The inhibition of tryptophan hydroxylase, not protein synthesis, reduces the brain trapping of α-methyl-l-tryptophan: an autoradiographic study

The effects of the tryptophan hydroxylase (TPH) inhibitor p-chlorophenylalanine (PCPA; 200mg/kg; 3 days), and of the protein synthesis inhibitor cycloheximide (CXM, 2mg/kg), on regional serotonin (5-HT) synthesis were studied using the alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) autoradiographic method. The objectives of these investigations were to evaluate the changes, if any, on 5-HT synthesis, as measured with alpha-MTrp method, following the inhibition of TPH by PCPA, or the inhibition of proteins synthesis by CXM. The rats were used in the tracer experiment approximately 24h after the last dose of PCPA was administered, and in the CXM experiments, they were used 30 min following a single injection of CXM. In both experiments, the control rats were injected with the same volume of saline (0.5 ml/kg; s.c.) and at the same times as the drug injections. The results demonstrate that trapping of alpha-MTrp, which is taken to be related to brain 5-HT synthesis, is drastically reduced (40-80%) following PCPA treatment. The inhibition of protein synthesis with CXM did not have a significant effect on the global brain trapping of alpha-MTrp and 5-HT synthesis. These findings suggest that the brain trapping of alpha-[14C]MTrp relates to brain 5-HT synthesis, but not to brain protein synthesis.     

Effects of acute and chronic administration of the serotonin1A agonist buspirone on serotonin synthesis in the rat brain

The effects of acute and chronic administration of buspirone, a serotonin 5-HT1A agonist, on the 5-HT synthesis rates in various rat brain structures were investigated using alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) and an autoradiographic method. In the acute treatment study, buspirone (10 mg/kg) was injected subcutaneously 30 min before alpha-[14C]MTrp administration (30 microCi over 2 min) into a femoral vein. In the chronic treatment study, buspirone was given in a sustained fashion (10 mg/kg/day) for 14 days using an osmotic minipump implanted subcutaneously. Rats were killed 60 and 150 min after alpha-[14C]MTrp administration (two-time point method). A single dose of buspirone induced a significant decrease of 5-HT synthesis throughout the brain with the exception of the pineal body. However, the chronic treatment with buspirone did not induce significant differences in 5-HT synthesis in the brain. There was no significant difference in plasma free tryptophan concentration between any of the groups. The unaltered 5-HT synthesis rates in the chronic treatment study likely reflect a normalization of this parameter due to a desensitization of 5-HT1A autoreceptors on the cell body of 5-HT neurons, which has been previously shown to occur following long-term treatment with 5-HT1A agonists.     

Effects of serotine receptors agonists, TFMPP and CGS12066B, on regional serotonin synthesis in the rat brain: an autoradiographic study

The effects of acute and repeat administration of the serotonin (5-HT)(1) agonists TFMPP [N -(3-trifluoromethyl)phenylpiperazine hydrochloride] and CGS12066B [7-trifluoromethyl-4- (4-methyl-1-piperazinyl)pyrrolo[1,2-a ]-quinoxaline dimaleate] were evaluated on 5-HT synthesis rates using the alpha-[(14) C]methyl-l-tryptophan (alpha-MTrp) autoradiographic method. In the acute treatment study, TFMPP (10 mg/kg) and CGS12066B (5 mg/kg) were injected intraperitoneally 30 min before an alpha-MTrp injection. In an acute study TFMPP reduced overall brain 5-HT synthesis, in the dorsal and median raphe, and in almost all of their projection areas, with the exception of the parietal, sensory-motor, and frontal cortices, the accumbens nucleus, and the caudate. Acute CGS12066B treatment did not have overall significant effect, but the rates did decrease in the cell body areas of 5-HT neurons. In a 7-day treatment with TFMPP (10 mg/kg/day) or CGS12066B (5 mg/kg/day), the 5-HT synthesis rates (24 h after last dose) decrease, with both compounds, in almost all of the nerve terminal structures. TFMPP reduced the synthesis in the dorsal and median raphe, while CGS12066B reduced it only in the dorsal raphe. This data suggests that after a 7-day treatment with TFMPP and CGS12066B, the rate of 5-HT synthesis in the dorsal raphe is restored and is reduced in many projection areas. The observed effects in the 7-day treatment could also be related to actions through the postsynaptic 5-HT(1B) sites and/or other 5-HT receptors since this compounds have limited selectivity.     

