Enzyme Tryptophan Hydroxylase Research Articles

The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons.

A fundamental question in developmental neurobiology is how a common neurotransmitter is specified in different neuronal types?. We describe cell-specific regulation of the serotonergic phenotype by the C. elegans POU-transcription factor UNC-86. We show that unc-86 regulates particular aspects of the terminal neuronal identity in four classes of serotonergic neurons, but that the development of the ADF serotonergic neurons is regulated by an UNC-86-independent program. In the NSM neurons, the role of unc-86 is confined in late differentiation; the neurons are generated but do not express genes necessary for serotonergic neurotransmission. unc-86-null mutations affect the expression in NSM of tph-1, which encodes the serotonin synthetic enzyme tryptophan hydroxylase, and cat-1, which encodes a vesicular transporter that loads serotonin into synaptic vesicles, suggesting that unc-86 coordinately regulates serotonin synthesis and packaging. However, unc-86-null mutations do not impair the ability of NSM to reuptake serotonin released from the ADF serotonergic chemosensory neurons and this serotonin reuptake is sensitive to the serotonin reuptake block drugs imipramine and fluoxetine, demonstrating that serotonin synthesis and reuptake is regulated by distinct factors. The NSM neurons in unc-86-null mutants also display abnormal neurite outgrowth, suggesting a role of unc-86 in regulating this process as well.

Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus.

Substance P (SP) is known to act at supraspinal sites to influence pain sensitivity as well as to promote anxiety. The effects of SP could be mediated in part by actions in the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN), adjoining mesencephalic cell groups that are strategically positioned to influence both nociception and mood. Previous studies have indicated that SP regulates both enkephalin and serotonin neurotransmission in these brain regions. To determine the mechanism underlying the effects of SP in the PAG and DRN, the distribution of the principal receptor for SP, the neurokinin 1 (NK1) receptor, was examined with respect to other neurotransmitter markers. PAG neurons that had NK1 receptor immunolabeling were interdigitated with and received contacts from enkephalin-containing neurons. However, only a few (16/144; 11%) neurons with NK1 receptor also contained enkephalin immunoreactivity after colchicine treatment. In the DRN, dendrites containing NK1 receptor were selectively distributed in the dorsomedial subdivision. The majority (132/137; 96%) of these dendrites did not contain immunoreactivity for the serotonin-synthesizing enzyme tryptophan hydroxylase. In contrast, neuronal profiles with NK1 receptor in both the PAG and the DRN often contained immunolabeling for glutamate. Light and electron microscopic examination revealed that 48-65% of cell bodies and dendrites with NK1 receptor were dually immunolabeled for glutamate. These data suggest that SP directly acts primarily on glutamatergic neurons in the PAG and DRN. To a lesser extent, enkephalin-containing neurons may be targeted. Through these actions, it may subsequently influence activity of larger populations of neurons containing enkephalin as well as serotonin. This circuitry could contribute to, as well as coordinate, effects of SP on pain perception and mood.

Up-regulation of tryptophan hydroxylase expression and serotonin synthesis by sertraline.

The neurotransmitter serotonin is involved in a variety of brain functions, and abnormal changes in serotonin neurotransmission are associated with an array of psychiatric disorders, including depression. Sertraline is a selective serotonin reuptake inhibitor (SSRI) and an effective antidepressant. Sertraline increases the serotonin concentration in the synaptic cleft by a short-term action; however, clinical improvement is observed only after several weeks, suggesting that the therapeutic effect may be caused by long-term alterations in serotonin transmission. We determined the effects of sertraline on serotonin synthesis in vivo and in vitro. Long-term treatment of rats with sertraline up-regulated mRNA and protein levels of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH), as determined by in situ hybridization and immunocytochemistry, respectively. In vitro studies using RBL-2H3 cells also showed an increase in mRNA and protein levels of TPH by sertraline, as determined by Northern blot and immunoblot analyses, respectively. This was accompanied by increases in the levels of TPH enzymatic activity and total serotonin. These data demonstrate that in addition to the known short-term action as an uptake blocker, sertraline also exerts a long-term effect on the serotonin neurotransmission by enhancing serotonin synthesis. A similar effect was observed with another SSRI, fluoxetine, but not with the non-SSRI chlorpromazine. The up-regulation of TPH gene expression by sertraline was attenuated by the protein kinase A (PKA) inhibitor N-[2-(p-bromocinnamylamine)-ethyl]-5-isoquinolinesulfonamine, suggesting that a mechanism involving the PKA signaling pathway might at least in part mediate the long-term therapeutic action.

