Luc Denoroy

Effects of sinoaortic baroreceptor denervation on blood pressure and PNMT activity in medulla oblongata and spinal cord of normotensive and genetically hypertensive rats.

The effects of sinoaortic denervation on arterial blood pressure and central activity of phenylethanolamine-N-methyl transferase (PNMT, the last enzyme in adrenaline biosynthesis), were compared in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHR-SP). Denervation of the arterial baroreceptors caused immediate increases in mean arterial blood pressure (MAP) in all three strains which were maximal at 90 min (32 mmHg in WKY, 51 mmHg in SHR and 80 mmHg in SHR-SP). Spinal cord PNMT activity increased above sham-operated levels in WKY at 90 min, but PNMT levels in SHR and SHR-SP, already significantly higher than in WKY, were not altered acutely after sinoaortic denervation. Over a seven day period after baroreceptor denervation, MAP rose by 15 mmHg in WKY and PNMT activity was about 100% greater in spinal cord and ventral medulla. In the two genetically hypertensive strains sinoaortic denervation failed to produce a further sustained rise in pressure or and PNMT activity in the ventral medulla or spinal cord. We suggest that increased activity of bulbospinal adrenaline neurons contribute to the sustained elevation in pressure seen in intact SHR and SHR-SP, as well as in WKY after denervation of arterial baroreceptors.

Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis.

Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

Biochemical evidence for an interaction between adrenaline and noradrenaline neurons in the rat brainstem

In this study, we sought to determine if there was an interaction between the C2 adrenaline-containing (A) neurons of the rat medulla oblongata and the noradrenaline-containing (NA) cell bodies of the locus coeruleus (LC). For this purpose, the biochemical response of the NA cell bodies of the LC after a lesion of the C2 region was studied by using as markers the in vitro activities of the catecholamine synthesizing enzymes: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). An increase in TH activity, not associated with any change in DBH or PNMT activity, was found in the LC (+104%, P less than 0.001) 4 days after a bilateral electrolytic lesion (3 mA for 5 s) of the C2 region. Conversely, the electrolytic lesioning of the neighboring A2 region of NA neurons did not modify the TH activity of the LC. These results suggest the existence of an ascending adrenergic inhibitory control of the NA cell bodies of the LC.

Brainstem PNMT Neurons and Experimental Hypertension in the Rat

The number of phenylethanolamine-N-methyl transferase (PNMT) cells visualised with immunohistochemical techniques in the medulla oblongata is increased by 20% in 4 week old spontaneously hypertensive rats (SHR) and stroke prone spontaneously hypertensive rats (SHR-SP). This is associated with a 50% increase in the activity of PNMT and a significant rise in the amount of PNMT enzyme protein present in the medulla and spinal cord of both 4 weeks old and 4 months old SHR and SHR-SP. Since previous experiments had demonstrated that sinoaortic denervation also increased spinal cord PNMT activity we subjected normotensive Wistar Kyoto control rats (WKY) and hypertensive SHR and SHR-SP to denervation and measured the changes in blood pressure and in PNMT activity. Mean arterial pressure rose immediately after denervation in all 3 strains of rats, with much greater rises in the SHR and SHR-SP than in WKY, but the increase in pressure was only sustained in the normotensive WKY, in which it remained elevated throughout the one week observation period. In a similar way, denervation of the arterial baroreceptors increased the activity of PNMT in the medulla and spinal cord of normotensive WKY controls, confirming the results of previous studies but was not able to increase the already elevated PNMT levels in the SHR and SHR-SP any further in these two tissues. We suggest that there is good evidence that PNMT neurons contribute to the maintenance and elevation of arterial pressure in both the neurogenic and genetic models of hypertension. It also seems likely that the activity of descending spinal PNMT neurons is more important in the maintenance of a sustained increase in pressure than in the induction of a transient rise.

Fluorescence Enhancement of a Meisenheimer Complex of Adenosine by γ-Cyclodextrin: A Thermodynamic and Kinetic Investigation

The fluorescent properties of a trinitrophenylated Meisenheimer complex of adenosine (TNP-Ado) in water were examined in the presence of alpha-, beta-, and gamma-cyclodextrins (CDs). The TNP-Ado complex exhibits minimal fluorescence in water, whereas addition of 10 mM alpha-CD, beta-CD, and gamma-CD enhances fluorescence by factors of 2, 7, and 110, respectively. The large enhancement by gamma-CD is attributed to its larger hydrophobic cavity, which is able to accommodate the TNP moiety of TNP-Ado. (1)H NMR spectra demonstrate 1:1 stoichiometry of the complex, which undergoes slow exchange on the NMR time scale. (1)H NMR and 2D ROESY spectra reveal substantial interaction of the TNP hydrogens with gamma-CD. Equilibrium constants were determined by fluorimetry from 10 to 20 degrees C by nonlinear curve fitting. Fluorescence is temperature dependent, with maximum fluorescence increasing with decreasing temperature. Complexation is exothermic with large negative entropy, consistent with formation of a tight complex between TNP-Ado and gamma-CD. Rate constants and activation parameters for both complexation and dissociation were determined by a combination of fluorimetry and 2D NMR exchange spectroscopy (EXSY).