Effects of pentadecapeptide BPC157 on regional serotonin synthesis in the rat brain: alpha-methyl-L-tryptophan autoradiographic measurements

A novel pentadecapeptide, BPC157, was recently reported to have a large spectrum of in vivo activities, from anti-ulcer to central action on the brain dopaminergic system. The mechanisms of these actions are not well understood. In this study, the evaluation of the effects of acute and repeated administration of BPC157 on serotonin (5-HT) synthesis in the rat brain is reported. The alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method was used to measure regional 5-HT synthesis rates. In the first series of experiments, a single dose treatment of BPC157 (10 microg/kg) administered intraperitoneally 40 min before the alpha-MTrp tracer injection significantly reduced the regional rate of 5-HT synthesis in the dorsal thalamus, hippocampus, lateral geniculate body and hypothalamus. 5-HT synthesis rates in the substantia nigra reticulate and medial anterior olfactory nucleus in BPC157 treated rats were significantly higher than in the control rats. No significant change in the synthesis rate was observed in the raphe nuclei. In the second series of experiments, following a 7-day treatment with BPC157 (10 microg/kg; s.c.), a significant reduction in the 5-HT synthesis rate was observed in the dorsal raphe nucleus, and significant increases were observed in the substantia nigra, lateral caudate, accumbens nucleus and superior olive. This data suggests that BPC157, a gut peptide, influences brain 5-HT synthesis in rats, but we cannot determine, from this data, the mechanism of this action.     

Selective 5-HT1B receptor agonist reduces serotonin synthesis following acute, and not chronic, drug administration: results of an autoradiographic study

The effects of acute and chronic administration of the serotonin (5-HT)1B agonist CP-93,129, on 5-HT synthesis rates were evaluated using the alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method. In the acute treatment study, CP-93,129 (7 mg/kg) was injected intraperitoneally 30 min before the alpha-MTrp injection (30 microCi over 2 min). A single dose of CP-93,129 caused a significant increase in the synthesis in the median raphe nucleus (MR) without a significant influence on the dorsal raphe nucleus (DR). There was a reduction in 5-HT synthesis in almost all of the projection areas. In the chronic treatment study, CP-93,129 was administered continuously (7 mg/kg/day) for 14 days using an osmotic minipump implanted subcutaneously. The chronic treatment with CP-93,129 did not produce a significant change in 5-HT synthesis in the raphe nuclei nor in the nerve terminal structures, except for the medial frontal bundle and the visual and sensory-motor cortices. The unaltered 5-HT synthesis rates in the chronic treatment study probably reflect a normalization of the synthesis as a result of the desensitization of 5-HT1B autoreceptors and/or heteroreceptors.     

Regional brain serotonin synthesis is increased in the olfactory bulbectomy rat model of depression: an autoradiographic study

Serotonin synthesis rates were evaluated using alpha-[14C]methyl-l-tryptophan (alpha-MTrp) autoradiographic methods in olfactory bulbectomized (OBX) rats. They were significantly (p < 0.05) increased in the frontal (50%) and parietal (40%) cortices, superior olive (over 30%), and the substantia nigra (30%) in the OBX rats as compared to the sham operated animals. There were also increases in 5-hydroxytryptamine (5-HT) synthesis in some limbic areas: the cingulate (32%), the medial forebrain bundle (58%), the hippocampus (13-25%) and the thalamus (22-40%). The largest increase in 5-HT synthesis after OBX was observed in the sensory-motor cortex (67%). 5-HT synthesis rates were significantly decreased in the dorsal and medial raphe nuclei, but there was no significant change the ventral tegmental area and the locus coeruleus following OBX. These results indicate that olfactory bulbectomy causes an imbalance in 5-HT synthesis in some projection areas by disproportionally increasing 5-HT synthesis rates in specific brain regions and making more 5-HT available for neurotransmission. This imbalance in 5-HT synthesis and the subsequent elevation of tissue 5-HT may be responsible for the creation of non-physiological circuitry which may, in part, be reflected in the symptoms resembling human depression.     

