Both acute and subchronic treatments with pindolol,a 5-HT1A and β1 and β2 adrenoceptor antagonist,elevate regional serotonin synthesis in the rat brain: An autoradiographic study

Antidepressant treatments, including those that increase serotonin (5-HT) neurotransmission, require several weeks or months until the onset of the therapeutic effect in depressed patients. The negative feedback on 5-HT transmission exhibited by the 5-HT_1A and 5-HT_1B autoreceptors has been postulated as a possible delaying factor. The aim of the present study was to assess the effect of the acute and subchronic treatment with pindolol, a 5-HT_1A/1B, β₁ and β₂ adrenoceptor antagonist, on 5-HT synthesis, one of the key parameters of 5-HT neurotransmission. Male Sprague–Dawley (SPD) rats (180–220 g) were treated with pindolol or an adequate volume of saline, administered either acutely (15 mg/kg i.p.; SPD-AC-SAL, SPD-AC-TR) or subchronically (15 mg/kg day i.p. for 7 days; SPD-SUBCHR-SAL, SPD-SUBCHR-TR). Thirty minutes following the single i.p. injection (acute experiment) or at the 8th day following the commencement of the subchronic treatment (subchronic experiment), 5-HT synthesis was measured using α-[¹⁴C]methyl-l-tryptophan autoradiography. The analysis of variance (ANOVA), followed by the Benjamini–Hochberg correction for multiple comparisons, revealed: (1) a significant increase of 5-HT synthesis in the SPD-AC-TR rats, relative to the SPD-AC-SAL rats in all brain regions examined except the substantia nigra – pars reticularis, dorsal subiculum, inferior olive, raphe magnus and raphe obscurus and (2) a significant increase of 5-HT synthesis in the SPD-SUBCHR-TR rats, relative to the SPD-SUBCHR-SAL rats in all brain regions except the median raphe, hypothalamus and raphe pontine. On the basis of these results, we hypothesized that the antagonism of the 5-HT_1A/1B receptors prevents the negative feedback mediated by these receptors on 5-HT synthesis, resulting in a persistent increase of 5-HT synthesis. The results accord with clinical reports on the utility of pindolol in the augmentation of antidepressant treatment.     

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.     

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.     

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.     

The inhibition of tryptophan hydroxylase, not protein synthesis, reduces the brain trapping of alpha-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.     

Both acute and subchronic treatments with pindolol,a 5-HT1A and β1 and β2 adrenoceptor antagonist,elevate regional serotonin synthesis in the rat brain: An autoradiographic study

Antidepressant treatments, including those that increase serotonin (5-HT) neurotransmission, require several weeks or months until the onset of the therapeutic effect in depressed patients. The negative feedback on 5-HT transmission exhibited by the 5-HT_1A and 5-HT_1B autoreceptors has been postulated as a possible delaying factor. The aim of the present study was to assess the effect of the acute and subchronic treatment with pindolol, a 5-HT_1A/1B, β₁ and β₂ adrenoceptor antagonist, on 5-HT synthesis, one of the key parameters of 5-HT neurotransmission. Male Sprague–Dawley (SPD) rats (180–220 g) were treated with pindolol or an adequate volume of saline, administered either acutely (15 mg/kg i.p.; SPD-AC-SAL, SPD-AC-TR) or subchronically (15 mg/kg day i.p. for 7 days; SPD-SUBCHR-SAL, SPD-SUBCHR-TR). Thirty minutes following the single i.p. injection (acute experiment) or at the 8th day following the commencement of the subchronic treatment (subchronic experiment), 5-HT synthesis was measured using α-[¹⁴C]methyl-l-tryptophan autoradiography. The analysis of variance (ANOVA), followed by the Benjamini–Hochberg correction for multiple comparisons, revealed: (1) a significant increase of 5-HT synthesis in the SPD-AC-TR rats, relative to the SPD-AC-SAL rats in all brain regions examined except the substantia nigra – pars reticularis, dorsal subiculum, inferior olive, raphe magnus and raphe obscurus and (2) a significant increase of 5-HT synthesis in the SPD-SUBCHR-TR rats, relative to the SPD-SUBCHR-SAL rats in all brain regions except the median raphe, hypothalamus and raphe pontine. On the basis of these results, we hypothesized that the antagonism of the 5-HT_1A/1B receptors prevents the negative feedback mediated by these receptors on 5-HT synthesis, resulting in a persistent increase of 5-HT synthesis. The results accord with clinical reports on the utility of pindolol in the augmentation of antidepressant treatment.     

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.     

