C1473G polymorphism in mouse tph2 gene is linked to tryptophan hydroxylase‐2 activity in the brain,intermale aggression,and depressive‐like behavior in the forced swim test

Tryptophan hydroxylase‐2 (TPH2) is the rate‐limiting enzyme of brain serotonin synthesis. The C1473G polymorphism in the mouse tryptophan hydroxylase‐2 gene affects the enzyme's activity. In the present study, we investigated the linkage between the C1473G polymorphism, enzyme activity in the brain, and behavior in the forced swim, intermale aggression, and open field tests using mice of the C57BL/6 (C/C) and CC57BR/Mv (G/G) strains and the B6‐1473C (C/C) and B6‐1473G (G/G) lines created by three successive backcrossings on C57BL/6. Mice of the CC57BR/Mv strain had decreased brain enzyme activity, aggression intensity, and immobility in the forced swim test, but increased locomotor activity and time spent in the central part of the open field arena compared with animals of the C57BL/6 strain. Mice of the B6‐1473G line homozygous for the 1473G allele had lower TPH2 activity in the brain, aggression intensity, and immobility time in the forced swim test compared with animals of the B6‐1473C line homozygous for the 1473C allele. No differences were found between the B6‐1473G and B6‐1473C mice in locomotor activity and time spent in the central part of the arena in the open field test. Thus, the C1473G polymorphism is involved in the determination of TPH2 activity and is linked to aggression intensity and forced‐swim immobility in mice. At the same time, the polymorphism does not affect locomotion and anxiety‐related behavior in the open field test. The B6‐1473C and B6‐1473G mice represent a valuable experimental model for investigating molecular mechanisms of serotonin‐related behavior. © 2008 Wiley‐Liss, Inc.     

Regional serotonin metabolism in the brain of transgenic mice lacking monoamine oxidase A.

The effect of a lack of the gene encoding monoamine oxidase A (MAO A) in transgenic Tg8 mice on the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin (5-HT) biosynthesis, and on the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the midbrain, hypothalamus, hippocampus, striatum, amygdala, and frontal cortex was studied. It was shown that mice with a genetic MAO A knockout differed from mice of the initial C3H/HeJ strain in having a higher level of 5-HT and a lower level of its metabolite, 5-HIAA, in all brain regions but the frontal cortex, where the changes were insignificant. Although the 5-HIAA/5-HT ratio in various brain regions differed considerably, the decrease of the 5-HT oxidative deamination index in Tg8 mice was similar in different brain regions (to 41-45% of control values), with the exception of the frontal cortex, where the decrease of the 5-HIAA/5-HT was somewhat smaller (to 54%). The presence of the remaining 45% +/- 1.9% of the control ratio value indicates rather effective oxidative deamination of 5-HT in MAO A knockout mice and explains the lack of severe behavioral and pathological consequences in MAO A genetic deficiency. An increase of TPH activity in mice lacking MAO A was found in the frontal cortex, hippocampus, and amygdala. No significant changes were found in the striatum, hypothalamus, and midbrain. The data show an effect of the MAO A gene mutation on TPH and indicate a uniform decrease of 5-HT catabolism in different brain regions except for the frontal cortex, which is somewhat more resistant to the lack of MAO A than other brain structures.     

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) enhances tryptophan hydroxylase activity in mouse striatum, but not in the frontal cortex

We measured serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) contents and tryptophan hydroxylase (TPH) activity in mouse striatum and frontal cortex after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (7 daily injections of 30 mg/kg). In the striatum, TPH activity was increased for at least 4 weeks after injection of MPTP, along with an increase in 5-HIAA. However, no significant change was observed in 5-HT, 5-HIAA or TPH activity in the frontal cortex. These results suggest that MPTP affects 5-HT through a change in TPH activity, specifically at nerve terminals in the striatum.     

