Nicole Sarda

Voltammetric detection of the release of 5-hydroxyindole compounds throughout the sleep-waking cycle of the rat

SummaryIn the present work, differential pulse voltammetry (DPV) measurements of the extracellular fraction of 5-hydroxyindole compounds were performed in rats under long-term chronic conditions. In the nucleus Raphe Dorsalis (n.RD), the voltammetric signal measured at +300 mv (peak 3) disappeared completely 70 to 90 min after injection of Clorgyline (10 mg/kg), a monoamine oxidase inhibitor type A (MAOI-A); the signal measured in such conditions is thus dependent upon extracellular 5-hydroxyindoleacetic acid (5-HIAA peak 3). Deprenyl, an MAOI type B, at the same dose, induced only a slight increase in peak 3 height; according to the fact that MAO-B is selectively located in the 5-HT neurons and since their inhibition does not decrease 5-HIAA peak 3 nor the endogenous 5-HIAA content as measured with High Performance Liquid Chromatography (HPLC), 5-HIAA measured with DPV in the extracellular fluid of untreated animals might come from 5HT released and metabolized by MAO-A outside the 5-HT neurons. In animals implanted for measurements of both voltammetric and polygraphic parameters, the 5-HIAA peak 3 measured mainly in the anterior and ventral part of the n.RD exhibited large increases in its height during slow-wave sleep (SWS: +39%) and paradoxical sleep (PS=+71%) as compared to the waking state (W=100%); these variations could reflect the dendritic release of 5-HT. In the Caudate nucleus (n.Cd) the same voltammetric signal presented reverse fluctuations, i.e. an increase during W and a decrease during SWS and PS. Intracerebroventricular administration of Corticotropin-Like Intermediate lobe Peptide (CLIP, 10 ng/2 μl) induced an increase in PS duration (+51%) preceded and accompanied by an increase in the n.RD 5-HIAA peak 3 height (+50%).

Detection of the release of 5-hydroxyindole compounds in the hypothalamus and the n. raphe dorsalis throughout the sleep-waking cycle and during stressful situations in the rat: a polygraphic and voltammetric approach

SummaryIn the present work, voltammetric method combined with polygraphic recordings were used in animals under long-term chronic conditions; the extracellular concentrations of 5-hydroxyindole compounds (5-OHles) and in particular 5-hydroxyindoleacetic acid (5-HIAA) were measured in the hypothalamus and in the nucleus Raphe Dorsalis (n.RD). The hypothesis that extracellular detection of 5-HIAA, in animals under physiological conditions, might reflect serotonin (5-HT) release is suggested by the following observations: — serotoninergic neurons are reported to contain only monoamine oxidase type B (MAO-B); — an inhibitor of such an enzyme, MDL 72145 (1 mg/kg), fails to decrease the extracellular 5-HIAA peak 3 height; — MAO type A is contained in non-5-HT cells or neurons; — only the inhibitor of this last type of enzyme (Clorgyline 2.5 mg/kg) induces a complete disappearance of the voltammetric signal. The 5-HIAA measured in the extracellular space thus comes from the 5-HT released and metabolized outside the 5-HT neurons. Throughout the sleep-waking cycle, 5-OHles release occurs following two different modes: 1 — during sleep, in the vicinity of the 5-HT cellular bodies in the n.RD; this release might come from dendrites and be responsible for the 5-HT neuronal inhibition occurring during sleep; 2 — during waking, at the level of the axonal nerve endings impinging on the hypothalamus; this release might be related to the synthesis of “hypnogenic factors”. Finally, we have observed that in the hypothalamus, 30 min. of immobilization-stress (IS) induces a larger increase of the voltammetric signal (+ 80%) than a painful stimulation of the same duration (+ 30%); the possible link between the 5-OHles release occurring in this area during an IS and the subsequent paradoxical sleep rebound is discussed.

