Regulation of expression and enzymatic activities of tyrosine and tryptophan hydroxylases in rat brain after acute electroconvulsive shock.

Acute electroconvulsive shock (ECS) causes a significant increase of protein synthesis in depressive patients and such an increase raises the possibility that the regulation of specific proteins and enzymatic activities in the brain might be one of the mechanisms required for the induction of long-term adaptive neurochemical changes after electroconvulsive therapy. In current studies, we investigated and compared simultaneously the short- and long-term effects of an acute ECS on the expression and enzymatic activities of both tyrosine and tryptophan hydroxylases (TH and TpOH, respectively) in different rat brain areas. Our results demonstrated that an acute ECS produced: (1) a long-lasting decrease in TH and TpOH protein levels in locus ceruleus (LC), ventral tegmental area (VTA) and in TpOH protein level in the raphe centralis (RC), maximal at 72 h, with concomitant changes in mRNA levels and enzymatic activities in the LC only; (2) large increase of TpOH protein levels in the frontal cortex (Cxf) (+145%) and increase of TH protein levels in the hippocampus (Hip) (+207%), maximal at 72 h and 7 days which was not accompanied by corresponding increase of in vivo enzymatic activities. Furthermore, a second ECS increased in vivo TpOH activity in the Cxf (+19%) while decreasing K(m) value (-50%) for tetrahydrobiopterin cofactor. A stability of the observed findings on TpOH activity in the Cxf after repeated ECS might be one of the mechanisms for the antidepressant effects of electroconvulsive therapy.

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

Biochemical approaches were used in freely moving rats to determine, under steady-state conditions, the brain/arterial plasma partition coefficients of L-tryptophan and alpha-[3H]methyl-L-tryptophan, from which the lumped constant for the alpha-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 alpha-methyl-L-tryptophan as tracer has limitations.

Early and prolonged widespread increase in brain protein synthesis following a single electroconvulsive shock in free-moving rats.

The autoradiographic method with l-[35S] methionine ([35S]Met) was used to determine the effect of a single electroconvulsive shock (ECS) on local rates of protein synthesis in the adult rat brain in free-moving conditions. We have estimated the relative contribution of methionine derived from protein breakdown to the intracellular precursor amino acid pool (tRNA pool) for protein synthesis. In steady-state conditions, we showed a large contribution (around 60%) of Met recycling into the precursor pool (lambda=0.37+/-0.11), after a single ECS. In all the 36 brain regions examined, apparent rates of protein synthesis were greatly increased (21-50%) 3 h after a single ECS indicating a generalized effect in rat brain. This ECS-induced activation of the overall rate of brain protein synthesis persisted for at least 24 h after cessation of ECS. This is consistent with the hypothesis that electroconvulsive therapy is associated with long-term molecular changes in neuronal activity.

Paradoxical metabolic response of the human brain to a single electroconvulsive shock.

Regional brain protein synthesis was evaluated with positron emission tomography (PET) and L-(S-[11C]methyl)methionine ([11C]MET) in depressive patients, before and 3 h after an electroconvulsive shock (ECS), when energy supply is restored, and in healthy volunteers. Depressive patients presented apparent lower protein synthesis than normals, in agreement with known reduction of cerebral activity. In contrast, ECS resulted in a significant increase (56%, P < 0.05) in global cortical protein synthesis. This paradoxical hyperactivation of cellular protein metabolism in response to seizures and the fact that synaptic activity is further reduced after electroconvulsive therapy (ECT), may provide new insights for understanding the mechanism of action of ECT.

Widespread increase in brain protein synthesis following acute immobilization stress in adult rat brain.

The autoradiographic method with [L-35S]methionine was used to determine the effects of a 2 h acute immobilization stress followed by a 4 h recovery on local rates of protein synthesis in the adult rat brain. Methionine incorporation into proteins was significantly increased (from 17 to 86%) in 37 out of the 39 analyzed brain structures. These results show that the stress-induced activation of the overall rate of brain protein synthesis may persist for at least 4 h after cessation of the stimulus even though the stress-related physiological variables have returned to basal levels. They suggest that increased protein synthesis may play a key role in the molecular events which lead to the neuronal plastic changes following an acute stress.

Cerebral protein synthesis alterations in response to acute and chronic immobilization stress in the rat.

The quantitative autoradiographic method with L-(35S)methionine was used to determine the effects of 1-acute (4h) and 2-chronic (14 days) immobilization stress followed by one week of recovery. Acute stress induced a significant decrease in methionine incorporation into proteins in 17 of the 35 brain structures examined (mean effect: -22%), and a significant increase in the prepositus hypoglossal nucleus (+23%). Chronic stress induced a significant decrease in methionine incorporation into proteins in 8 of the 35 structures analyzed. Only 4 structures were similarly affected in both these conditions. Our results indicate that stress-induced specific molecular changes in brain are also associated with changes in more general molecular components of cellular metabolism.

Decrease of brain phospholipid synthesis in free-moving n-3 fatty acid deficient rats.