Comparison of regional serotonin levels and turnover in the brain of naturally high and low aggressive rats

Rats, matched by weight and goal approach, were tested for aggressiveness using a food-competition paradigm. Those winning and those losing all of their matches were designated as high and low aggressive, respectively. A third group of randomly selected rats never fought and were used as a control for the fighting experience. Half of the rats were used to determine serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels in discrete brain regions. The other half were used to estimate 5-HT turnover in the same brain regions.High and low aggressive rats did not differ with regard to 5-HT or 5-HIAA levels, but high aggressive rats showed faster 5-HT turnover than low aggressive rats. The turnover differences reached statistical significance in only two of the 3 brain regions examined: (a) all structures (minus olfactory bulbs) rostral to the level of the anterior commissure, and (b) the brain stem (hypothalamus-midbrain-medulla). The observed differences cannot be attributed to differing behavioral experiences since the high and low aggressive groups pooled did not differ from the no-fighting control.These findings are discussed with respect to the involvement of serotonin in rat intraspecies aggression.     

Intermale aggression in corticotropin-releasing factor receptor 1 deficient mice

The anxiogenic neuropeptide, corticotropin-releasing factor (CRF), has a complex effect on intermale aggression. CRF receptor 1 (CRFR1) is the primary receptor for CRF and in this study, we examined in detail isolation-induced intermale aggression in CRFR1 deficient mice. All mice contained a mixed 50:50 inbred/outbred background to improve aggressive performance. Mice were isolated for 4 weeks prior to 2 consecutive days of aggression testing using the resident-intruder paradigm. Mice were also tested for anxiety on the elevated plus maze. Relative to littermate wild-type (WT) controls, CRFR1-mutant mice exhibited normal levels of intermale aggression over the 2 test days in terms of percentage showing aggression, number of attacks, time aggressive, and latency to first attack. In terms of sites of attacks on intruders, CRFR1-deficient mice attacked the ventral portion of the mid-section (including belly) significantly less frequently than WT males on test day 1, but these differences did not reach significance on test day 2. No other differences in sites of attacks were observed. Tail rattling also did not differ between groups. Importantly, KO males showed decreased anxiety relative to WT mice (consistent with previous reports) as evidenced by spending significantly more time on the open arms and significantly less time on the closed arms of the elevated plus maze. Plus maze performance did not correlate with any measure of levels of aggression, suggesting a dissociation between altered levels of anxiety and aggressive performance. Taken together, the results suggest that the activation CRFR1 is not necessary for the normal production of isolation-induced intermale aggression.     

Effect of brain monoamine precursors on stress-induced behavioral and neurochemical changes in aged mice

Male CF-1 mice aged 22 months showed approximately the same level of motor activity and aggressive behavior as 3-month-old mice under control (no stress) conditions, or 45 min following cold swim stress. Increasing brain catecholamine activity by dietary l-tyrosine treatment had no effect on these two age groups either under control conditions or after stress. In contrast, 30-month-old mice showed lower motor activity under control conditions which was raised significantly by supplementation of the diet with l-tyrosine. However, marked reductions in activity and aggression following stress were observed in the 30-month-old animals and these deficits were not reversed by l-tyrosine treatment prior to stress. Reduction in motor activity was greatest in stressed, 30-month-old mice on l-tyrosine supplemented diets. Compared to 3-month-old mice, the 30-month-old animals had lower brain tyrosine following dietary l-tyrosine treatment, lower brain tryptophan, norepinephrine (NE), dopamine (DA) and DOPAC, but higher HVA, serotonin (5-HT) and 5-HIAA levels. Under both control (no stress) and stress conditions, l-tyrosine pretreatment decreased brain 5-HT in the young animals, but increased 5-HT in the old mice. After stress the 30-month-old animals evidenced only slight increases in levels of blood corticosterone. Brain tyrosine was reduced by stress in the young animals but increased by stress in the old animals. Stress-induced decreases in brain NE and increased in serotonin and 5-HIAA levels were observed in both age groups. These results are consistent with hypotheses concerning age-related alterations in brain monoamine functions and adrenocortical control mechanisms.     