CHANGES IN THE SLEEP-WAKE CYCLE ARCHITECTURE AND CORTICAL NITRIC OXIDE RELEASE DURING AGEING IN THE RAT

Changes in sleep-wake states and nitric oxide release were examined in aged rats versus young-adult ones. Sleep-wake recordings and nitric oxide measurements were taken from animals chronically equipped with polygraphic and voltametric electrodes. Animals were examined in baseline conditions and in response to a 24-hour paradoxical sleep deprivation. In aged rats, basal amount of paradoxical sleep is decreased during the light phase versus young-adult animals. After paradoxical sleep deprivation, a paradoxical sleep rebound occurs with an amount and intensity that are less marked in aged animals than in young-adult rats. The amplitude of the circadian distribution for wakefulness, slow-wave sleep and paradoxical sleep amounts is reduced with age. Finally, delta-slow-wave sleep and theta-paradoxical sleep power spectra are attenuated either in baseline conditions or after paradoxical sleep deprivation in aged animals. It is also reported that cortical nitric oxide release exhibits a circadian rhythm with higher amplitude in aged rats than in young-adult ones. However, after paradoxical sleep deprivation, a limited overproduction of nitric oxide is obtained compared with young-adult ones. These results, evidencing the dynamics of the nitric oxide changes occurring in relation to the sleep-wake cycle, point out the homeostatic paradoxical sleep regulation as an age-dependent process in which the nitric oxide molecule is possibly involved.

Biochemical and autoradiographic measurements of brain serotonin synthesis rate in the freely moving rat: a reexamination of the alpha-methyl-L-tryptophan method.

Abstract: Biochemical approaches were used in freely moving rats to determine, under steady‐state conditions, the brain/arterial plasma partition coefficients of L‐tryptophan and α‐[3H]methyl‐L‐tryptophan, from which the lumped constant for the α‐methyl‐L‐tryptophan method of estimating the rate of brain serotonin synthesis is calculated. The lumped constants were significantly different in the various structures examined: 0.149 ± 0.003 in the raphe dorsalis, 0.103 ± 0.002 in the raphe centralis, 0.087 ± 0.003 in the reticular formation, and 0.62 ± 0.08 in the pineal gland. From these data we proposed a two‐compartment model to calculate the rate of serotonin synthesis by quantitative autoradiography using a three‐time point experiment. Rates of synthesis for the raphe dorsalis and the reticular formation (620 ± 57 and 80 ± 35 pmol/g of tissue/min, respectively) were similar to those measured simultaneously by biochemical means, but rates were 50% higher for the raphe centralis (568 ± 90 vs. 381 ± 31 pmol/g of tissue/min). The lack of dynamic equilibrium of the tracer between plasma and tissue pools may explain the discrepancy between the two methods. Our findings did not confirm previous data, indicating that the application of the autoradiographic method to measure the rate of brain serotonin synthesis using α‐methyl‐L‐tryptophan as tracer has limitations.

Sleep wake profile and EEG spectral power in young or old senescence accelerated mice

Changes occurring with age in cortical EEG and sleep-wake states architecture were examined in senescence accelerated prone (SAMP8) or senescence resistant (SAMR1) mice (age: 2 and 12 months) under baseline conditions or after a 4 h sleep deprivation (SD). In baseline conditions, an increase in slow wave sleep (SWS) amount (21-24%) occurs at the expense of the wakefulness (W) in old SAMP8 and SAMR1 mice versus young animals. In these conditions, SWS latency is reduced (67-72%). Moreover, in SAMP8 and SAMR1 mice, aging deteriorates paradoxical sleep (PS) architecture with more pronounced changes in SAMP8 (amount: -63%; episode duration: -44%; latency: +286%; circadian component loss; and EEG theta (theta) peak frequency (TPF): -1 Hz). During the 4 h recovery subsequent to a 4 h sleep deprivation, old SAMP8 mice exhibit an enhanced sensitivity resulting in SWS (+62%) and PS (+120%) rebounds, a characteristic of this inbred strain. Results obtained are discussed in line with the age-related learning and memory impairments existing in SAMP8 animals. In particular, the reduced cognitive performances described in old SAMP8 might be linked to the TPF deterioration during PS.