Morphofunctional characteristics of serotonin-like neurons in the hypothalamus of rats in ontogeny

An attempt has been made to reveal 5-HT immunopositive (IP) neurones in the hypothalamus of intact foetuses (18th day of gestation) and neonatal (9-day) rats under normal conditions and after their treatment with drugs involved into 5-HT metabolism or into regulation of its uptake by serotoninergic neurones. 5-HTIP cells were not observed in intact animals as well as after L-tryptophan treatment, whereas two large colonies of these neurones were found in the anterio-lateral hypothalamus and dorsomedial nucleus after subsequent injections of monoamine oxidase inhibitor, pargyline, and amino acid precursor of 5-HT synthesis, L-tryptophan. Significantly less intensive reaction was observed after injections of another precursor of 5-HT synthesis, 5-hydroxytryptophan, or pargyline only. Immunostaining evoked by pargyline or L-tryptophan can be prevented by preliminary injections of fluoxetine, a specific inhibitor of 5-HT uptake by serotoninergic neurones. These data suggest that the immunostaining of hypothalamic neurones is due to their capacity to take up specifically 5-HT from the environment rather than to its intraneuronal synthesis from L-tryptophan. However, 5-HT synthesis from 5-hydroxytryptophan in the same cells may also take place. The uptake of extracellular 5-HT by catecholaminergic neurones is absent, since nomifensine, a specific inhibitor of this uptake, does not affect immunostaining.     

TRYPTOPHAN AND 5-HYDROXYTRYPTAMINE METABOLISM IN THE IMMATURE RAT BRAIN DURING RECOVERY FROM ASPHYXIA

Abstract— The turnover of cerebral 5-HT in the 5-day-old rat during recovery from asphyxia was assessed in the brain by determining 5-hydroxyindoleacetic acid concentration, and by following the accumulation of 5-HT after inhibition of MAO. Marked increases in turnover were found to persist for up to 3 h after resuscitation and this increase was accompanied by increases in brain tryptophan, plasma total tryptophan and the fraction of the plasma total tryptophan that was not bound to albumin. None of these parameters were different from those in control animals 24 h after resuscitation. Brain and plasma tyrosine levels remained unaltered by the treatment at all times investigated.     

Monoamine Synthesis and Concentration in Neonatal Rat Brain During Hypercapnia and Recovery

One-day-old rats were exposed to a gas mixture of 15% CO2-21% O2-64% N2 for a 30-min period. Monoamine synthesis in whole brain was measured during, and at various intervals after, hypercapnia by estimating the accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) after inhibition of aromatic L-amino-acid decarboxylase with NSD 1015. Endogenous concentrations of tyrosine, dopamine (DA), noradrenaline (NA), tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured at the same intervals. Exposure to CO2 induced an increased synthesis of catecholamines and 5-HT. Further, an increase in DA concentration was seen during hypercapnia, while NA and 5-HT were unchanged. After the CO2 exposure the increased in vivo synthesis rates of catecholamines and 5-HT were rapidly normalized, as was the endogenous DA concentration. A slight increase in 5-HT and 5-HIAA concentrations was seen immediately after CO2 exposure. These results indicate that in neonatal animals, hypercapnia induces changes in central monoamine neurons, primarily an increased synthesis. These alterations may be relevant to some physiological changes seen during CO2 exposure, such as the alteration in central respiratory performance.     

Both acute and chronic buspirone treatments have different effects on regional 5-HT synthesis in Flinders Sensitive Line rats (a rat model of depression) than in control rats

The main objective of this investigation was to evaluate the effects of buspirone, a 5-HT_1A agonist with some partial agonist properties and also an antidepressant, on regional 5-HT synthesis in Flinders Sensitive Line (FSL) rats (“depressed”), and to compare the effects to the Flinders Resistant Line (FRL) control rats (not “depressed”). In addition results were compared to those previously reported in normal Sprague–Dawley (SPD) rats (normal control). Serotonin synthesis in both FSL and FRL rats was measured following acute and chronic treatments with buspirone. Both of these strains were derived from the SPD rats. No direct comparison was done between the FSL saline and FRL saline groups, or the FSL buspirone and FRL buspirone groups, because the objective of the studies was to evaluate effects of buspirone in these two strains. The results show that acute treatment with buspirone elevates 5-HT synthesis throughout the brain in the FRL rats. In the FSL rats, there were reductions in some brain regions (e.g., dorsal and median raphe, amygdala, anterior olfactory nucleus, substantia nigra reticulate), while in other regions, there were increases in the synthesis observed (e.g., frontal, parietal, visual and somatosensory cortices, ventral hippocampus). In 20 out of the 30 brain regions investigated in the FSL rats, there was no significant change in the synthesis following acute buspirone treatment. During the chronic treatment, buspirone produced a significant reduction of 5-HT synthesis in 15 out of 30 brain regions in the FRL rats. In the FSL rats, buspirone produced a significant elevation of the synthesis in 10 out of 30 brain regions. In both the FSL and FRL rats, buspirone produced rather different effects than those reported previously for SPD (normal) rats. The acute effect in the FSL rats was somewhat similar to the effect reported previously for the SPD rats, while in the FRL rats, the acute buspirone treatment produced an effect observed previously in treatments with 5-HT_1A antagonists suggesting an action of buspirone as partial agonist in FRL rats. The data suggest that with respect to 5-HT synthesis, FRL rats differ from SPD rats (a natural control; normal rats) and, as such, indicate that when the effects related to the serotonergic system (e.g., influence of serotonergic drugs) are studied in the FSL rats and compared to those in the FRL rats, any conclusions drawn may not reflect differences relative to a normal rat.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

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.     