A comparative study of aggression related changes in brain serotonin in CBA, C57BL, and DBA mice

Spontaneous aggression was induced in 3 strains of male mice (CBA, C57BL, and DBA) by a new technique of socially intermixing them for 14 days and daily measuring the amount of scarring each received. In all strains, serotonin (5-HT) was lower in the ventromedial (VMH) and supraoptic (SO) hypothalamic areas and amygdala in the more aggressive as compared to the controls or less aggressive animals. The correlation coefficients between the amount of physical scarring and brain 5-HT in these areas ranged from 0.50 to 0.91 (P less than 0.05 to P less than 0.01). Hippocampal (HPC) 5-HT was also lower in the more aggressive animals in CBA mice. Also the most aggressive strain (CBA) tended to have the lowest SO, VMH, and amygdala 5-HT concentration. In all 3 strains the concentration of brain 5-HT from lowest to highest was: frontal cortex less than HPC less than amygdala less than VMH less than SO. The data suggest that there is an inverse relationship between hypothalamic and limbic 5-HT and aggressiveness. Also the data suggest that the technique of intermixing 3 strains of mice successfully induced spontaneous aggression which peaked around 14 days and was reliable and could be quantified.     

Elevated stress sensitivity in corticotropin-releasing factor receptor 2 deficient mice decreases maternal, but not intermale aggression

Maternal aggression is a form of aggression towards intruders by lactating females that is critical for defense of offspring. During lactation, fear and anxiety are reduced, the CNS is less responsive to the anxiogenic neuropeptide, corticotropin-releasing factor (CRF), and central injections of CRF inhibit maternal aggression. Together, these previous findings suggest that decreased CRF neurotransmission during lactation supports normal maternal aggression expression. Recent work indicates that mice deficient in CRF receptor 2 (CRFR2) display increased anxiety-like behaviors, have a hypersensitive stress response, and overproduce CRF. In this study, we examined both maternal and intermale aggression in wild-type (WT) and CRFR2-deficient mice. CRFR2-mutant mice exhibited significant deficits in maternal aggression on postpartum Day 4 relative to WT mice in terms of percentage displaying aggression, mean number of attacks, and mean time in aggressive encounters. However, time sniffing male intruder, pup retrieval, number of pups, and performance on the elevated plus maze were similar between genotypes. In contrast, intermale aggression did not differ between genotype in any measure on any of three consecutive test days. For neither form of aggression did sites of attacks on the intruder differ between genotype. Taken together, the results suggest that differences in stress sensitivity and the overproduction of CRF of the knockout (KO) mice specifically affects maternal, but not intermale aggression.     

Behavioral effects of pubertal anabolic androgenic steroid exposure in male rats with low serotonin

The goal of this study was to assess the interactive effects of chronic anabolic androgenic steroid (AAS) exposure and brain serotonin (5-hydroxytryptamine, 5-HT) depletion on behavior of pubertal male rats. Serotonin was depleted beginning on postnatal day 26 with parachlorophenylalanine (PCPA 100 mg/kg, every other day); controls received saline. At puberty (P40), half the PCPA-treated rats and half the saline-treated rats began treatment with testosterone (T, 5 mg/kg, 5 days/week). Behavioral measures included locomotion, irritability, copulation, partner preference, and aggression. Animals were tested for aggression in their home cage, both with and without physical provocation (mild tail pinch). Brain levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were determined using HPLC. PCPA significantly and substantially depleted 5-HT and 5-HIAA in all brain regions examined. Chronic T treatment significantly decreased 5-HT and 5-HIAA in certain brain areas, but to a much lesser extent than PCPA. Chronic exposure to PCPA alone significantly decreased locomotor activity and increased irritability but had no effect on sexual behavior, partner preference, or aggression. T alone had no effect on locomotion, irritability, or sexual behavior but increased partner preference and aggression. The most striking effect of combining T+PCPA was a significant increase in attack frequency as well as a significant decrease in the latency to attack, particularly following physical provocation. Based on these data, it can be speculated that pubertal AAS users with low central 5-HT may be especially prone to exhibit aggressive behavior.     

Manipulations of dietary tryptophan: Effects on mouse killing and brain serotonin in the rat

Maintaining rats on a tryptophan-free diet for 4--6 days induced mouse killing in non-killer rats, and significantly facilitated killing in killer rats, as indicated by shorter latencies to kill the mice. The killing responses were similar in topography to the natural killing responses. These changes in killing behavior did not appear to be due to generalized changes in irritability. The increased killing after maintenance on a tryptophan-free diet was accompanied by a 26% reduction in brain serotonin (5-HT) and a 29% reduction in brain 5-hydroxyindoleacetic acid (5-HIAA). When the tryptophan-free diet was supplemented with L-tryptophan (0.5 or 2%), brain 5-HT and 5-HIAA were increased above control levels, and the rat's killing response appeared normal both in terms of latency and topography, similar to that seen in control chow fed animals. While rats consumed less of the tryptophan-free and tryptophan supplemented diets, control subjects deprived of chow such that they lost as much weight as rats fed the tryptophan-free diet, did not show changes in killing behavior. These results are consistent with the hypothesis that central serotonergic systems exert inhibitory control over mouse killing behavior in rats.     