Acute flesinoxan treatment produces a different effect on rat brain serotonin synthesis than chronic treatment: an alpha-methyl-l-tryptophan autoradiographic study

5-HT(1A) receptor agonists display anxiolytic and anti-depressant properties in clinical studies. In this study, we used the alpha-[(14)C]methyl-l-tryptophan (alpha-MTrp) autoradiographic method to evaluate the effects of the 5-HT(1A) agonist, flesinoxan, on regional 5-HT synthesis in the rat brain, following acute or a 14-day continuous treatment. In the first series of experiments, flesinoxan (5mg/kg; i.p.) was administered 40min before the alpha-MTrp. It resulted in a significant increase of the arterial blood oxygen partial pressure (pO(2)) and a reduction of the regional rate of 5-HT synthesis throughout the brain, with the exception of a few regions (medial geniculate body and thalamus). In the second series of experiments, flesinoxan (5mg/kgday) was administered for 14 days, using an osmotic minipump implanted subcutaneously. When compared to rats treated with saline, there was an overall significant (p<0.05) reduction in the synthesis (one-sample two-tailed t-test). However, there was no significant influence on the 5-HT synthesis rate in the dorsal and median raphe nuclei and the majority of their projection areas. A significant (p<0.05) reduction was observed in the nucleus raphe magnus, medial caudate, ventral thalamus, amygdala, ventral tegmental area, medial forebrain bundle, nucleus accumbens, medial anterior olfactory nucleus and superior olive. The unaltered 5-HT synthesis rates in a large majority of regions following the 14-day treatment of flesinoxan may reflect the normalization (implies to not be different from salne treated control) of synthesis due to a desensitization of 5-HT(1A) autoreceptors on the cell body of 5-HT neurons as well as at postsynaptic sites, which is known to occur following long-term treatment with 5-HT(1A) agonists. It is of some importance to note that the normalization of the synthesis occurred in the majority of the brain limbic structures, the brain areas implicated in affective disorders and the corresponding successful treatments, as well as in the cortical regions, which are implicated in mood. However, there were some terminal regions (e.g., accumbens, anterior olfactory, lateral thalamus, raphe magnus and obscurus) in which the chronic flesinoxan treatment resulted in a significant reduction of synthesis, suggesting that there was not a full desensitization across the brain of the receptors controlling 5-HT synthesis.     

Increasing blood oxygen increases an index of 5-HT synthesis in human brain as measured using alpha-[(11)C]methyl-L-tryptophan and positron emission tomography

The main objective of this investigation was to test the hypothesis that brain serotonin (5-HT) synthesis, as measured by trapping of alpha-[(11)C]methyl-L-tryptophan (alpha-MTrp) using positron emission tomography (PET), can be modulated by changes in blood oxygen. The study involved six healthy participants (three male and three female), who breathed a 15% or 60% oxygen mixture starting 15 min before the injection of tracer and continuing during the entire acquisition period. Participants were injected with up to 12m Ci of alpha-MTrp. Two sets of PET images were acquired while the participants were breathing each of the oxygen mixtures and, after reconstruction, all images were converted into brain functional images illustrating the brain trapping constant K(*) (microL/g/min). The K(*) values were obtained for 12 regions of interest outlined on the magnetic resonance images. The K(*) values obtained at high and low blood oxygen content were compared by paired statistics using Tukey's post hoc correction. As there were no difference in plasma tryptophan concentrations, these K(*) values are directly related to regional 5-HT synthesis. The results showed highly significant increases (50% on average) in brain serotonin synthesis (K(*) values) at high (mean value of 223+/-41 mmHg) relative to low (mean value 77.1+/-7.7 mmHg) blood oxygen levels. This suggests that tryptophan hydroxylase is not saturated with oxygen in the living human brain and that increases in blood oxygen can elevate brain serotonin synthesis.     