Amines of the mucosal mast cell of the gut in normal and nematode infected rats

Infection with the nematode N. brasiliensis is accompanied by a marked increase of the number of mucosal mast cells (MMC) and the mucosal content of histamine and 5-hydroxytryptamine (5-HT). We compared amine levels, determined by ion exchange and high performance liquid chromatography (HPLC) with numbers of MMC and enterochromaffin cells (ECC). Furthermore, we measured 5-HT cytofluorometrically in individual MMC and ECC. The cellular distribution of 5-HT was studied immunohistochemically. Our results corroborate previous findings that histamine is stored in MMC. Quotients between histamine content and numbers of MMC decreased throughout the period of worm expulsion, followed by a recovery, suggesting a histamine release during this defense reaction. The HPLC analysis gave no evidence for a storage of dopamine in MMC. ECC and MMC of normal and infected rats showed a formaldehyde induced fluorescence and 5-HT immunoreactivity. The formaldehyde induced fluorescence of MMC from normal rats was about 10% that of ECC, but MMC exceeded ECC three times by numbers. These findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC. ECC numbers did not change during the infection and their content of 5-HT was unchanged, as judged by cytofluorometry. The cytofluorometric measurements showed that the intensity of the monoamine fluorescence from the MMC of infected animals was about three times as high as that of controls. It was concluded that the increased tissue levels of 5-HT was due to both an increase in MMC numbers and an increase in the 5-HT content of individual MMC. The results suggest a different role for histamine and 5-HT in the defense reaction towards the nematode infection.     

Cerebral Markers of the Serotonergic System in Rat Models of Obesity and After Roux-en-Y Gastric Bypass

Food intake and body weight are regulated by a complex system of neural and hormonal signals, of which the anorexigenic neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) is central. In this study, rat models of obesity and weight loss intervention were compared with regard to several 5-HT markers. Using receptor autoradiography, brain regional-densities of the serotonin transporter (SERT) and the 5-HT(2A) and 5-HT(4) receptors were measured in (i) selectively bred polygenic diet-induced obese (pgDIO) rats, (ii) outbred DIO rats, and (iii) Roux-en-Y gastric bypass (RYGB)-operated rats. pgDIO rats had higher 5-HT(4) and 5-HT(2A) receptor binding and lower SERT binding when compared to polygenic diet-resistant (pgDR) rats. The most pronounced difference between pgDIO and pgDR rats was observed in the nucleus accumbens shell (NAcS), a brain region regulating reward aspects of feeding. No differences were found in the 5-HT markers between DIO rats, chow-fed control rats, and DIO rats experiencing a weight loss. The 5-HT markers were also similar in RYGB and sham-operated rats except for a downregulation of 5-HT(2A) receptors in the NAcS. The higher receptor and lower SERT binding in pgDIO as compared to pgDR rats corresponds to what is reported in overweight humans and suggests that the dysfunctions of the 5-HT system associated with overeating or propensity to become overweight are polygenically determined. Our results support that the obesity-prone rat model has high translational value and suggests that susceptibility to develop obesity is associated with changed 5-HT tone in the brain that may also regulate hedonic aspects of feeding.     

Pentylenetetrazol: Effect on carbaryl-induced changes of serotonin metabolism in pons-medulla of rat brain

Oral administration of carbaryl to adult male albino rats produced a dose dependent increase in the steady state level of 5-hydroxytryptamine (5-HT) at 1.00 h in pons-medulla (PM). 5-Hydroxyindole acetic acid (5-HIAA) concentration was significantly elevated only in response to a higher dose of this pesticide under similar conditions. A time course study with carbaryl and pentylenetetrazol (PTZ) showed a characteristic elevation of the steady state level of 5-HT in PM, but the 5-HIAA level was significantly elevated at 0.5 h only after carbaryl treatment. No significant change of the 5-HIAA level was evident after administration of PTZ alone or in combination with carbaryl. Tryptophan concentration was significantly elevated in PM at 0.5 h after carbaryl treatment and at 1.0 h after carbaryl + PTZ treatment. No significant change of tryptophan concentration was evident after the administration of PTZ alone under similar conditions. Measurement of (1) pargyline induced (a) accumulation of 5-HT and (b) depletion of 5-HIAA levels, and (2) probenecid-induced accumulation of 5-HIAA level in presence and absence of carbaryl and revealed that carbaryl accelerated the synthesis as well as the breakdown of 5-HT, whereas PTZ alone or in combination with carbaryl accelerated the synthesis of 5-HT without affecting its catabolism. The potency of this pesticide in elevating the pargyline-induced accumulation of 5-HT is in the order of carbaryl + PTZ greater than PTZ congruent to carbaryl. These results suggest that the carbaryl-induced increase in the synthesis of 5-HT is potentiated, and the turnover is reduced, in PM when PTZ is administered to the carbaryl-intoxicated rats.     