Measurement of 5-hydroxytryptamine synthesis and metabolism in selected discrete regions of the rat brain using high performance liquid chromatography and electrochemical detection: Pharmacological manipulations

High performance liquid chromatography coupled with electrochemical detection (LCEC) was employed to measure 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophan (5-HTP) in the suprachiasmatic (SCN), medial preoptic (MPO) and arcuate (AN) nuclei as well as the median eminence (ME) and striatum (ST) of individual rat brains. Biochemical estimations of changes in 5-HT neuronal activity were made by measuring: (1) concentrations of 5-HT and 5-HIAA and (2) the rate of 5-HT synthesis (5-HTP accumulation following the administration of NSD 1015, an inhibitor of aromatic L-amino acid decarboxylase) after the administration of pharmacological agents known to influence these neurons. Pargyline increased the concentration of 5-HT and decreased the concentration of 5-HIAA while probenecid increased the concentration of 5-HIAA in all 5 brain regions. At both 2 and 24 hours after reserpine the concentration of 5-HT decreased, 5-HIAA increased or did not change, and the rate of 5-HT synthesis increased. In most of the brain regions blockers of 5-HT neuronal uptake (fluoxetine, chlorimipramine) did not influence 5-HT or 5-HIAA concentrations dramatically, but increased the rate of 5-HT synthesis. L-tryptophan generally increased the concentrations of 5-HT and 5-HIAA as well as the rate of accumulation of 5-HTP in all regions except the ME where 5-HIAA and 5-HTP concentrations both were unaffected. These results reveal that the method using LCEC is sensitive enough to detect pharmacologically-induced changes in 5-HT metabolism and synthesis in discrete regions of rat brain. The drugs examined in the present study generally caused similar changes in 5-HT dynamics in all 5 brain regions examined.     

Dietary restriction changes behaviours in brain-derived neurotrophic factor heterozygous mice: role of serotonergic system

Accumulating evidence has suggested that brain-derived neurotrophic factor (BDNF) plays a role in eating behaviours, and that BDNF-heterozygous (+/-) mice exhibit abnormal behaviours (e.g. obesity, anxiety and aggression). The present study was undertaken to determine whether or not dietary restriction (DR) alters the behaviours in BDNF(+/-) mice, as DR has been shown to exert a number of beneficial effects on the brain. Eight-week-old male wild-type (+/+) and BDNF(+/-) mice were divided into two groups, ad libitum (AL) diet group and DR group, for 16 weeks. After carrying out a behavioural evaluation, we determined the BDNF mRNA levels, as well as mRNA levels for subtypes (5-HT(1A), 5-HT(1B), 5-HT(2A) and 5-HT(2C)) of the 5-HT receptor and 5-HT transporter (5-HTT), protein levels of BDNF and concentrations of 5-HT and 5-HIAA in the hypothalamus, hippocampus and frontal cortex. DR significantly ameliorated behaviours including obesity, anxiety and aggression in BDNF(+/-) mice. The concentrations of 5-HT and 5-HIAA in the frontal cortex, and 5-HT in the hippocampus, of BDNF(+/-) mice were significantly lower than those of wild-type mice. Interestingly, DR significantly increased the levels of 5-HT and 5-HIAA in the frontal cortex of BDNF(+/-) mice. These findings suggest that DR may alter the behaviours in BDNF(+/-) mice, and that the 5-HT system may be implicated in the beneficial effects of DR on these behaviours.     