Acute and Chronic D-Fenfluramine Treatments Have Different Effects on Serotonin Synthesis Rates in the Rat Brain: An Autoradiographic Study

The effects of acute and chronic treatments with D-fenfluramine on the regional rates of serotonin (5-hydroxy-tryptamine; 5-HT) synthesis were investigated using the -[¹⁴C]methyl-L-tryptophan (-[¹⁴C]MTrp) autoradiographic method. In the first series of experiments, acute D-fenfluramine treatment (5 mg/kg; i.p.) given 20 min before the tracer injection significantly (p < 0.05) decreased 5-HT synthesis in the dorsal raphe, and significantly (p < 0.05) increased the rates in the cerebral cortices and caudate nucleus, when compared to the rates in the control rats (saline treated). In a second series of experiments, following a 7-day treatment with D-fenfluramine (5 mg/kg/day; i.p.), a significant (p < 0.05) decrease of 5-HT synthesis, in the dorsal raphe was observed, and significant (p < 0.05) increases were observed in the hypothalamus, the dorsal thalamus, the medial and lateral geniculate body and some brain stem regions (locus ceruleus, inferior and superior colliculus). No significant changes were observed in the cerebral cortices.     

[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.     

Brain 5-HT synthesis in the Flinders Sensitive Line rat model of depression: an autoradiographic study

Alterations of serotonin (5-HT) levels and serotonergic transmission have been associated with depression. 5-HT synthesis is an important factor of serotonergic neurotransmission that may also be altered in depression. Many studies of the relationships between brain serotonergic functions and affective disorders have been performed in different animal models. In this study, brain regional 5-HT synthesis was examined using the alpha-[(14)C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method in a genetic rat model of depression, Flinders Sensitive Line (FSL) rats, and was compared to both the Flinders Resistant Line (FRL) rats and the control Sprague-Dawley (SD) rats. The plasma concentration of free tryptophan in the FSL rats was not significantly different (p > 0.05; ANOVA and post-hoc Bonferroni correction) when compared to that of the FRL and SD rats. The FSL rats had significantly lower 5-HT synthesis (one sample two-tailed t-test on the ratio) than both the FRL and SD rats (the mean ratios were 0.78 +/- 0.12 and 0.73 +/- 0.15, respectively). Overall, the 5-HT synthesis in the FRL rats was not significantly different (p > 0.05) from that in the SD rats (one sample two-tailed t-test on the ratio and the mean ratio was 0.93 +/- 0.13). Studies of individual brain structures, such as the raphe nuclei and their many terminal areas, including the nucleus accumbens, cingulate and frontal cortex, hippocampus, amygdala, and thalamus revealed significant reductions (typically 25-50%) in 5-HT synthesis in the FSL rats compared to the non-depressive FRL and SD rats. These results suggest that significantly reduced 5-HT synthesis in the raphe nuclei and limbic areas in FSL rats may contribute to their depressive features.     