Central and peripheral catecholamine synthesizing enzymes during the development of two-kidney, one clip hypertension in rats.

The activities of the catecholamine synthesizing enzymes have been determined in discrete brain areas and in peripheral tissues of rats, at different times after clipping the left renal artery (two-kidney Goldblatt hypertension, 2KGH) and in sham operated animals. Three days after clipping the only enzymatic change was a slight decrease in plasma dopamine-beta-hydroxylase (DBH) activity. Ten days after clipping no change in enzymatic activity was found at the central level. However, the DBH and the phenylethanolamine-N-methyltransferase (PNMT) activities were increased in the adrenal medulla (+49.0%, P less than 0.001 and +36.6%, P less than 0.001, respectively) and DBH activity was also increased in the superior cervical ganglia (+22.8%, P less than 0.01). These data suggest that sympathetic hyperactivity is present in 2KGH rats when hypertension is established. In addition, as this type of hypertension does not alter the PNMT activity in brainstem areas, it seems that the alterations in PNMT activity reported for genetically hypertensive rats are unlikely to be secondary to the elevated blood pressure.

Enhanced Cocaine-Associated Contextual Learning in Female H/Rouen Mice Selectively Bred for Depressive-Like Behaviors: Molecular and Neuronal Correlates

Background: Major depression has multiple comorbidities, in particular drug use disorders, which often lead to more severe and difficult-to-treat illnesses. However, the mechanisms linking these comorbidities remain largely unknown. Methods: We investigated how a depressive-like phenotype modulates cocaine-related behaviors using a genetic model of depression: the Helpless H/Rouen (H) mouse. We selected the H mouse line for its long immobility duration in the tail suspension test when compared to non-helpless (NH) and intermediate (I) mice. Since numerous studies revealed important sex differences in drug addiction and depression, we conducted behavioral experiments in both sexes. Results: All mice, regardless of phenotype or sex, developed a similar behavioral sensitization after 5 daily cocaine injections (10 mg/kg). Male H and NH mice exhibited similar cocaine-induced conditioned place preference scores that were only slightly higher than in I mice, whereas female H mice strikingly accrued much higher preferences for the cocaine-associated context than those of I and NH mice. Moreover, female H mice acquired cocaine-associated context learning much faster than I and NH mice, a facilitating effect that was associated to a rapid increase in striatal and accumbal brain-derived neurotrophic factor levels (BDNF; up to 35% 24 h after cocaine conditioning). Finally, when re-exposed to the previously cocaine-associated context, female H mice displayed greater Fos activation in the cingulate cortex, nucleus accumbens, and basolateral amygdala. Conclusions: Our data indicate that neurobiological mechanisms such as alterations in associative learning, striato-accumbal BDNF expression, and limbic-cortico-striatal circuit reactivity could mediate enhanced cocaine vulnerability in female depressive-like mice.

Efferent mechanisms responsible for the bradycardia produced by lesions coinciding with the A1 group of central catecholamine neurons in the conscious rabbit.

1. The effector mechanisms responsible for the bradycardia evoked by bilateral lesions of the brainstem coinciding with the A1 catecholamine cells were analyzed in four groups of rabbits. Sham or lesion operations were carried out in animals with intact cardiac effectors, with cardiac sympathetic block induced by propranolol, with cardiac vagal block induced by methylscopolamine and with total cardiac autonomic block induced by the use of both drugs together.

Biochemical Approaches for Glutamatergic Neurotransmission

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system that gets accumulated into synaptic vesicles by vesicular glutamate transporters (vGluTs). Three isoforms of vGluTs have been cloned (vGluT1, vGluT2, and vGluT3), and shown by biochemical studies to selectively transport glutamate. The cloning of vGluTs has facilitated the anatomical and functional analysis of glutamatergic neurons, and while there are commercially available specific antibodies against vGluTs that label axonal terminals of glutamatergic neurons, the vGluT1 and vGluT2 proteins are undetectable in most cell bodies of glutamatergic neurons. Thus cellular detection of transcripts encoding vGluT1 or vGluT2 is so far the only available and reliable method to label cell bodies of glutamatergic neurons in wild-type animals. However, advances in viral cell-specific tagging of vGluTs neurons in transgenic mice has greatly facilitated the identification and manipulation of glutamatergic neurons. We will describe a multidisciplinary approach including neuronal track tracing, in situ hybridization, immuno-fluorescence, and immuno-electron microscopy that has allowed us the identification of glutamatergic neurons within the ventral tegmental area.