Bernard Renaud

Distribution of alpha-1 and alpha-2 binding sites in the rat locus coeruleus

Precise anatomical distribution of alpha-1 and alpha-2 adrenergic binding sites has been investigated in the rat locus coeruleus (LC) using quantitative radioautography of brain sections incubated with 3H-prazosin or 3H-idazoxan. Distribution patterns of 3H-prazosin (alpha-1 sites) and 3H-idazoxan (alpha-2 sites) were heterogeneous and different along a postero-anterior axis in the LC. Comparison between distribution of alpha-2 binding sites and noradrenergic (NA) cellular density suggests that at least a fraction of these sites might be localized on NA perikarya or dendrites in this structure. Quantitative estimations of the binding parameters along this postero-anterior axis in the LC have revealed that the heterogeneous distributions of alpha-1 and alpha-2 binding sites are due not only to variations in the maximal densities of sites but also to variations in the affinities of these sites for their respective ligand.

Baroreflex-linked variations of catecholamine metabolism in the caudal ventrolateral medulla: an in vivo electrochemical study

In vivo electrochemical recordings of the metabolism of catecholamines were obtained in the caudal ventrolateral medulla in anesthetized rats submitted to various experimental changes in systemic arterial pressure. Hypertension induced with phenylephrine and reversal of hypovolemia decreased the catechol metabolic activity. In contrast, controlled or hypovolemic hypotension, induced respectively with sodium nitroprusside or blood withdrawal (30% of blood volume), reversibly elicited the opposite pattern. This was suppressed by deafferentation. The changes in catechol metabolic activity in response to hypovolemia were accompanied by similar trends of variations of plasma vasopressin levels. By contrast with the increased catechol metabolic activity secondary to hypotension induced by either prazosin, sodium nitroprusside or hypovolemia, clonidine elicited a decrease in catechol metabolic activity. These data show a dynamic and specific involvement of the metabolism of catecholamines themselves promoted by changes in systemic arterial pressure. This pattern of functioning of catechol metabolism in the caudal ventrolateral medulla appears to be negatively related to systemic arterial pressure changes, a finding which does not fit with the proposed vasodepressor role of the A1-group.

Microdialysis monitoring of variations in extracellular levels of serotonin, GABA and excitatory amino acids in the frontal cortex of awake rats in response to a single peripheral or central administration of dexfenfluramine

The effects of a single dexfenfluramine (D-fen) administration on the release of endogenous serotonin (5-hydroxytryptamine, 5-HT), excitatory (glutamate, Glu, aspartate, Asp) and inhibitory (gamma-aminobutyric acid, GABA) amino acids from the frontal cortex were studied by using in vivo microdialysis in freely-moving rats. Extracellular levels of these neurotransmitters were measured with HPLC coupled to electrochemical detection or with capillary electrophoresis coupled to laser-induced fluoresence detection (CE-LIFD). In a first study, single intraperitoneal administration of D-fen (0.5, 1.3, 5 and 10 mg/kg) increased extracellular 5-HT levels in a dose-dependent manner (maximal increase by 982% over baseline for the highest dose) while changes in Glu, Asp or GABA never reached statistical significance. In a second study, 73 nM of D-fen applied locally through the frontocortical dialysis probe, at a flow rate of 1.5 microliters/min in 30 microliters of perfusion fluid for 20 min, increased extracellular 5-HT and Asp levels [the maximal increases were to 1804% and 280% of the respective basal values (100%)] without altering extracellular levels of Glu and GABA. Thus, the order of magnitude of the changes induced by systemic administration or local infusion of D-fen on frontocortical extracellular levels of several neurotransmitters (5-HT > > Asp > GABA = Glu) demonstrate that D-fen, an indirect serotoninergic agonist, mainly increases 5-HT release while producing slight (Asp) or no (Glu, GABA) short-term in vivo variations in amino acid extracellular levels in the rat frontal cortex.

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

Abstract: The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (< 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.

In vivo microdialysis study of the extracellular 3,'4-dihydroxyphenylacetic acid in the rat locus ceruleus : topographical and pharmacological aspects

Abstract: In vivo microdialysis coupled with HPLC and electrochemical detection was used to monitor extracellular levels of 3, 4‐dihydroxyphenylacetic acid (DOPAC) of the locus ceruleus (LC) in halothane‐anesthetized rats. The identity of DOPAC was confirmed by experiments showing that the chromatographic peak was totally suppressed after inhibition of monoamine oxidase by pargyline. Histological examinations allowed to relate the quantity of DOPAC measured in the dialysates with the localization of the probe implantation site. We found that the DOPAC concentration was inversely proportional to the distance between the probe and the lateral border of the LC. Regardless of the caudorostral level of the nucleus, concentrations were maximal when the axis of the probe was 100 μM from the lateral border of the LC and decreased by 53% when this distance reached 300 μM. Activation of LC noradrenergic neurons by systemic administration of the α2‐antagonist piperoxane increased by 100% DOPAC concentrations in LC dialysates. These results suggest that the DOPAC measured by microdialysis could be considered an indicator of the functional state of LC noradrenergic neurons.