Cerebrospinal fluid 5-hydroxyindolacetic acid and homovanillic acid: reciprocal relationships with impulsive aggression in human subjects

While the relationship between cerebrospinal fluid 5-HIAA (CSF 5-HIAA) and aggression is typically reported as inverse, studies of some groups of aggressive individuals demonstrate a positive (or no) relationship, between these two variables. It is possible that simultaneous examination of both CSF 5-HIAA and CSF homovanillic acid (HVA), which co-vary in human subjects may clarify differences in reported findings in different groups of aggressive individuals. CSF 5-HIAA and CSF HVA concentrations were simultaneously examined in 60 healthy human subjects (40 with personality disorder and 20 healthy controls) and were correlated with measures of aggression and impulsivity. CSF 5-HIAA concentrations correlated positively, and CSF HVA concentrations correlated inversely, with a composite measure of impulsive aggression in all subjects as well as in the personality disordered subjects. The CSF 5-HIAA findings are consistent with those demonstrating reduced post-synaptic 5-HT receptor responsiveness to 5-HT agent challenge and suggest differences in the pathophysiology between different groups of subjects with aggressive behavior, particularly with regard to severity of aggressive behavior.     

Aggression and monoamines: effects of sex and social rank in zebrafish (Danio rerio)

Social defeat is a common model for studies on depression. However, such models are most often used to study aggression in males and sex differences in depression may therefore be overseen. This study investigated the potential of the zebrafish (Danio rerio) as a model for male and female aggression. In addition, effects on the brain serotonergic and dopaminergic neurotransmitter systems after agonistic interaction are well studied in many species, but not in zebrafish. We wanted to explore whether the zebrafish follows the same patterns as many other species. Therefore, the effects of agonistic interaction on brain monoaminergic activity were studied in adult male and female wild-type zebrafish. The fish interacted in pairs with one of the same sex for five days during which agonistic behaviour was quantified daily. Clear dominant/subordinate relationships developed in all pairs, both in males and females. The frequency of aggressive acts increased over time but did not differ between male and female pairs. Further, we found that dyadic agonistic interaction resulted in elevated brain serotonergic activity in subordinate zebrafish, as indicated by elevated hindbrain 5-hydroxyindoleacetic acid to serotonin ratios (5-hydroxyindolacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT) ratios). We also observed a sex difference in forebrain dopamine levels and forebrain 5-HIAA/5-HT ratios, with females displaying higher concentrations of dopamine but lower 5-HIAA/5-HT ratios than males.These results suggest that zebrafish is a suitable model for studies on female aggression and sex differences in brain monoaminergic neurotransmission.     

Rat brain serotonin: Biochemical and functional evidence for a sex difference

Summary Male and female rats were compared with respect to brain serotonin (5-HT) levels, synthetic capacity, receptor sensitivity, and CNS functions. Levels of whole brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were higher in females. The accumulation of 5-HT after treatment with the monoamine oxidase inhibitor pargyline alone and in combination with the 5-HT precursor L-tryptophan was greater in females than in males. 5-HT increased and 5-HIAA decreased to the same extent in both sexes after administration of the 5-HT agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The temperature fall after all drug treatments was greater in females, but the 5-HT behavioural syndrome was more pronounced in females merely after pargyline plus tryptophan; the behavioural response after 8-OH-DPAT did not differ between the sexes. These results are indicative of sex differences in the brain 5-HT neuronal systems. They are discussed in relation to differences between males and females in sexual behaviour, aggression and affective disorders.     

Estradiol modulation of central monoamine activity in female mountain spiny lizards

Although estradiol (E2) mediates many behaviors in females, relatively little is known about its role in female aggression. Previous studies in female mountain spiny lizards indicated that female aggression is modulated by ovariectomy and sex steroid hormone replacement and that expression of aggressive behavior is accompanied by changes in serotonin activity. This study examines if E2 modulates the activity of serotonin and other central monoamines. Free-living females were caught and housed in the laboratory and received one of 3 treatments: sham surgery (SHAM), ovariectomy plus empty implant (OVEX), or sham surgery plus a long lasting E2 implant (E2-IMP). After 3 weeks of treatment, selected brain areas were examined for levels of monoamines and their metabolites. Changes in monoamine activity were most pronounced in the septum where levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) were higher in E2-IMP females relative to SHAM, and levels of 5-HIAA were higher in OVEX females relative to SHAM. Changes in dopamine (DA) activity were also found, with increased DA concentration and decreased ratio of forebrain:brainstem HVA concentrations in E2-IMP relative to SHAM females. These results suggest that the actions of E2 on aggression might be mediated, in part, by dose-dependent effects on 5-HT activity in the septum.     