The autoradiographic method with [14C]-docosahexaenoic acid ([14C]22:6 n-3) was used to determine whether a diet deficient in n-3 fatty acids, inducing a decrease in 22:6 n-3 circulating level, was associated with changes in local rates of phospholipid synthesis in the rat brain. As compared with rats fed a normal diet (peanut plus rapeseed oil), a n-3 fatty acid deficiency [peanut oil group (P group)] induced a generalized decrease (-35 to -76%) of 22:6 n-3 incorporation rates into phospholipids in all the regions examined. This effect was confirmed by using [3H]22:6 n-3 infusion by biochemical analysis and quantifications corrected for the contribution of docosahexaenoate derived from lipid store recycling to the unesterified pool, taken as the precursor pool for phospholipid synthesis in the whole brain. In normal or n-3 fatty acid-deficient rats, the values of the brain-to-plasma 22:6 n-3 specific activity ratio (psi) were similar (0.03), indicating that a considerable endogenous source of 22:6 n-3 (97%), likely derived from phospholipid degradation, dilutes the specific activity of the tracer coming from plasma. Using the specific activity of 22:6 n-3 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis. The results also demonstrate that the pattern of 14C or 3H distribution in brain lipids was not modified by the n-3 fatty acid-deficient diet. The major lipids labeled were phospholipids, particularly phosphatidylethanolamine. Nevertheless, the unesterified 22:6 n-3 concentrations in plasma and brain were significantly reduced (eight-and threefold, respectively) in the P group. In addition, the proportion of 22:6 n-3 in the brain total lipid fraction, total phospholipids, and phosphatidylcholine, -ethanolamine, and -serine was significantly decreased in n-3 fatty acid-deficient rats. This was partially compensated for by an increase in the 22:5 n-6 level. These results are discussed in relation to the limitation of 22:6 n-3 use to quantify, by the quantitative autoradiographic method, changes in local rates of phospholipid synthesis in rat brain.

Triiodothyronine does not affect the average incorporation of L-[35S]methionine in rat brain structures.

The autoradiographic method with L-[35S]methionine was used to examine the effect of acute administration of L-triiodothyronine on local rates of brain protein synthesis in free-moving adult rats. Triiodothyronine was given intraperitoneally at doses of 12.5 or 25 micrograms kg-1. It did not modify the rate of plasma methionine incorporation in the 40 brain regions examined, despite a 4- to 8-fold increase of plasma free triiodothyronine levels. Biochemical analysis confirmed that triiodothyronine (25 micrograms kg-1) had no apparent effect on the overall rate of protein synthesis in the brain as a whole. These results suggest that changes in the circulating levels of thyroid hormones do not exert a general and direct metabolic effect in brain of intact adult rats.

n-3 fatty acid deficiency increases brain protein synthesis in the free-moving adult rat.

The autoradiographic method with L-[35S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.

Effects of an acute immobilization stress upon proopiomelanocortin (POMC) mRNA levels in the mediobasal hypothalamus: a quantitative in situ hybridization study.

The aim of this study was to examine by quantitative in situ hybridization the effects of an acute stress on the expression of the POMC gene in the mediobasal hypothalamus (MBH) of the rat. In control animals, the highest levels of POMC mRNA were observed in the posterior periventricular region of the MBH. Lower levels were found in the anterior and posterior arcuate nucleus. At the end of a one hour immobilization, a small decrease (-8%) was observed in the periventricular region only. Four hours after the end of immobilization, increases in POMC mRNA levels were detected in the anterior part (7%), in the posterior part (25%) and in the periventricular region (13%) of the MBH. These results suggest that MBH POMC-derived peptides might be an important component in the central response to stress.

Evidence for brain docosahexaenoate recycling in the free-moving adult rat: implications for measurement of phospholipid synthesis.

The specific activity (SA) of unesterified docosahexaenoic acid (22:6 n-3) in the brain and arterial plasma was measured after constant intravenous infusion of [3H] 22:6 n-3 in the free-moving rat. Within 40-105 min, an apparent steady state of labeled unesterified 22:6 n-3 in plasma and in brain was reached. However, the values of the brain to plasma 22:6 n-3 SA ratios ranged from 0.03 to 0.05, indicating that an isotopic equilibrium between brain and plasma was not attained. This suggests that a considerable endogenous source of unesterified 22:6 n-3 (95-97%) (likely derived from lipid metabolism) dilutes the SA of the tracer coming from plasma. Using the SA of 22:6 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis.

Adrenalectomy-induced increase of brain protein synthesis is antagonized by corticosterone replacements in free-moving rats.

The autoradiographic method with L-[35S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of methionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.

Vasopressin mRNA in the cerebellum and circumventricular organs: a quantitative in situ hybridization study.

To answer the question as to whether vasopressin is synthesized in brain structures other than the supraoptic and paraventricular nuclei of the hypothalamus, vasopressin mRNA was detected by in situ hybridization in the pituitary, cerebellum, dentate gyrus, habenula and circumventricular organs. The highest levels (0.3-0.2 pmol/g), measured by quantitative autoradiography, were observed in the pituitary intermediate lobe and the granular layers of the cerebellum and dentate gyrus. Lower levels (0.15-0.08 pmol/g) were found in the medial habenula, adenohypophysis, area postrema, pineal, subfornical and subcommissural organs.