Role of serotonin (5-HT) in the antidepressant-like properties of neuropeptide Y (NPY) in the mouse forced swim test

Neuropeptide Y (NPY) is thought to be implicated in depressive disorders. The mouse forced swim test (FST) is an animal model widely used as a predictor of the efficacy of antidepressant drugs. The present study was undertaken to explore the possible contribution of endogenous serotonin (5-HT) systems in the behavioral effects elicited by NPY in this model. The selective serotonin re-uptake inhibitor (SSRI), fluoxetine, was also tested for comparison. 5-HT was depleted prior to testing by the administration of the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg, i.p., each day for 3 days; control mice received saline-vehicle over the same period). On the fourth day, mice received NPY (3 nmol, I.C.V.), fluoxetine (16 mg/kg, i.p.) or saline injections before testing in the FST. Both NPY and fluoxetine significantly reduced immobility time in saline-treated control animals. Pre-treatment with PCPA significantly blocked the effects of fluoxetine in the FST, confirming the role of endogenous 5-HT. Similarly, pre-treatment with PCPA also significantly attenuated the anti-immobility effects of NPY, thus suggesting a role for 5-HT in the effects of NPY in the FST. Quantitative receptor autoradiography revealed increases in specific [125I][Leu31, Pro34]PYY sites that were sensitive to BIBP3226 (Y1-like sites) in various brain regions. Specific [125I]GR231118 and [125I]PYY(3-36) binding levels were not changed following PCPA treatment, suggesting that depletion of endogenous 5-HT resulted in an apparent increase in the level of Y1 sites in their high-affinity state. Taken together, these results suggest a role for 5-HT-related systems in the antidepressant-like properties of NPY.     

p-Chlorophenylalanine Changes Serotonin Transporter mRNA Levels and Expression of the Gene Product

Abstract: After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p -chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [³H]-citalopram binding and synaptosomal [³H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by ∼20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [³H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.     

Effects of desipramine on regional serotonin synthesis in the rat brain: acute and chronic autoradiographic studies

Various studies have implicated the involvement of noradrenaline (NA) and/or serotonin (5-hydroxytryptamine (5-HT)) in the pathogenesis and treatment of depression. The aim of the present study was to investigate the effects of acute and 7 days of administration of desipramine, a NA re-uptake inhibitor, on the rate of 5-HT synthesis in the rat brain. The study was done by an autoradiographic method using alpha-[14C]-methyl-L-tryptophan as a tracer. The acute (10mg/kg, i.p., 2h before i.v. infusion of the tracer) or 7 days of desipramine (10mg/kg per day, i.p.) did not affect plasma tryptophan (Trp) concentrations, as compared to control (saline treated) rats. Acute treatment with desipramine decreased the rate of 5-HT synthesis in the brain regions that contain 5-HT cell bodies between 19 and 28%, and increased the rate of 5-HT synthesis in the majority of areas containing 5-HT terminals between 21 and 65%. In contrast to the acute treatment, a 7-day administration increased 5-HT synthesis rates in the dorsal raphe (24%), but decreased it in raphe magnus (35%), superior olive (45%), caudate (31%), superior (38%) and inferior (53%) colliculus, and in the auditory cortex (35%). This suggests that the effect of desipramine on 5-HT synthesis rate is time-dependent and differs in the cell bodies and structures containing 5-HT nerve terminals.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

In vivo regulation of the serotonin-2 receptor in rat brain

Serotonin-2 (5-HT-2) receptors in brain were measured using [3H]ketanserin. We examined the effects of amitriptyline, an antidepressant drug, of electroconvulsive shock (ECS) and of drug-induced alterations in presynaptic 5-HT function on [3H]ketanserin binding to 5-HT-2 receptors in rat brain. The importance of intact 5-HT axons to the up-regulation of 5-HT-2 receptors by ECS was also investigated, and an attempt was made to relate the ECS-induced increase in this receptor to changes in 5-HT presynaptic mechanisms. Twelve days of ECS increased the number of 5-HT-2 receptors in frontal cortex. Neither the IC50 nor the Hill coefficient of 5-HT in competing for [3H]ketanserin binding sites was altered by ECS. Repeated injections of amitriptyline reduced the number of 5-HT-2 receptors in frontal cortex. Reserpine, administered daily for 12 days, caused a significant increase in 5-HT-2 receptors, but neither daily injections of p-chlorophenylalanine (PCPA) nor lesions of 5-HT axons with 5,7-dihydroxytryptamine (5,7-DHT) affected 5-HT-2 receptors. However, regulation of 5-HT-2 receptors by ECS was dependent on intact 5-HT axons since ECS could not increase the number of 5-HT-2 receptors in rats previously lesioned with 5,7-DHT. Repeated ECS, however, does not appear to affect either the high-affinity uptake of [3H]5-HT or [3H]imipramine binding, two presynaptic markers of 5-HT neuronal function. 5-HT-2 receptors appear to be under complex control. ECS or drug treatments such as reserpine or amitriptyline, which affect several monoamine neurotransmission systems including 5-HT, can alter 5-HT-2 receptors. While depleting 5-HT alone (5,7-DHT or PCPA) does not alter [3H]ketanserin binding to 5-HT-2 receptors, intact 5-HT axons are necessary for the adaptive up-regulation of the receptor following ECS.     

