Fetal brain serotonin synthesis and catabolism is under control by mother intake of tryptophan.

Previous morphological studies reported that serotonergic neurons appear in rats in the second half of prenatal life. Initially the biochemical differentiation of these neurons before birth was studied. Both serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) was detected in the fetal brain on day 15 of gestation. During prenatal development an increase was detected in the brain levels of 5-HT (200% higher on day 19 than on day 15) and 5-HIAA (700% higher on day 19 than on day 15). Oral administration of tryptophan to pregnant rats induced a dose-related increase of tryptophan concentration in different fetal tissues, including brain. The increase in tryptophan tissue concentration was detected for low doses (50 mg/kg) and remained unsaturated after administration of high doses (1000 mg/kg). This observation suggests that the placental barrier is not effective to block the influx of high levels of tryptophan to the fetus. Tryptophan concentration in the brain is 300% higher than in the carcass and 600% higher than in the placenta. These data suggest a mechanism to assume a role in concentrating of tryptophan in the brain. Finally, it was found that an increase in brain tryptophan induced changes in both serotonin and 5-HIAA brain levels, but did not modify tyrosine, dopamine or norepinephrine levels. Thus, under physiological conditions, tryptophan hydroxylase activity in prenatal brain is probably not saturated by its substrate tryptophan.     

Footshock-induced changes in brain catecholamines and indoleamines are not mediated by CRF or ACTH

Stressful treatments have long been associated with increased activity of brain catecholaminergic and serotonergic neurons. An intracerebroventricular (icv) injection of the corticotropin-releasing factor (CRF) also activates brain catecholaminergic neurons. Because brain CRF-containing neurons appear to be activated during stress, it is possible that CRF mediates the catecholaminergic activation. This hypothesis has been tested by assessing the responses in brain catecholamines and indoleamines to footshock in mice pretreated icv with a CRF receptor antagonist, and in mice lacking the gene for CRF (CRFko mice). Consistent with earlier results, icv administration of CRF increased catabolites of dopamine and norepinephrine, but failed to alter tryptophan concentrations or serotonin catabolism. A brief period of footshock increased plasma corticosterone and the concentrations of tryptophan and the catabolites of dopamine, norepinephrine and serotonin in several brain regions. Mice injected icv with 25 microg alpha-helical CRF(9-41) prior to footshock had neurochemical responses that were indistinguishable from controls injected with vehicle, while the increase in plasma corticosterone was slightly attenuated in some experiments. CRFko mice exhibited neurochemical responses to footshock that were indistinguishable from wild-type mice. However, whereas wild-type mice showed the expected increase in plasma corticosterone, there was no such increase in CRFko mice. Similarly, hypophysectomized mice also showed normal neurochemical responses to footshock, but no increase in plasma corticosterone. Hypophysectomy itself elevated brain tryptophan and catecholamine and serotonin metabolism. Treatment with ACTH icv or peripherally failed to induce any changes in cerebral catecholamines and indoleamines. These results suggest that CRF and its receptors, and ACTH and other pituitary hormones, are not involved in the catecholamine and serotonin responses to a brief period of footshock.     

Acute effects of heroin and morphine on newly synthesized serotonin in rat brain.

Heroin and morphine, in acute intraperitoneal doses of 2 and 10 mg/kg respectively, produced significant increments in the formation of newly formed brain serotonin from tritiated (³H)-L-tryptophan to ³H-serotonin. Opiate analgesia, Straub tail sign and catatonia, were observed during the increase in the synthesis of serotonin. The transport of radio-labelled tryptophan into the rat brain was not increased by the acute injection of the opiates, but brain levels of ³H-serotonin and of its main metabolite, 5-hydroxyindoleacetic acid, were significantly elevated. These opiates do not interfere with the accumulation of serotonin or with the transport of its metabolite in serotonergic neurons after inhibition of monoamine oxidases with Pargyline. An increase in the activity of tryptophan hydroxylases was more pronounced in the forebrain than in the brain stem. Stimulation of newly synthesized serotonin is probably mediated by an increase in tryptophan hydroxylase activity and not by an increase in the transport of tryptophan into the brain.     

Dietary tryptophan does not alter the function of brain serotonin neurons

The hypothesis that alterations in dietary tryptophan modify the functional activity of brain serotonin-containing neurons was tested by recording the electrophysiological activity of single serotonergic cells in awake, behaving cats after meal ingestion of diets containing varying proportions of tryptophan and the neutral amino acids that compete with tryptophan for uptake into the brain. The data revealed that while the various diets produced significant changes in brain serotonin and its major metabolite, 5-hydroxyindoleacetic acid, there was no change in the activity of serotonin-containing dorsal raphe cells following meal ingestion. Furthermore, a pulse injection of tritiated labeled tryptophan following the various diets produced no significant change in the release of tritiated serotonin into the lateral ventricles, while tritiated 5-hydroxyindoleacetic acid was significantly increased. These data suggest that dietary tryptophan does not alter the functional activity of central serotonergic neurons, in contrast with current popular beliefs that such dietary manipulations alter brain function.     

Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay

Hypotensive responses to tryptophan and 5-hydroxytryptophan infusions were studied in normotensive male Sprague-Dawley rats. Results showed that 5-hydroxytryptophan but not tryptophan lowered pressure in a dose dependent way in direct relation to the production of brain serotonin and 5-HIAA. Intrinsic release of serotonin from brain was also studied during periods of induced hypertension and hypotension. Brain monoamine responses to blood pressure changes induced by intravenous phenylephrine and nitroprusside were measured in dorsal raphe nucleus and nucleus tractus solitarius by in vivo electrochemistry. Results showed that 5-HIAA was increased during drug induced hypertension and during reflex hypertension which followed a period of hypotension. These changes were blocked by sinoaortic denervation indicating that these central serotonergic neurons are responding to increased pressure sensed by baroreceptors. Therefore, serotonin has a role in blood pressure regulation as a pharmacologic agent and as a neurotransmitter in homeostatic control of pressure.     

Brain Serotonin Content: Physiological Regulation by Plasma Neutral Amino Acids

When plasma tryptophan is elevated by the injection of tryptophan or insulin, or by the consumption of carbohydrates, brain tryptophan and serotonin also rise; however, when even larger elevations of plasma tryptophan are produced by the ingestion of protein-containing diets, brain tryptophan and serotonin do not change. The main determinant of brain tryptophan and serotonin concentrations does not appear to be plasma tryptophan alone, but the ratio of this amino acid to other plasma neutral amino acids (that is, tyrosine, phenylalanine, leucine, isoleucine, and valine) that compete with it for uptake into the brain.     

Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.     

Effects of disulfiram and coprine on rat brain tryptophan hydroxylation in vivo

The effects of disulfiram and coprine on brain tryptophan hydroxylation, and on the brain-levels of serotonin and 5-hydroxyindole-3-acetic acid, were studied in 45 and 235 days old rats. Both drugs were found to affect the parameters measured. Disulfiram increased the rate of tryptophan hydroxylation and the serotonin level in young rats, while these parameters appeared to be unaffected in old disulfiram-treated rats. In contrast, coprine increased the rate of tryptophan hydroxylation and possibly also the serotonin level in old rats while no significant effects were seen in young coprine-treated rats. Regarding the 5-hydroxyindole-3-acetic acid concentration, this appeared to be increased by disulfiram in both age-groups, while no significant effects were found with coprine. The lack of similarity in the action of disulfiram and coprine, which are both potent aldehyde dehydrogenase inhibitors, suggests that the effects found were not caused by an impaired metabolism of monoamine-derived biogenic aldehydes.     

EFFECTS OF IMMOBILIZATION AND FOOD DEPRIVATION ON RAT BRAIN TRYPTOPHAN METABOLISM

Abstract— Withdrawal of food or immobilization both led to changes in rat brain tryptophan metabolism. Brain tryptophan and 5-hydroxyindolylacetic acid concentrations both increased while changes in 5-hydroxytryptamine were much smaller. Changes were greater upon withdrawal of food. The brain tryptophan change did not appear merely to reflect an overall increase of brain amino acid concentrations, brain tyrosine concentration being only slightly increased by food withdrawal and significantly decreased upon immobilization. Plasma tryptophan did not increase. The changes in brain indole metabolism were not abolished by adrenalectomy. Results are discussed in relation to the regulation of brain serotonin metabolism.     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

Effect of dietary tryptophan on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid in rainbow trout

In order to determine the effect of dietary tryptophan level on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid levels, juvenile rainbow trout (Salmo gairdneri) were raised for 16 weeks on semipurified diets containing 0.06%, 0.16%, 0.21%, 0.26%, 0.39%, or 0.59% tryptophan. After 14 weeks, feed intake was depressed in fish fed the diets containing 0.06% or 0.16% tryptophan. No further differences in feed intake were noted between the remaining treatments. In addition, body weight was lower in fish fed diets containing 0.06%, 0.16%, or 0.21% tryptophan compared with fish fed higher levels. After 16 weeks of feeding the test diets, plasma tryptophan levels were found to be directly related to dietary tryptophan levels. Similarly, increased dietary levels of tryptophan resulted in increased brain levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid. These results demonstrate that in rainbow trout, as in mammals, altered dietary levels of tryptophan result in alterations in plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid.     

