Species differences in the distribution of central 5-HT1 binding sites: a comparative autoradiographic study between rat and guinea pig

In this study, we compared the localization of central 5-HT₁ binding sites of rat and guinea pig. The 5-HT_1B sites were absent in the guinea pig brain. Good correlations were found between species in the regional distribution of 5-HT₁ sites labelled with [³H]5-HT(r = 0.73), 5-HT_1A sites labelled with [³H]8-OH-DPAT (r = 0.87), and 5-HT_1B versus 5-HT_1D sites labelled with [³H]5-HT in the presence of ipsapirone and DOI (r = 0.76). Despite the overall similarities, species differences were observed in many brain regions. The CA1/CA2 fields of the hippocampus and the dorsal subiculum displayed significantly more 5-HT_1A receptor binding in guinea pig than in rat. Conversely, the 5-HT_1A binding in dorsolateral septum, cingulate cortex and laminae IV-V of the neocortex, was more pronounced in rat. Areas almost exclusively containing 5-HT_1B or 5-HT_1D sites, such as the ventral pallidum, globus pallidus and substantia nigra, expressed markedly more [³H]5-HT binding in rat as compared to guinea pig, while the opposite occurred in claustrum, dorsal endopiriform nucleus, lateral geniculate nucleus, and superficial grey layer of the superior colliculus. The implications of the species differences are illustrated by the binding of [³H]eltoprazine. The distribution of [³H]eltoprazine binding sites showed a good correlation with that of the 5-HT_1B sites in rat (r = 0.89), and with that of the 5-HT_1A sites in guinea pig (r = 0.97). The data give rise to the possibility that differences in the presence and distribution of 5-HT₁ receptor sites are related to species differences in behavioral, neurochemical and physiological responses to drugs with 5-HT₁ receptor affinity.     

Autoradiographic evidence for the heterogeneity of 5-HT1 sites in the rat brain

The distribution of the binding sites of a new, potent agonist of serotonin (5-HT), 8-OH-N,N-dipropoyl-2-aminotetralin (PAT), was studied in the rat brain with the quantitative autoradiographic technique utilizing tritium-sensitive LKB film. The localization of [³H]PAT binding sites was very similar to that of [³H]5-HT binding sites, except in some discrete regions (choroid plexus, striatum, area preoptica lateralis, subiculum, and substantia nigra), which exhibited very low levels of labeling with [³H]PAT and high levels with [³H]5-HT. These results indicate that 5-HT₁ receptors are heterogeneous, and that [³H]PAT recognizes only a 5-HT₅ subclass (called 5-HT_1A).     

Cortical and striatal variations in drug competition studies with putative 5-hydroxytryptamine1D binding sites

The ability of 5-hydroxytryptamine (5-HT), 5-car☐ytryptamine (5-CT) and sumatriptan to compete for [³H]5-HT binding sites in various brain tissues was analyzed in bovine, guinea pig, pig and human cortex and caudate. Radioligand binding conditions were designed to allow for the selective labeling of putative 5-HT_1D binding sites. 5-HT competed monophasically with putative 5-HT_1D binding sites in each of the 8 tissues studied. By contrast, both 5-CT and sumatriptan,competed with markedly shallow displacement curves in each of the 8 tissues. In the case of sumatriptan, complete displacement of [³H]5-HT could not be achieved even at concentrations as high as 2000 times its IC₅₀ value. These data indicate that 10⁻⁵ M 5-HT should not be used to define specific binding to 5-HT_1D receptors in radioligand binding assays. Instead, 5-HT_1D receptor binding sites should be redefined as [³H]5-HT-labeled binding sites displaced by 10⁻⁵ M sumatriptan.     

Autoradiographic mapping of 5-HT1B and 5-HT1D receptors in the post mortem human brain using [H]GR 125743