Developmental regulation of tryptophan hydroxylase messenger RNA expression and enzyme activity in the raphe and its target fields

Tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin and during development, brain serotonin levels and tryptophan hydroxylase activities increase. Increased tryptophan hydroxylase activity could result from alterations in tryptophan hydroxylase messenger RNA levels, translation, and/or post-translational regulation. Tryptophan hydroxylase messenger RNA levels in the dorsal raphe nucleus increased 35-fold between embryonic day 18 and postnatal day 22, measured by quantitative in situ hybridization, then decreased by 40% between postnatal days 22 and 61. These changes correlated with tryptophan hydroxylase enzyme activities in the raphe nuclei as expected, but not in cortical or hippocampal targets. Tryptophan hydroxylase messenger RNA expression in the nucleus raphe obscuris increased 2.5-fold between postnatal days 8 and 22 but did not correlate with enzyme activity in the spinal cord. Using an in vitro model of serotonergic raphe neuron differentiation, serotonergic differentiation was associated with an increase in both tryptophan hydroxylase promoter activity and protein expression. In vivo, tryptophan hydroxylase messenger RNA levels per single cell and per brain section were correlated during development up to postnatal day 22, but not beyond for both the dorsal raphe nucleus and nucleus raphe obscuris. Between postnatal days 22 and 61 single cell levels of tryptophan hydroxylase messenger RNA in the dorsal raphe nucleus did not change yet the levels per brain section significantly decreased by 40%. During the same period in the nucleus raphe obscuris, tryptophan hydroxylase messenger RNA levels per single cell signifcantly increased by 30% yet levels per brain section did not change. Comparison of tryptophan hydroxylase messenger RNA levels per cell and per brain section indicated a serotonergic loss between postnatal days 22 and 61 in both the dorsal raphe nucleus and nucleus raphe obscuris and may reflect either a loss of neurotransmitter phenotype or cell death. This study is the first to characterize the expression of brain tryptophan hydroxylase messenger RNA during rat development. In addition, this study is the first to report the activity of tryptophan hydroxylase in the spinal cord and hippocampus in the embryonic and neonatal rat. Together, the data provide a better understanding of the intricate relationship between patterns of tryptophan hydroxylase messenger RNA expression and enzyme activity.

Mechanisms governing the differentiation of a serotonergic phenotype in culture

In this study, we explored whether a serotonergic (5-HT) phenotype could be novelly induced in the phenotypically plastic neurons of the developing striatum. We found that the 5-HT biosynthetic enzyme tryptophan hydroxylase (TPH) was expressed in nearly 10% of neurons following treatment with an extract derived from adult raphe tissue. This effect was mimicked by co-treatment with a growth factor (aFGF, bFGF or BDNF; but not GDNF, IGF-1, EGF or TGF) and the neurotransmitter 5-HT (but not GABA, dopamine, glutamate) and/or a protein kinase activator (IBMX, forskolin, TPA). Treatment with combined factors (aFGF+5-HT+IBMX+forskolin+TPA) yielded the greatest level of TPH induction (15.6%). Moreover, TPH was enzymatically active (112.8±36 pmol/mg per h) and produced detectable levels of 5-HT (2.12±0.30 ng) and its metabolite 5-HIAA (4.24±0.11 ng) in maximally stimulated cultures. These findings demonstrate that it is possible to promote the differentiation of serotonergic phenotypic traits in developing brain neurons in culture.

The aromatic amino acid hydroxylases.

The enzymes phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute the family of pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its respective amino acid substrate using molecular oxygen and a tetrahydropterin as substrates. Recent advances have provided insights into the structures, mechanisms, and regulation of these enzymes. The eukaryotic enzymes are homotetramers comprised of homologous catalytic domains and discrete regulatory domains. The ligands to the active site iron atom as well as residues involved in substrate binding have been identified from a combination of structural studies and site-directed mutagenesis. Mechanistic studies with nonphysiological and isotopically substituted substrates have provided details of the mechanism of hydroxylation. While the complex regulatory properties of phenylalanine and tyrosine hydroxylase are still not fully understood, effects of regulation on key kinetic parameters have been identified. Phenylalanine hydroxylase is regulated by an interaction between phosphorylation and allosteric regulation by substrates. Tyrosine hydroxylase is regulated by phosphorylation and feedback inhibition by catecholamines.