Serotonin synthesis inhibition or receptor antagonism reduces pregnancy-induced nocturnal prolactin secretion

During early pregnancy, two surges of prolactin (PRL) designated as nocturnal (N) and diurnal (D) are displayed by the rat. We previously reported the positive influence of serotonin (5-HT) in regulating the D surge. Its role in the N surge remained inconclusive due to the contradictory results obtained with the 5-HT synthesis inhibitor parachlorophenylalanine (PCPA) and 5-HT2 receptor antagonists. This study further characterizes the involvement of 5-HT in regulating the N surge. The effectiveness of different doses of ketanserin (KET), a 5-HT2 receptor antagonist, to reduce plasma PRL levels during the surge was established. Sub-threshold (1 mg/kg BW) or just maximally effective (10 mg/kg BW) doses of KET were administered to rats that had been pre-treated with PCPA (250 mg/kg BW) for 24h. The lower dose of KET was ineffective in reducing the N surge even though less 5-HT was available due to PCPA treatment 24h earlier. The higher dose was effective in blocking the surge. Subsequently, the effect of one compared to two injections of PCPA 24 hours apart on plasma PRL levels and concentrations of 5-HT, dopamine (DA) and their respective metabolites 5-hydroxy-indoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) in the medial basal hypothalamus (MBH) and the medial dorsal hypothalamus (MDH) was studied. Two injections of PCPA but not one abolished the N PRL surge. Levels of 5-HT and 5-HIAA were significantly (p less than .005) reduced following either one or two injections of PCPA. Nevertheless, there was a greater (50 fold) decrease in 5-HIAA following 2 injections compared to one injection (10 fold), resulting in lower 5-HT turnover as indicated by lower 5-HIAA/5-HT ratios. Levels of DA in the MBH were reduced significantly only following two injections of PCPA, suggesting that the lack of effect of PCPA after one injection on the N surge was not due to a decrease in DA.     

Perturbation of Rat Brain Serotonergic Systems Results in an Inverse Relation Between Substance P and Serotonin Concentrations Measured in Discrete Nuclei

Since substance P (SP) has been demonstrated to coexist with serotonin (5-HT) in the same population of neurons in the descending raphe system, we have studied the possibility of interactions between these neurotransmitters in other brain areas. Brain nuclei were punched from frozen 300-micron slices of rat brain and extracted with 0.1 M HCIO4 or 2 M acetic acid prior to assay, respectively, of 5-HT content by HPLC with electrochemical detection or SP content by specific radioimmunoassay. Ten days after injection of rats with the 5-HT neurotoxin P-chloroamphetamine (PCA, 10 mg/kg, B.W., i.p.) or 3 days after 5-HT synthesis blockade with p-chlorophenylalanine (PCPA, 300 mg/kg, B.W., i.p.), the 5-HT content of all brain nuclei studied was reduced by means of, respectively, 50% and 81%. In PCA-treated animals, the SP content of the periaqueductal grey matter was significantly increased; PCPA treatment caused, in addition, large increases in the SP content of five other brain nuclei. Blockade of 5-HT receptors by methysergide (15 mg/kg for 5 days) did not significantly change 5-HT levels or turnover, but resulted in 50-200% increases in the SP content of 10 of the 28 brain nuclei studied. Significant decreases in the SP content of numerous areas were seen following treatments (pargyline 30 mg/kg, alone or in combination with 5-hydroxytryptophan, 60 mg/kg) that simultaneously increased 5-HT levels. These results illustrate the modulation of distinct SP-containing systems of the rat brain by perturbation of central serotoninergic pathways and indicate a reciprocal relationship between the SP and 5-HT concentrations of numerous brain nuclei, in particular n. striae terminalis, n. raphe dorsalis, n. accumbens, n. septi, substantia grisea centralis, and n. raphes medianus.     