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

Abstract: The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady‐state conditions, the specific activity of L‐[35S]methionine in the tRNA‐bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L‐[35S]‐Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyIadenosine (3H]CHA), R‐N6‐[3H]phenylisopropyladenosine ([3H]R‐PIA), and 5′‐N‐ethylcarboxamido[3H]adenosine ([3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A‐1‐selective ligands (CHA and R‐PIA) had ±10% specific binding. By contrast, [3H]NECA, a nonselective A‐l/A‐2 li‐gand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 M) or R‐PIA (50 μM). To characterize this binding, we sed the N‐ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 ± 10 nM and an apparent Bmax of 1,060 ± 239 fmol/ mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylyliinidodi‐ phosphate (100 μM) (‐25%), a result suggesting the involvement of an N‐protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA ± 2‐chloroadenosine ±S‐adenosyl‐L‐homocysteine ± CHA. Binding was also displaced by 3‐isobutyl‐l‐meth‐ylxanthine (IC50= 23.6 μM). These findings are consistent with the selective labeling by [3H]NECA of an A‐2‐type adenosine receptor in rat pineal membranes.

Changes occurring in cortical NO release and brain NO‐synthases during a paradoxical sleep deprivation and subsequent recovery in the rat

Variations occurring in cortical nitric oxide (NO) release were analysed with a voltametric method in rats (i) placed in control conditions, (ii) while being paradoxical sleep deprived (PSD), or (iii) recovering from a PSD. Activities of neuronal (nNOS) and inducible (iNOS) NO‐synthases as well as nNOS expression were also determined in several brain regions. In baseline conditions, circadian variations in nNOS expression and activity were maximal during the dark period and minimal during the light one for all the structures analysed (frontal cortex, pons and medulla). In the same way, cortical NO release occurred through a circadian rhythm exhibiting maxima and minima during dark and light periods, respectively. In the same experimental conditions, iNOS activity did not exhibit time‐dependent changes. The correlative changes observed in baseline conditions between NO release, nNOS expression and activity within the frontal cortex were disrupted during PSD and subsequent recovery. Still again, iNOS activity remained unchanged. Results obtained point out that the tight coupling existing in control conditions between nNOS expression‐activity and NO release is disrupted by a PSD and remains affected during the subsequent 24 h recovery. Their significance is discussed.

Effect of an n‐3 Fatty Acid‐Deficient Diet on the Adenosine‐Dependent Melatonin Release in Cultured Rat Pineal

Abstract: We studied the effect of a diet deficient in n‐3 fatty acids on the adenosine‐dependent melatonin release from cultured rat pineal gland after stimulation by 5′‐N‐ethylcarboxamidoadenosine (NECA), an A2 adenosine agonist. Experiments were conducted with 2‐month‐old rats raised on semipurified diets containing either peanut oil (n‐3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n‐3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n‐3‐deficient rats. This was compensated for partially by an increase in 22:4 n‐6 and 22:5 n‐6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N‐acetylserotonin and melatonin release in the medium, was lower after stimulation by 10‐5 mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n‐6/n‐3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane‐bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.

Docosahexaenoic acid (cervonic acid) incorporation into different brain regions in the awake rat

A quantitative method is presented to examine the localization, in individual brain regions of awake rats, of docosahexaenoic acid (22:6 n-3 or cervonic acid), the main polyunsaturated fatty acid of the nervous system together with arachidonic acid. Following the intravenous injection of 10 microCi [14C]22:6 n-3 (around 0.2 mumol/rat). 0.11-0.28% of the initial radioactivity was located in specific brain areas after detection from 10 to 240 min. Brain regional radioactivity determined by quantitative autoradiography indicated that 60 min after injection, [14C]22:6 n-3 concentrations ranged from 13.75 nCi/g of tissue in inferior olive to 5.59 nCi/g in frontal cortex. The results indicate a higher incorporation into the auditory system: inferior colliculus, central cochlear nucleus, lateral lemniscus, into neuroendocrine structures: paraventricular and supraoptic nuclei, and into certain circumventricular organs such as the pineal gland and neurohypophysis. Analysis of the Bligh and Dyer lipid extracts of rat brain revealed that 60 min after injection, 80-85% of the radioactivity was in choline and ethanolamine phosphoglycerides. These observations suggest that intravenous injection of [14C]22:6 n-3 may be used to study the brain lipid compartmental metabolism in vivo in order to visualize alterations of structural lipid components.