Excess TPH 17 779C allele in surviving cotwins of monozygotic twin suicide victims

Genetic factors contribute to the risk of psychopathology in many psychiatric conditions, but the specific genes are yet to be identified. Neurotransmitter alterations are implicated in the etiology of psychopathology based, in part, on studies of neurotransmitter receptors and their biosynthetic or degradative enzymes in postmortem tissue. Identification of the altered receptors and enzymes serves to identify candidate genes of potential etiological significance. Polymorphisms in these genes can contribute to alterations in protein function in vivo that are part of the neurochemical underpinnings of psychopathologies such as major depressive disorder, psychoses, alcoholism, personality disorders, aggressive–impulsive traits, or suicidal behavior. Altered serotonergic function is implicated in the etiology and pathogenesis of several major psychiatric conditions. In particular, there is much evidence for an association of lower serotonergic function and suicidal behavior. Thus genes related to the serotonergic system are candidate genes worthy of study as part of the genetic diathesis for suicidal behavior. This review examines the following polymorphisms in the serotonin biosynthetic enzyme tryptophan hydroxylase (TPH; A779C substitution), the serotonin transporter (5-HTT, 5-HTTLPR allele), the 5-HT1B receptor (G861C, C129T substitution) and the 5-HT2A receptor (T102C) for their relationship to suicidal behavior. For the TPH gene, we found the less common U or A allele variant of the A779C polymorphism was associated with suicide attempt. Other studies have found the U allele to be associated with aggression and lower serotonergic function in vivo. A 44 base pair insertion/deletion in the 5′ flanking promoter region of the 5-HTT gene may result in less 5-HTT expression and 5-HTT binding. We examined 220 cases postmortem and found no association between the promoter genotype and 5-HTT binding. We also found no association with major depressive disorder (MDD), suicide or pathological aggression, despite finding significantly fewer 5-HTT sites in the prefrontal cortex of depressed and/or suicide cases. In genomic DNA samples from 178 unrelated subjects, we detected two polymorphisms for the 5-HT1B receptor at nucleotides 861 and 129. However, no association between either polymorphism and depression, suicide, aggression, or alcoholism was observed. There are two common polymorphisms for the 5-HT2A receptor gene in humans. The results of studies of 5-HT2A receptor gene polymorphisms do not indicate significant major associations with suicidal behavior. In contrast, the 5-HT2A receptor itself is reported to be increased in suicide. Functional polymorphisms involving the promoter region that affect gene expression may explain this finding. Studies of candidate genes related to serotonergic function in brain are increasingly used to establish genetic alterations contributing to psychiatric illness. The most meaningful studies combine the study of candidate genes with direct measures of related proteins as well as psychopathology.

Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat

In the present work we confirm that gestational malnutrition effects body and brain composition and results in an activation of the synthesis of the brain neurotransmitter 5-hydroxytryptamine. These results also demonstrate more activity of the rate-limiting enzyme tryptophan hydroxylase in the malnourished fetal and postnatal brain. However, the activity of this enzyme remains increased in the brain of nutritionally recovered animals accompanied by an increase in the synthesis of 5-hydroxytryptamine. We therefore suggest that, in the nutritionally recovered animal, the mechanism of activation of this biosynthetic path in the brain may be not dependent on the increased availability of free L-tryptophan observed in malnourished animals, but might be due to a specific change in the enzyme complex itself. This hypothesis is supported by the fact that plasma free and brain L-tryptophan return to normal in the recovered animal.

