Monitoring nitric oxide (NO) in rat locus coeruleus: differential effects of NO synthase inhibitors.

A porphyrinic microsensor combined with in vivo voltammetry was used to monitor extracellular nitric oxide (NO) in the locus coeruleus (LC) of anaesthetized rats. Administration of N omega-nitro-L-arginine p-nitro-anilide (100 mg/kg, i.p) or 7-nitro indazole (30 mg/kg, i.p.), which both inhibit preferentially neuronal NO synthase (NOS), induced a marked decrease in the NO oxidation peak height. On the other hand, N omega-nitro-L-arginine methyl ester (L-NAME) (200 mg/kg, i.p.), a less selective NOS inhibitor, failed to decrease the NO signal. Moreover, intra LC administration of NMDA, known to activate LC noradrenergic neurones, increased the NO signal. This study demonstrates the usefulness of in vivo voltammetry to monitor basal levels of NO and their changes in the LC. Differential effects of NOS inhibitors show that their central activity need to be assessed through in situ measurement of NO before using these inhibitors as neuropharmacological tools.

In vitro study of the catecholaminergic metabolism of locus coeruleus neurones by differential normal pulse voltammetry.

The aim of the present work was to measure, by voltammetry, the catecholaminergic metabolic activity of rat locus coeruleus (LC) neurones in brain slices. For this new experimental approach, we used an optimized protocol of slice preparation intended to prevent neuronal damages due to brain ischaemia. Our results show that the LC neurones exhibit in vitro a stable spontaneous catecholaminergic metabolic activity and that, as in vivo, 3,4-dihydroxyphenylacetic acid (DOPAC) is likely to be the main contributor to the recorded signal. This catecholaminergic metabolic activity can be pharmacologically altered by administering carbachol and clonidine to the superfusion fluid. We also determined the values of bath temperature and superfusion flow rate providing, in our methodological conditions, an optimal catecholaminergic metabolic activity. Finally, we took advantage of both the direct accessibility to the LC and the compactness of this nucleus to determine the spatial resolution of differential normal pulse voltammetry. In conclusion, the study of the subregional mechanisms controling the catecholaminergic metabolism in LC neurones can be performed in brain slices by differential normal pulse voltammetry.

In vivo voltammetry and microdialysis monitoring of monoamine metabolism in the rat brainstem neurons.

Two "in vivo" techniques allow the monitoring of extracellular levels of monoamines and related compounds in selected rat brainstem regions: voltammetry and microdialysis. "In vivo" voltammetry has a high regional selectivity: for example, we have been able to perform a subregional study and to show that the increase in extracellular DOPAC induced by 30 min-hypotension was twice as larg in the rostral as in the caudal rat locus coeruleus. The spatial resolution, as expressed by x 1/2 (see text), is 4 times better for voltammetry (50 microns) than for microdialysis (190 microns). Another advantage of voltammetry is its excellent time resolution. However, microdialysis has a much better biochemical specificity than voltammetry. Furthermore it allows some enzymatic activities, such as tyrosine hydroxylase, to be measured almost continuously in catecholaminergic brain nuclei. From a functional point of view, the results of our experiments (alpha 2 ligand administration, arterial hypotension or stress) illustrate the respective interest and complementarity of these two "in vivo" techniques. Their current developments will lead to a better temporal and biochemical resolution combined with an increase in the number of substances analyzed "in vivo", including peptides and nitric oxide.

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.

Relationship between tyrosine hydroxylase content and noradrenergic cell reactivity to piperoxane: an in vivo voltammetric approach in the rat locus coeruleus.

A previous electrochemical study showed that the increase in the tyrosine hydroxylase (TH) content of the locus coeruleus (LC) produced by RU24722 administration was associated with a relative decrease in the catecholaminergic metabolic reactivity of this nucleus to a hypotensive stimulus. Since alpha 2 receptors participate in the regulation of the activity of both LC neurons and TH, the aim of the present work was to evaluate the possible involvement of the autoinhibition mediated by alpha 2 autoreceptors in the inverse relationship between the reactivity of the LC and its TH content. Our study was divided into two successive steps: (i) the electrochemical measurement of the in vivo metabolic activation of LC cells in response to alpha 2-adrenergic receptor blockade, and (ii) the evaluation of the quantity of TH every 100 microns along the caudorostral axis in each recorded LC. The capacity of TH protein to be activated was evaluated by the measurement, using differential normal pulse voltammetry, of the in vivo variations of the extracellular 3,4-dihydroxyphenylacetic acid concentrations in response to six cumulated doses of the alpha 2-antagonist piperoxane. The corresponding dose-response curves, determined in control- and RU24722-treated rats, were expressed as a function of the quantity of TH contained either in the whole recorded LC or in the 100 microns-wide coronal interval surrounding the recording site. It was established that the slopes of the dose-response curves were significantly (P < 0.01) and inversely related to the quantity of TH at the level of the recording site. This result suggests that the negative control of the catecholaminergic metabolic reactivity in a restricted area of the LC could be directly or indirectly dependent on the level of expression of TH protein in this particular area.