Acute effects of combining citalopram and pindolol on regional brain serotonin synthesis in sham operated and olfactory bulbectomized rats

The olfactory bulbectomized (OBX) rat is considered to be a good model of the pathology of human depression and also of the functional actions of antidepressant drug therapy. It has been proposed that antidepressant effects of selective serotonin reuptake inhibitors (SSRIs) can be accelerated by blocking 5-HT_1A/B autoreceptors with pindolol. The underlying mechanism is thought to involve acute unrestricting of 5-HT release and, consequently, relatively enhanced 5-HT turnover throughout the forebrain serotonergic networks. The effect of this combination on 5-HT turnover in sham operated or OBX rats can be assessed at the level of 5-HT synthesis, a very important presynaptic step in serotonergic neurotransmission, using the α-[¹⁴C]methyl-l-tryptophan autoradiography method. In sham rats, acute citalopram (20 mg/kg) treatment increased synthesis at almost all serotonergic terminal regions but slightly decreased synthesis at serotonergic cell body regions (i.e. dorsal and median (not significant) raphe; ～16%). Combining pindolol (10 mg/kg) with citalopram further increased synthesis at many regions in sham rats (relative to treatment with only citalopram). In OBX rats, citalopram decreased synthesis at a few terminal regions and greatly decreased synthesis at the dorsal and median raphe (～45%; relative to OBX rats treated with saline). Combining pindolol with citalopram greatly increased synthesis at almost all regions in OBX rats (relative to treatment with only citalopram). These results suggest that acute citalopram effects result in elevated terminal 5-HT synthesis, but these effects are restrained by 5-HT_1A/B autoreceptor feedback to different degrees in sham and OBX rats. Moreover, 5-HT_1A/B autoreceptor feedback is stronger in OBX rats and may underlie the delay of SSRI effects in OBX rats and, correspondingly, in human depression. Pindolol acceleration and augmentation of SSRI antidepressant therapy for human depression may be mediated by attenuation of 5-HT_1A/B autoreceptor feedback, permitting unhindered SSRI effects on serotonergic terminals.     

Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.     

The effects of 5-hydroxytryptophan and 5-hydroxytryptamine on dopamine synthesis and release in rat brain striatal synaptosomes.

The effects of 5-hydroxytryptophan (5-HTP) and serotonin (5-HT) on dopamine synthesis and release in rat brain striatal synaptosomes have been examined and compared to the effects of tyramine and dopamine. Serotonin inhibited dopamine synthesis from tyrosine, with 25% inhibition occurring at 3 μM-5-HT and 60% inhibition at 200 μM. Dopamine synthesis from DOPA was also inhibited by 5-HT, with 30% inhibition occurring at 200 μ. At 200 μM-5-HTP, dopamine synthesis from both tyrosine and DOPA was inhibited about 70%. When just the tyrosine hydroxylation step was measured in the intact synaptosome, 5-HT, 5-HTP, tyramine and dopamine all caused significant inhibition, but only dopamine inhibited soluble tyrosine hydroxylase [L-tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] prepared from lysed synaptosomes. Particulate tyrosine hydroxylase was not inhibited by 10 μM-5-HT, but was about 20% inhibited by 200 μM-5-HT and 5-HTP. At 200 μM both 5-HT and 5-HTP stimulated endogenous dopamine release. These experiments suggest that exposure of dopaminergic neurons to 5-HT or 5-HTP leads to an inhibition of dopamine synthesis, mediated in part by an intraneuronal displacement of dopamine from vesicle storage sites, leading to an increase in dopamine-induced feedback inhibition of tyrosine hydroxylase, and in part by a direct inhibition of DOPA decarboxylation.     

TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5-HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non-linear uptake of l -tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5-hydroxytryptamine (5-HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l [¹⁴C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5-HT synthesis. When the cerebral tryptophan level reached 1 ± 10 -4 , substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan-5-hydroxylase) but not of the indoleamine-2,3-dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5-hydroxyindoles from the brain. One hour after intraperitoneal injection of l -kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l -tryptophan to l -kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.