Nutritional recovery does not reverse the activation of brain serotonin synthesis in the ontogenetically malnourished rat

In the present work we confirm that gestational malnutrition affects body and brain composition and results in an activation of the synthesis of the brain neurotransmitter 5-hydroxytryptamine. These results also demonstrate more activity of the rate-limiting enzyme tryptophan hydroxylase in the malnourished fetal and postnatal brain. However, the activity of this enzyme remains increased in the brain of nutritionally recovered animals accompanied by an increase in the synthesis of 5-hydroxytryptamine. We therefore suggest that, in the nutritionally recovered animal, the mechanism of activation of this biosynthetic path in the brain may be not dependent on the increased availability of free l-tryptophan observed in malnourished animals, but might be due to a specific change in the enzyme complex itself. This hypothesis is supported by the fact that plasma free and brain l-tryptophan return to normal in the recovered animal.

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.

Tryptophan hydroxylase is expressed by photoreceptors in Xenopus laevis retina.

Serotonin has important roles, both as a neurotransmitter and as a precursor for melatonin synthesis. In the vertebrate retina, the role and the localization of serotonin have been controversial. Studies examining serotonin immunoreactivity and uptake of radiolabeled serotonin have localized serotonin to inner retinal neurons, particularly populations of amacrine cells, and have proposed that these cells are the sites of serotonin synthesis. However, other reports identify other cells, such as bipolars and photoreceptors, as serotonergic neurons. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the serotonin synthetic pathway, was recently cloned from Xenopus laevis retina, providing a specific probe for localization of serotonin synthesis. Here we demonstrate that the majority of retinal mRNA encoding TPH is present in photoreceptor cells in Xenopus laevis retina. These cells also contain TPH enzyme activity. Therefore, in addition to being the site of melatonin synthesis, the photoreceptor cells also synthesize serotonin, providing a supply of the substrate needed for the production of melatonin.

Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro

Many aspects of retinal physiology are controlled by a circadian clock including at least two steps in the melatonin synthetic pathway: the activity of the enzyme, N-acetyltransferase (NAT), and mRNA levels of the rate-limiting enzyme tryptophan hydroxylase (TPH). Light and dopamine (through D2-like dopamine receptors) can phase shift the clock, and can also acutely inhibit NAT activity, resulting in supressed melatonin synthesis. In this paper, we show that eyecups cultured in constant darkness maintain a clock-controlled rhythm in TPH mRNA, with low levels in early day, rising to a peak in early night. Both eyecups and isolated retinas, cultured in light during the day, also exhibit a similar increase in TPH mRNA levels, indicating that this expression is not acutely inhibited by light. Treatment with light or quinpirole (D2 dopamine receptor agonist) in early night, at a time and dose that acutely inhibits NAT activity, does not change levels of TPH mRNA. Addition of eticlopride (D2 dopamine receptor antagonist) during the day, also has no effect on the normal daytime increase in TPH message levels. Therefore, TPH mRNA level is controlled by a circadian clock located within the eye, but acute effects of light or dopamine are not detected.

Lack of effect of antidepressant drugs on the levels of mRNAs encoding serotonergic receptors, synthetic enzymes and 5HT transporter

Serotonergic transmission is thought to be central to the aetiology of depression and the therapeutic actions of antidepressant drugs, and the latters' delayed effect has given rise to the hypothesis that an adaptive change may be involved, possibly at the level of gene expression. We have examined this hypothesis by treating rats over a time course of up to 32 days with either imipramine, mianserin, fluvoxamine, citalopram, amoxapine or saline and measuring the levels of mRNAs encoding the 5HT 1A, 5HT 1B, 5HT 1C and 5HT 2 receptors, the enzymes tryptophan hydroxylase and aromatic amino acid decarboxylase, and the 5HT transporter. None of the treatments gave rise to significant changes in any of the mRNA levels at any time point. These results suggest that the reported changes in 5HT receptor numbers do not occur as a result of changes in the abundance of their encoding mRNAs, and that changes to the latter is not central to the therapeutic effects of antidepressant drugs.