Effect of tryptophan and selected analogues on body temperatur of endotoxin-poisoned mice

The effect of endotoxin on the body temperature of mice was studied in animals housed without bedding at an environmental temperature of 15 C. Rectal temperatures were measured during the initial 3 to 5 hr of exposure. Doses of endotoxin ranging from 0.01 to 1 ld(50), as determined for mice maintained at 25 C, produce a hypothermia in proportion to dose. Concurrent injection of tryptophan magnified this response in a dose-dependent manner. Cyproheptadine, an antiserotonin drug, antagonized both the hypothermia produced by serotonin alone, and the augmentation of hypothermia produced by tryptophan in endotoxin-poisoned mice. alpha-Methyltryptophan, an analogue of the amino acid that is known to induce tryptophan pyrrolase, also antagonized the increased hypothermia produced by tryptophan. These data support a previous suggestion that inhibition of tryptophan pyrrolase in endotoxin-poisoned mice has the effect of funneling injected tryptophan into the serotonin pathway.     

Altered precursor availability and metabolism of monoamines in the brain caused by the injection of physiologic saline to suckling rats: On the reliability of saline “controls” in neurochemical studies

The effect of subcutaneous injections of saline (0.9% NaCl, 10–40 μl/g b. wt) to 5- and 20-day old rats on the concentrations of tyrosine (Tyr) and tryptophan (Trp) in the serum and the brain and on the levels of biogenic amines and their metabolites in the developing brain at 6 h p.i. is described. At day 5 the concentration of Tyr in the blood was decreased (dose-dependent), but the brain concentrations of Tyr and of its amine-metabolites, dopamine (DA), norepinephrine (NE), homovanillic acid (HVA) and dihydroxyphenylacetate (DOPAC) were unaffected. In contrast, in the 20-day old rat, serum Tyr was unaffected by the saline injections, but the Tyr concentration in the brain decreased markedly at the highest saline dose. The concentrations of NE (only at maximum dose) and of DA (independent on the amount of saline injected) were elevated in the brains of saline injected 20-day old rats. The concentrations of Trp and indoles were more affected at day 5 than at day 20: slightly decreased concentration of Trp in the serum but markedly increased concentrations of brain Trp (only at maximum dose), elevated serotonin (5-HT, independent on the amount of saline injected) and 5-hydroxyindoleacetic acid (5-HIAA, at maximum dose) in the brain. If the maximum dose of 40 μl/g body weight was injected to suckling rats repeatedly during the whole suckling period (in 12 h intervals), some effects caused by one single injection of 40 μl/g disappeared (Tyr—depletion in blood or brain, increase in brain NE, DA and Trp), but other additional effects appeared (decreased DA and increased DOPAC, decreased 5-HT and 5-HIAA). The results show that saline injections do cause characteristic, age-dependent alterations of precursor availability as well as of the rate of synthesis and degradation of catecholamine and 5-HT. Repeated treatments have different effects than one single treatment on the precursor availability and the metabolism of monoamines. These alterations must be taken into account if the effects of certain “specific” treatments are compared and discussed in relation to saline “controls”.     

Interaction of diet and drugs in the regulation of brain 5-hydroxyindoles and the response to painful electric shock.

The long-term consumption of a tryptophan-poor, corn diet by rats decreases electroshock response thresholds. This hyperalgesia appears to be related directly to diet-induced reductions in the brain concentrations of the putative neurotransmitter, serotinin. Rehabilitating corn-fed animals by feeding them the corn diets supplemented with tryptophan restores brain serotonin and pain thresholds to normal; similarly, injecting the tryptophan-deficient, corn-fed animals with fluoxetine, a drug that blocks the uptake of serotonin into brain neurons, also restores the electroshock response thresholds to control levels. The tryptophan hydroxylase inhibitor, $\text{p}$-chlorophenylalanine, increases the hyperalgesia to electroshock in corn-fed rats and further reduces brain serotonin concentrations. Injection of the amino acid valine, on the other hand, produces hyperalgesia and decreases brain serotonin in casein-fed rats but not in animals fed the corn diet. These data lend support to the hypothesis that serotonin neurons may mediate the sensitivity or reactivity to painful stimuli.     