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.

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.

Comparison of the effects of chronic water deprivation and hypertonic saline ingestion on cerebral protein synthesis in rats.

The effects of 3 days water deprivation and 3 days with 2% (w/v) NaCl in drinking water on local rates of methionine incorporation into brain proteins were compared by means of a quantitative autoradiographic method with L-[35S]methionine. The two conditions of chronic dehydration resulted in large increases in the rate of methionine incorporation in the supraoptic (SON), magnocellular paraventricular (mPVN) and arcuate nuclei of the hypothalamus, and in the subfornical organ (SFO). Significant increases of lower amplitude occurred as a result of both treatments in the anteroventral third ventricle area, parvocellular paraventricular nucleus and locus coeruleus. Water deprivation caused larger increases of protein synthesis than hypertonic saline ingestion in the SON, mPVN and SFO. These results indicate that following chronic dehydration, increases in protein synthesis occur mainly in forebrain areas involved in the regulation of water balance, whereas no major changes in protein synthesis occur in brainstem areas involved in the control of blood volume and pressure.

Progressive increases of protein synthesis in the circumventricular organs during chronic dehydration in rats.

The quantitative autoradiographic method with L-(35S)methionine was applied to investigate the effect of chronic dehydration on rates of protein synthesis in circumventricular organs (CVOs). Water deprivation for 1, 2 and 3 days causes progressive increases of protein synthesis in the subfornical organ (SFO), the area postrema, the organum vasculosum laminae terminalis and the neurohypophysis. Chronic salt ingestion with 2% NaCl in drinking water for 3 days resulted in increases of protein synthesis in the CVOs similar to those found after 3 days water deprivation, with only one exception, the SFO, in which the rise in protein synthesis was of lower amplitude after 3 days salt ingestion as compared to 3 days water deprivation. These results suggest that several circulating factors related to intracellular dehydration and the high plasma levels of the neurohormones vasopressin and oxytocin are probably important determinants of the rise of protein synthesis in circumventricular organs. Alternatively, the elevated level of blood-borne angiotensin II may well explain the higher metabolic response of the SFO following water deprivation compared to salt ingestion.

Inhibition of methionine incorporation into brain proteins after the systemic administration of p-chlorophenylalanine and L-5-hydroxytryptophan.

The effects of p-chlorophenylalanine (p-CPA) and L-5-hydroxytryptophan (L-5-HTP) on local rates of plasma methionine incorporation into brain proteins were investigated by a quantitative autoradiographic method. The sequential i.v. administration of p-CPA (280 mg/kg, 42 h before the measurement) and L-5-HTP (60 mg/kg, 40 min before the measurement) resulted in an average 82% decrease of plasma methionine incorporation. The two treatments given separately also reduced the rates of plasma methionine incorporation in all the brain areas examined by 33 and 50%, respectively for p-CPA and L-5-HTP. These results indicate that: (1) p-CPA and L-5-HTP, two drugs which affect brain serotonin production in opposite ways, both produce large and general decreases of brain protein synthesis; (2) the administration of L-5-HTP does not restore the p-CPA-induced inhibition of brain protein synthesis but induces further decreases of protein synthesis. These results suggest that the reduction of brain protein synthesis in p-CPA-treated rats is mainly related to high circulating levels of p-CPA and phenylalanine; and that brain serotonin is not the only factor involved in the widespread metabolic changes observed. Such profound alterations of brain metabolism should be considered when interpreting the behavioral and neurochemical effects of p-CPA and L-5-HTP.

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.

[Brain protein synthesis in awake rats: equilibrium of L-methionine between plasma and direct precursor pool]. / Biosynthèse des protéines cérébrales chez le rat vigile: équilibre de la L-méthionine entre le plasma et le compartiment précurseur direct.

After 1 hr. of continuous infusion of L-(35S)methionine the specific activities of L-methionine in plasma and tissue-free and tRNA-bound L-methionine in brain were in the same range. This result indicates that, under steady-state conditions, dilution of the precursor pool for protein synthesis (L-methionyl-tRNA) by L-methionine derived from a source other than plasma can be considered as negligible.

Decreased protein synthesis in hypothalamic nuclei following L-5-hydroxytryptophan in intact and p-chlorophenylalanine-pretreated rats.

The rate of protein synthesis was estimated in individual hypothalamic nuclei by a quantitative autoradiographic technique with L-[35S]methionine. The i.v. administration of 60 mg/kg L-5-hydroxytryptophan (40 min before) resulted in a 45-55% decrease of overall protein synthesis rate in all the hypothalamic nuclei examined. In rats pretreated (42 h before) with a single i.v. injection of 280 mg/kg p-chlorophenylalanine, a drug which is known to deplete brain serotonin concentration, the administration of 60 mg/kg L-5-hydroxytryptophan resulted in a 50-75% decrease of protein synthesis rates in the hypothalamic nuclei. These results suggest that the systemic administration of large doses of L-5-hydroxytryptophan may inhibit protein synthesis in hypothalamic nuclei directly or indirectly after the conversion of this compound to serotonin.