Increase of tryptophan hydroxylase enzyme protein by dexamethasone in adrenalectomized rat midbrain

Using two specific anti-peptide antibodies (WH-66 and WH-412) against tryptophan hydroxylase (TPH or WH), a single principle band from the midbrain raphe area was seen (approximately 49 kDa) in immunoblots. Densitometric comparison of the immunoreactivity of the 49 kDa band was greater (50-75%) in immunoblots of midbrain raphe samples from adrenalectomized (ADX) rats given dexamethasone (DEX) in their drinking water (10 mg/liter) for 12-96 hr. No difference from ADX brains was seen in the 49 kDa band after only 4 hr of exposure to DEX in the drinking water. Immunocytochemical staining with WH-66 of sections from rat brainstem showed specific cellular staining in all of the serotonergic raphe nuclei but not in substantia nigra or locus coeruleus. More cellular staining of WH-66-immunoreactive (WH-66-IR) cells was observed in the dorsal and median raphe nuclei in ADX rats given DEX for 72 hr, especially in the perikarya and in the primary dendrites. Quantification of staining per cell soma with an automatic image analyzer indicates that amount of WH-66-IR in neurons from both the lateral wing subdivision of the dorsal raphe nucleus and in the supralemniscal nucleus, B-9, was 80% higher in the ADX+DEX compared to ADX animals. Interestingly, morphometric analysis of these same cells showed a corresponding increase of 37-80% in somal area. It is suggested that a part of the increase in TPH/WH staining may be a consequence of cellular hypertrophy due to DEX treatment of the ADX rats.

Chronic cocaine administration increases CNS tyrosine hydroxylase enzyme activity and mRNA levels and tryptophan hydroxylase enzyme activity levels.

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striatum and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.

Acute effects of reserpine on tryptophan hydroxylase activity and mRNA in rat brain

The present study examined changes in tryptophan hydroxylase (TPH) activity in the rat brainstem 1–4 days after i.p. reserpine administration. TPH enzyme activity was significantly decreased day 1 posttreatment and followed by a small but significant increase on days 2–4. Maximal TPH activity on day 4 was elevated only 42% above control level. This small increase in TPH activity was not preceded by any change of in situ hybridization signal of TPH message on day 1. In contrast, adrenal tyrosine hydroxylase (TH) activity in these same reserpinized animals was 2.5-fold higher than control on 4 day posttreatment. In addition, TH message was correspondingly increased day 1 posttreatment. Taken together, these results indicate a trend of small increase in TPH activity and no alteration in message after reserpine treatment unlike catecholaminergic systems.

Inhibition of catechol- O-methyltransferase (COMT) as well as tyrosine and tryptophan hydroxylase by the orally active iron chelator, 1,2-dimethyl-3-hydroxypyridin-4-one (L1, CP20), in rat brain in vivo

The orally active iron chelator, 1,2-dimethyl-3-hydroxypyridin-4-one (L1, CP20) proposed for reduction of iron overload in hemoglobinopathic patients, was studied in rats with respect to its ability to interfere with dopamine (DA) and serotonin (5-HT) metabolism. At 100 mg/kg i.p., it reduced the levels of DA, 5-HT, 5-hydroxyindoleacetic acid and particularly homovanillic acid in the rat striatum for several hours. These effects were shown to result from concomitant inhibition of catechol- O-methyltransferase (COMT; EC 2.1.1.6), tyrosine [tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating) (EC 1.14.16.2)] and tryptophan hydroxylase [tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating) (EC 1.14.16.4)], with similar time-courses. COMT was inhibited with a threshold dose of about 1 mg/kg i.p. and an ed 50 of about 10 mg/kg i.p. as determined by the conversion of exogenous l-dihydroxyphenylalanine ( l-DOPA) to its O-methylated derivative. Tyrosine and tryptophan hydroxylase activities as measured by the accumulation of DOPA and 5-hydroxytryptophan, respectively, after central decarboxylase inhibition, were inhibited in striatum and cortex, with threshold doses of 3–10 mg/kg and ed 50s of about 20–30 mg/kg i.p. or p.o. While COMT inhibition by L1 is probably related to the structural similarity of the latter drug with the normal enzyme substrates, tyrosine and tryptophan hydroxylase inhibition is more likely due to coordination to iron bound to these enzymes. Desferrioxamine at 100 mg/kg i.p. did not show comparable effects. It is not known whether this relates to poor brain and/or cell penetration, or whether multidentate chelators are less suitable as inhibitors of aromatic amino acid hydroxylases

3H]paroxetine binding and serotonin content of rat cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region following p-chlorophenylalanine and p-chloroamphetamine treatment.