Mice selected for differences in sensitivity to a benzodiazepine receptor inverse agonist vary in intermale aggression

Brain γ-aminobutyric acid (GABA) levels are involved in intermale aggression in mice. It was therefore expected that animals genetically selected for their sensitivity to the convulsive effects of methyl β-carboline-3-carboxylate (β-CCM; BS, β-CCM sensitive, and BR, β-CCM resistant), a benzodiazepine (BZ) inverse agonist that specifically binds to the BZ site on the GABA-A receptor complex, would differ in their levels of aggressive behavior. Using two different aggression tests, in two independent experiments, we showed that BS mice are more aggressive than BR animals. The precise mechanisms underlying the observed line differences in β-CCM sensitivity and aggression remain to be determined. Received: 18 January 1999 / Accepted: 24 June 1999 / Published online: September 1 1999     

Tryptophan metabolism in developing chick brain.

The normal patterns of the occurrence of tryptophan, 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and monoamine oxidase (MAO) activity in chick brain during development were investigated. Tryptophan level in the brain on the 7th day of embryonic development was rather high and decreased gradually until the 17th day, but increased markedly from the 17th day to hatching, then increased slightly from hatching to the 14th day after hatching. 5-HT, 5-HIAA and MAO were already present on the 7th day of incubation, increased gradually with a temporary peak on the 14th day, and increased remarkably from the 17th day to hatching, after which they reached a plateau and remained unchanged from the 7th to the 14th day after hatching. Since the increase between the 17th day and hatching was observed in the patterns of tryptophan, 5-HT, 5-HIAA and MAO activity, the critical period of tryptophan metabolism in the brain during development seems to exist in this period. In order to examine the formation of 5-HT from tryptophan in vivo, newly hatched chicks and chicks 2 weeks after hatching were injected intraperitoneally with L-tryptophan and brain 5-HT was measured. 5-HT was increased markedly, even in newborn chick brain. This might indicate that the activity of tryptophan hydroxylase in newborn chick brain would be as high as that in adult chick brain.     

Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice

The relative contribution of the two tryptophan hydroxylase (TPH) isoforms, TPH1 and TPH2, to brain serotonergic system function is controversial. To investigate the respective role of TPH2 in neuron serotonin (5-HT) synthesis and the role of 5-HT in brain development, mice with a targeted disruption of Tph2 were generated. The preliminary results indicate that in Tph2 knockout mice raphe neurons are completely devoid of 5-HT, whereas no obvious alteration in morphology and fiber distribution are observed. The findings confirm the exclusive specificity of Tph2 in brain 5-HT synthesis and suggest that Tph2-synthesized 5-HT is not required for serotonergic neuron formation.     

Tryptophan hydroxylase inhibition increases preprotachykinin mRNA in developing and adult medullary raphe nuclei.

In order to study the regulation of co-localized monoamine and peptide neurotransmitters in the medullary raphe nuclei (MRN), we determined whether inhibition of serotonin (5-HT) synthesis affected levels of preprotachykinin (PPT; the prohormone precursor of substance P) mRNA in the MRN. Adult rats received p-chlorophenylalanine (pCPA), an irreversible inhibitor of tryptophan hydroxylase (TPH), via Alzet minipumps. TPH activity was inhibited by 70-80% for 3 weeks following pump implantation. During this period Northern mRNA analysis revealed that PPT mRNA levels in the MRN were increased 1.5-2-fold. The pCPA-induced increase was specific for PPT mRNA since no change was detected in mRNA coding for neuron-specific enolase (NSE; a constitutive neuronal protein) or 28 S ribosomal RNA. To determine whether fetal inhibition of 5-HT synthesis affected development of PPT mRNA in the MRN, pregnant rats were administered pCPA via Alzet minipump implanted on embryonic day 8. In pCPA-treated litters TPH activity was decreased by 60-70% from E16 to postnatal day 3 (P3), returning to control levels by P8. Northern mRNA analysis revealed that PPT mRNA levels increased 2.4-fold of control levels at P1. Infusion of pCPA for one week resulted in an earlier increase in PPT mRNA levels, suggesting that birth was not required to elicit the surge in PPT message. These results support the hypothesis that alterations in 5-HT metabolism have regulatory consequences for co-localized substance P formation in the MRN.