greatest in the red muscle fibres and least in the white muscle fibres. The white muscle fibres of I. punctatus stained slightly more for lipid than the white fibres of I. nebulosus which may be correlated with a greater aerobic capacity related to lifestyle and possibly innervation.