The agents p-chlorophenylalanine (PCPA) and p-chloroamphetamine (PCA) deplete brain serotonin (5-HT) levels by two different mechanisms; PCPA inhibits the enzyme tryptophan hydroxylase, whereas PCA has a neurotoxic action on certain 5-HT neurons. The parameters of [3H]paroxetine binding to homogenates prepared from the cerebral cortex of rats treated with PCPA, PCA, or saline; vehicle were investigated. The tissue concentrations of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were also determined by HPLC in the same brain samples. After PCPA treatment, neither the maximum binding capacity (Bmax) nor the dissociation constant (KD) of [3H]paroxetine for the 5-HT uptake recognition site differed from controls despite a substantial reduction in the concentration of 5-HT and 5-HIAA. In contrast, significant changes in both the Bmax and KD values were observed in the cerebral cortex of rats treated with PCA. Furthermore, [3H]paroxetine binding and tissue concentrations of 5-HT and 5-HIAA were measured in the following different regions of the rat brain: cingulate, parietal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum; ventral mesencephalic tegmentum; and midbrain raphe nuclei region after administration of PCPA, PCA, or saline vehicle. There was an excellent correlation between regional 5-HT levels and specific [3H]paroxetine binding in control and PCA-treated rats although this correlation was lost after PCPA treatment. Under these conditions, the 5-HT innervation remains unchanged whereas the concentration of 5-HT and 5-HIAA is greatly reduced. Thus, [3H]paroxetine binding appears to provide a reliable marker of 5-HT innervation density within the mammalian CNS.

Effect of serotonin depletion by p-chlorophenylalanine upon discriminative behaviours

1. 1. Para-chlorophenylalanine ( p-CPA), a competitive inhibitor of the serotonin (5-HT) synthesis enzyme tryptophan hydroxylase, was administered to rats at a dosage (100 mg/kg daily for 3 days) that depletes 5-HT. 2. 2. Different groups of these rats were previously trained to discriminate the interoceptive stimuli produced by amphetamine, cathinone, 3,4-methylenedioxymethamphetamine (MDMA), N-ethyl-3,4-methylenedioxyamphetamine (MDE), fenfluramine or yohimbine, and the effect of p-CPA pretreatment upon their discriminative performance was compared with the effect of saline (control) pretreatment. 3. 3. p-CPA was shown to have no effect upon the dopaminergically-mediated stimuli produced by the stimulants amphetamine and cathinone or upon yohimbine performance. 4. 4. p-CPA significantly decreased discriminative performance with the serotonergic releasing drugs MDMA, MDE and fenfluramine. This decrease in discriminative performance returned to pre- p-CPA (criterion) levels at a time (9–12 days) when 5-HT has been reported to replete to normal brain concentrations. 5. 5. It is concluded that p-CPA pretreatment lowers brain 5-HT and, in turn, significantly decreases the ability of rats to discriminate centrally active drugs whose interoceptive cueing stimuli are mediated by 5-HT neurons.

Injected tryptophan increases brain but not plasma tryptophan levels more in ethanol treated rats

In previous studies, long term treatment with ethanol has been shown to enhance brain 5-hydroxytryptamine 5-(HT) metabolism by increasing the activity of the regulatory enzyme tryptophan hydroxylase and or availability of circulating tryptophan secondarily to an inhibition of hepatic tryptophan pyrrolase. In the present study ethanol treatment given for two weeks decreased hepatic apo-tryptophan pyrrolase but not total tryptophan pyrrolase activity in rats. Tryptophan levels in plasma and brain did not increase significantly. But there was a marked increase of 5-HT but not 5-hydroxyindoleacetic acid (5-HIAA) concentration in brain, suggesting a possible increase in the activity of tryptophan hydroxylase. The effect of a tryptophan load on brain 5-HT metabolism was therefore compared in controls and ethanol treated rats. One hour after tryptophan injection (50mg/kg i.p.) plasma concentrations of total and free tryptophan were identical in controls and ethanol treated rats, but the increases of brain tryptophan 5-HT and 5-HIAA were considerably greater in the latter group. The results are consistent with long term ethanol treatment enhancing brain serotonin metabolism and show that brain uptake/utilization of exogenous tryptophan is increased in ethanol treated rats and may be useful to understand the role and possible mechanism of tryptophan/serotonin involvement in mood regulation.