24h withdrawal following repeated administration of caffeine attenuates brain serotonin but not tryptophan in rat brain: Implications for caffeine-induced depression

Caffeine injected at doses of 20, 40 and 80 mg/kg increased brain levels of tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat brain. In view of a possible role of 5-HT in caffeine-induced depression the effects of repeated administration of high doses of caffeine on brain 5-HT metabolism are investigated in rats. Caffeine was injected at doses of 80 mg/kg daily for five days. Control animals were injected with sahne daily for five days. On the 6th day caffeine (80 mg/kg) injected to 5 day sahne injected rats increased brain levels of tryptophan, 5-HT and 5-HIAA. Plasma total tryptophan levels were not affected and free tryptophan increased. Brain levels of 5-HT and 5-HIAA but not tryptophan decreased in 5 day caffeine injected rats injected with sahne on the 6th day. Plasma total and free tryptophan were not altered hi these rats. Caffeine-induced increases of brain tryptophan but not 5-HT and 5-HIAA were greater in 5 day caffeine than 5 day sahne injected rats. The findings are discussed as repeated caffeine administration producing adaptive changes in the serotonergic neurons to decrease the conversion of tryptophan to 5-HT and this may precipitate depression particularly in conditions of caffeine withdrawal.     

Effect of parachlorophenylalanine,a brain serotonin depletor,on the prolactin cells of the eel pituitary

Summary Parachlorophenylalanine (pCPA), an inhibitor of tryptophan hydroxylase depleting brain serotonin in higher vertebrates, was injected into freshwater eels. After 4 or 6 injections (200 mg/kg/day) or 10 injections (100 and 140 mg/kg/day) plasma electrolyte values were not modified. Prolactin (PRL) cells appear less active, with increased granulation after 6 and 10 injections. Their cell height (P < 0.01) and their nuclear area (P < 0.001) are reduced. As injections of 5-hydroxytryptophan stimulate PRL cells, these findings suggest that a serotoninergic system may participate in the regulation of PRL cell activity. Brain serotonin depletion probably decreases granule release in PRL cells, a result comparable to the lowering action of pCPA on the plasma PRL level in some mammals.     

Pharmacological manipulation of serotonin levels in the nervous system of the opisthobranch mollusc Tritonia diomedea

Serotonin-related disorders can be treated by manipulating serotonin synthesis with the serotonin precursor 5-hydroxytryptophan (5-HTP) or other pharmacological agents. The mollusc Tritonia diomedea is a model for investigating the effects of altering serotonin content on the functions of identified neurons. We used high-performance liquid chromatography and immunohistochemistry to examine the amount and localization of 5-HTP, serotonin, and the serotonin breakdown product 5-hydroxyindolacetic acid (5-HIAA) in the Tritonia brain after various pharmacological treatments. Exposure to 5-HTP (2 mM for 30 min-1 h) caused an immediate and massive increase in total 5-HTP content, which lasted more than 20 h, and the widespread appearance of 5-HTP immunoreactivity in neurons. Serotonin levels rose gradually, but only a restricted number of additional neurons displayed serotonin immunoreactivity. 5-HTP treatment also caused an increase in the total amount of 5-HIAA and the appearance of 5-HIAA immunoreactivity throughout the brain. Treatment with the synthesis cofactor tetrahydrobiopterin, the initial precursor tryptophan, or serotonin itself had no persistent effect on total serotonin content. The amino acid decarboxylase inhibitor hydroxybenzylhydrazine (NSD-1015) also had no effect on the total serotonin content, although it caused an accumulation of 5-HTP. Thus, serotonin levels in the brain of T. diomedea appear to be maintained by a homeostatic mechanism that can be disrupted by 5-HTP.     

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.     

Participation of the brain serotoninergic system in creating the stress reactivity of the hypophyseal-adrenal axis

The concentration of corticosteroids in the blood of rats was shown to increase in response to the immobilization stress at an earlier age than the brain serotonin metabolism changes. The level of corticosteroids in blood increased in response to the intraperitoneal serotonin injection also earlier than the reaction to the serotonin injection in the brain lateral ventricle sets up. The increase of the reaction of hypophysial-suprarenal system to stress during the period from the 12th till the 16th day of postnatal development coincided with the changes in serotonin metabolism in the brain stem and the reaction to serotonin injection in the brain lateral ventricle. It is suggested that the system of serotonin brain neurons connected with the hypophysial-suprarenal complex matures later tran the serotonin receptors in the periphery; the reaction to immobilization may be realized at the early developmental stages without the participation of brain serotonin.     

Neurotransmitter-Mediated Regulation of Brain Aromatase: Protein Kinase C- and G-Dependent Induction

Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α₁-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α₁-agonist phenylephrine than in control neurons, whereas prazosin, an α₁-antagonist, suppressed this increase, and ligands for α₂- or β-adrenergic receptors did not exert any influence. The profile of α₁-adrenergic receptor subtypes during actual development in vivo suggested that the α_1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α₁ (possibly α_1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.