Implication of 5-HT2A receptors in the genetic mechanisms of the brain 5-HT system autoregulation

Brain serotonin (5-HT) system has been implicated in pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT2A receptor is involved in the mechanisms of stress-induced psychopathology and impulsive behavior. Here, we investigated the role of 5-HT2A receptor in the autoregulation of the brain 5-HT system. The chronic treatment with agonist of 5-HT2A receptor DOI (1.0 mg/kg, i.p./14 days) produced considerable decrease of 5-HT2A receptor-mediated "head-twitches" in AKR/J mice indicating desensitization of 5-HT2A receptors. Chronic DOI treatment failed to alter 5-HT2A receptor gene expression in the midbrain, hippocampus and frontal cortex. At the same time, the increase in the expression of the gene encoding key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), the increase in TPH2 activity and 5-HT levels and decreased expression of serotonin transporter (5-HTT) gene was found in the midbrain of DOI-treated mice. The results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and the implication of 5-HT2A receptor in the autoregulation of the brain 5-HT system.     

Flesinoxan challenge suggests that chronic treatment with paroxetine in rats does not desensitize receptors controlling 5-HT synthesis

It has been proposed that the desensitization of 5-HT_1A (5-hydroxytryptamine; serotonin) receptors following chronic therapy with selective serotonin reuptake inhibitors (SSRIs) is necessary for their therapeutic efficacy. Stimulation of the 5-HT_1A receptors decreases serotonin (5-HT) synthesis and release, but it is not clear if the receptors are fully desensitized following chronic SSRI treatment. The main objective of this study was evaluation of ability of 5-HT_1A receptors to modulate 5-HT synthesis after 14-day paroxetine treatment. 5-HT_1A receptor sensitivity following chronic administration of the SSRI paroxetine was assessed by the ability of an acute challenge with the 5-HT_1A agonist, flesinoxan, to modulate 5-HT synthesis in the rat brain. The rates of 5-HT synthesis were measured using the α-[¹⁴C]methyl-l-tryptophan autoradiographic method. The rats were treated for 2 weeks with paroxetine (10 mg/(kg day), s.c., delivered by osmotic minipump). After this treatment, the rats received an acute challenge with flesinoxan (5 mg/kg, i.p.), while the control rats were injected with the vehicle. Forty minutes following the flesinoxan injection, the tracer, α-[¹⁴C]methyl-l-tryptophan, was injected over 2 min. 5-HT synthesis rates were calculated from autoradiographically measured tissue tracer concentrations and plasma time–activity curves. The results demonstrated that the acute flesinoxan challenge produced a significant decrease in 5-HT synthesis rates throughout the rat brain. The greatest decrease was observed in the ventral hippocampus, somatosensory cortex and the ascending serotonergic cell bodies. In comparison with data reported on an acute challenge with flesinoxan in naïve rats (rats without any other treatment), the results presented here suggest a greater effect of flesinoxan on synthesis reduction in rats chronically treated with paroxetine. The results also suggest that the 5-HT receptors were not fully desensitized by paroxetine treatment, and that the stimulation of 5-HT_1A receptors with an agonist is still capable of reducing 5-HT synthesis.