Pharmacological and functional evidence for extracellular 3,4-dihydroxyphenylacetic acid as an index of metabolic activity of the adrenergic neurons: An in vivo voltammetry study in the rat rostral ventrolateral medulla

Catecholamine metabolism was studied in vivo in the C1 adrenergic area of the rostral ventrolateral medulla oblongata in rats, using differential normal pulse voltammetry coupled with an activated carbon fiber microelectrode. Pharmacological evidence indicates that 3,4-dihydroxyphenylacetic acid, the major dopamine metabolite, is responsible for the electrochemical signal appearance in the C1 group, and that it reflects the catecholamine synthesis rate, as previously reported in the locus coeruleus. Indeed, 3,4-dihydroxyphenylacetic acid was estimated to be formed from 77% of the intracellular dopamine, since its synthesis was increased by only 23%, after blockade of the dopamine-beta-hydroxylase activity. Neuronal activation by retrograde electrical stimulation increased the electrochemical signal, as well as hemorrhage and hypotension, suggesting that the level of extracellular 3,4-dihydroxyphenylacetic acid is a good biochemical index of the C1 adrenergic cellular activity in baseline conditions and during cellular activation.

Decrease in the Reactivity of Locus Coeruleus Neurons to Hypotension after an Increase in their Tyrosine Hydroxylase Content: A Subregional In Vivo Voltammetry Study in the Rat

The aim of the present work was to determine if noradrenergic neurons of the anterior and the posterior subregions of the locus coeruleus exhibit a difference in reactivity in response to sodium nitroprusside‐induced arterial hypotension, and if the pharmacological induction of tyrosine hydroxylase by RU24722 modifies the reactivity of locus coeruleus neurons to this hypotensive stimulus. Previous findings have demonstrated that administration of RU24722 increases the concentration of tyrosine hydroxylase in the rat locus coeruleus by two different mechanisms in the anterior and in the posterior locus coeruleus subregions. The goal of the present study was to measure in vivo the changes in catecholaminergic metabolism in the locus coeruleus after treatment with RU24722 using differential normal pulse voltammetry (DNPV). In vehicle‐treated rats, arterial hypotension increased catecholaminergic metabolism with the same pattern in the two locus coeruleus subregions. However, the changes in the magnitude of the catechol oxidation current throughout the recording period were significantly smaller in the posterior subregion (P < 0.001). In the RU24722‐pretreated rats, there was a 39% increase in tyrosine hydroxylase and dihydroxyphenylacetic acid in the locus coeruleus. The functional reactivity to hypotension measured by DNPV was significantly decreased (P < 0.001) in both the anterior and posterior locus coeruleus subregions with RU24722 treatment. Therefore, this study suggests that the response of locus coeruleus cells to a hypotensive stimulus depends upon the intracellular tyrosine hydroxylase concentration both in the basal condition and during pharmacological induction of tyrosine hydroxylase gene expression.

Central and peripheral changes in catecholamine-synthesizing enzyme activities after systemic administration of the neurotoxin DSP-4

In brain regions containing noradrenergic (NA) cell bodies or terminals, DSP-4 induces changes in the activity of catecholamine-synthesizing enzymes which suggest that central NA neurons are lesioned by this neurotoxin. In contrast, the lack of change in the same enzymatic activities in an area containing mostly adrenergic (A) neurons (C2 region), favors the hypothesis of a resistance of the A neurons to DSP-4. Furthermore, the enzymatic changes observed in peripheral organs suggest a peripheral activation of the NA cell bodies in response to lesioning of the sympathetic terminals by DSP-4.

Effects of chronic β-blockers treatment on catecholamine synthesizing enzymes in spontaneously hypertensive rats

Abstract In the present work the effects of two β-blocking agents (Propranolol: 40 mg/kg/day and S 2395: 20 mg/kg/day) on the activity of central and peripheral catecholaminergic (CA) structures were studied in SHR after 55 days of oral treatment. These effects were assessed by measuring the activity of tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine- N -methyl-transferase (PNMT) in different structures dissected out from treated and control SHR. At the peripheral level, the only significant change was a decrease in the DBH activity of the adrenal medulla in propranolol-treated SHR. The plasma DBH activity was not altered. In the pons medulla, propranolol and S 2395 increased the TH activity of the A 6 , C 1 and C 2 region, and propranolol decreased the DBH activity in the A 6 and the C 2 region. The PNMT activity of these 3 neuronal groups remained unchanged. In the hypothalamus, TH and DBH activity exhibited no consistent changes. On the other hand, the PNMT activity was significantly reduced by propranolol in the anterior hypothalamus and in the median eminence but not in the posterior hypothalamus. The effects of β-blockers on the activity of central and peripheral CA structures were markedly different from those observed in similar conditions after chronic treatment with hydralazine, a peripheral vasodilator. Therefore, it was concluded that the enzymatic changes observed were not a consequence of the treatment induced decrease in blood pressure, but might more probably reflect a specific action of β-blockers on the central and peripheral CA structures of the SHR.

Microdialysis Monitoring of 3,4‐Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on‐line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4‐dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl‐DOPA, which was shown to be more stable than NSD 1015 or Ro 4‐4602 in the perfusion fluid. A 1‐h perfusion of a 10−4 mol/L of difluoromethyl‐DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α‐methyl‐p‐tyrosine. Systemic administration of the α2‐antagonist piperoxane before difluoromethyl‐DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady‐state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.