The distribution of 5-HT_1B and 5-HT_1D receptors in the human post mortem brain was examined using whole hemisphere autoradiography and the radioligand [³H]GR 125743. [³H]GR 125743 binding was highest in the substantia nigra and the globus pallidus. Lower levels were detected in the striatum, with the highest densities in the ventromedial parts. In the amygdala, the hippocampus, the septal region and the hypothalamus, lower [³H]GR 125743 binding was observed, reflecting low densities of 5-HT_1B/1D receptors. In the cerebral cortex, binding was similar in most regions, although restricted parts of the medial occipital cortex were markedly more densely labeled. Binding densities were very low in the cerebellar cortex and in the thalamus. Two methods were used to distinguish between the two receptor subtypes, the first using ketanserin to block 5-HT_1D receptors and the second using SB 224289 to inhibit 5-HT_1B receptor binding. The autoradiograms indicated that in the human brain, the 5-HT_1B receptor is much more abundant than the 5-HT_1D receptor, which seemed to occur only in low amounts mainly in the ventral pallidum. Although [³H]GR 125743 is a suitable radioligand to examine the distribution of 5-HT_1B receptors in the human brain in vitro, the selectivities of ketanserin and SB 224289 are not sufficiently high to give definite evidence for the occurrence of the 5-HT_1D receptor in the human brain.     

Differential effects of serotonin (5-HT) lesions and synthesis blockade on neuropeptide-Y immunoreactivity and 5-HT1A, 5-HT1B/1D and 5-HT2A/2C receptor binding sites in the rat cerebral cortex

The present study was aimed at comparing the effects of serotonin (5-HT) synthesis blockade using chronic administration of p-chlorophenylalanine (PCPA) and 5,7-dihydroxytryptamine injections of variable volume (3 vs. 6 μl) on the density of NPY immunoreactive (Ir) neurons and binding of [³H]8-OH-DPAT, S-CM-G[¹²⁵I]TNH₂ and [¹²⁵I]DOI to 5-HT_1A, 5-HT_1B/1D, and 5-HT_2A/2C receptors in rat cortical regions. Three weeks after large but partial (89% depletion in 5-HT tissue concentration) lesions of 5-HT neurons no changes in neither NPY immunoreactivity nor 5-HT receptor binding were detected. The complete 5,7-DHT lesions produced increases in the number of NPY-Ir neurons in the upper regions of the cingular (134%), frontal (140%) and parietal cortex (48%) and corresponding decreases in 5-HT_2A/2C binding (16–26%). No changes in 5-HT_1A and 5-HT_1B/1D binding were observed after lesions of this kind. After PCPA treatment, decreases in NPY-Ir neurons density (22–40%) and increases in 5-HT_1A and 5-HT_1B/1D receptor binding sites (20–50%) were distributed in both upper and deeper cortical regions. The lack of effect of the partial lesion suggests that spared 5-HT neurons may exert compensatory mechanisms up to a large extent. The changes in NPY immunoreactivity and 5-HT_2A/2C binding detected in the upper regions of the cortex after complete 5-HT lesions probably result from local cellular rearrangements, whereas blocking 5-HT synthesis has more widespread influence on NPY neurons and on 5-HT_1A and 5-HT_1B/1D receptor subtypes. Moreover, decreases in DOPAC concentrations detected only after complete lesions suggest that the involvement of catecholaminergic transmission may also differentiate 5,7-DHT and PCPA treatments. Altogether, these data suggest that different receptor subtypes might be involved in 5-HT–NPY relationships.     

Increased binding at 5-HT1A, 5-HT1B, and 5-HT2A receptors and 5-HT transporters in diet-induced obese rats

5-Hydroxytryptamine (5-HT, serotonin), synthesized in midbrain raphe nuclei and released in various hypothalamic sites, decreases food intake but the specific 5-HT receptor subtypes involved are controversial. Here, we have studied changes in the regional density of binding to 5-HT receptors and transporters and the levels of tryptophan hydroxylase, in rats with obesity induced by feeding a palatable high-energy diet for 7 weeks. We mapped binding at 5-HT receptor subtypes and transporters using quantitative autoradiography and determined tryptophan hydroxylase protein levels by Western blotting. In diet-induced obese (DiO) rats, specific binding to 5-HT_1A receptors ([³H]8-OH-DPAT) was significantly increased in the dorsal and median raphe by 90% (P<0.01) and 132% (P<0.05), respectively, compared with chow-fed controls. 5-HT_1B receptor binding sites ([¹²⁵I]cyanopindolol) were significantly increased in the hypothalamic arcuate nucleus (ARC) of DiO rats (58%; P<0.05), as were 5-HT_2A receptor binding sites ([³H]ketanserin) in both the ARC (44%; P<0.05) and lateral hypothalamic area (LHA) (121%; P<0.05). However, binding to 5-HT_2C receptors ([³H]mesulgergine) in DiO rats was not significantly different from that in controls in any hypothalamic region. Binding to 5-HT transporters ([³H]paroxetine) was significantly increased (P<0.05) in both dorsal and median raphe, paraventricular nuclei (PVN), ventromedial nuclei (VMH), anterior hypothalamic area (AHA) and LHA of DiO rats, by 47%–165%. Tryptophan hydroxylase protein levels in the raphe nuclei were not significantly different between controls and DiO rats. In conclusion, we have demonstrated regionally specific changes in binding to certain 5-HT receptor subtypes in obesity induced by voluntary overeating of a palatable diet. Overall, these changes are consistent with reduced 5-HT release and decreased activity of the 5-HT neurons. Reduction in the hypophagic action of 5-HT, possibly acting at 5-HT_1A, 5-HT_1B and 5-HT_2A receptors, may contribute to increased appetite in rats presented with highly palatable diet.     

Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord

The distribution of 5-HT₁, 5-HT_1A, 5-HT_1B and 5-HT₂ receptors in the rat spinal cord was investigated with quantitative autoradiography. Receptors were labeled respectively with [³H]serotonin (5-[³H]HT],8-hydroxy-2-[N-dipropylamino-³H]tetralin (8-OH-[³H]DPAT), [¹²⁵I]iodocyanopindolol and [³H]ketanserin. It is shown that 5-HT₁, 5-HT_1A and 5-HT_1B receptors are distributed within the spinal cord according to a rostro-caudal gradient. Both 5-HT₁ and 5-HT_1A receptors are mainly present in the dorsal horn and 5-HT_1B is present throughout the spinal cord, exhibiting high densities in the caudal-most part of the dorsal in lamina X and in the sacral parasympathetic area. On the other hand, 5-HT₂ receptors are shown mostly in the thoracic sympathetic area and in the thoracic ventral horn; the dorsal horn exhibits few 5-HT₂ receptors. The differential involvement of 5-Ht through different receptors in nociception, autonomous nervous system control and motility are discussed.     

In Vitro Autoradiographic Localization of 5-HT1A Receptor-Activated G-Proteins in the Rat Brain

Serotonin 5-HT_1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of G-proteins can be determined by agonist-stimulated [³⁵S]GTPγS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT_1A receptor activation of G-proteins using 8-OH-DPAT-stimulated [³⁵S]GTPγS binding in membranes and brain sections. In hippocampal membranes, 8-OH-DPAT stimulated [³⁵S]GTPγS binding by twofold, with an ED₅₀ value of 25 nM. 5-HT₁ antagonists, but not 5-HT₂ antagonists, increased the ED₅₀ of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [³⁵S]GTPγS binding showed that 8-OH-DPAT induced the formation of high affinity [³⁵S]GTPγS binding sites with a K_D for GTPγS of 3.2 nM. [³⁵S]GTPγS autoradiography, performed in brain sections with the 5-HT_1A agonist 8-OH-DPAT, revealed high levels of 5-HT_1A-stimulated [³⁵S]GTPγS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT_1A-stimulated [³⁵S]GTPγS binding in sections was blocked by the addition of the 5-HT₁ antagonist methiothepin. These results show that the use of agonist-stimulated [³⁵S]GTPγS autoradiography for the 5-HT_1A receptor system should provide new information regarding signal transduction in specific brain regions.     

Electrophysiological evidence for excitatory 5-HT2 and depressant 5-HT1A receptors on neurones of the rat midbrain tectum

It has been claimed that the aversive behaviour induced by electrical stimulation of the midbrain tectum (MT) has validity as an animal model of panic attack. A great deal of evidence obtained from behavioural studies suggests that 5-HT₂ mechanisms phasically inhibit the substrates of aversion in the MT. In order to test this hypothesis we employed the technique of microiontophoresis of drugs onto neurones of the MT to assess the identity of the receptors mediating the effects of 5-hydroxytryptamine (5-HT). The results obtained show that the majority of 5-HT responsive cells in MT are cells excited by 5-HT (72%). These cells were silent or showed very low spontaneous firing activity, whereas cells depressed by 5-HT showed high spontaneous firing activity at baseline. The 5-HT_1A receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone and gepirone caused consistent reduction in the firing rate of cells depressed by 5-HT while they did not change the firing activity of cells excited by 5-HT. The excitatory effects induced by 5-HT on MT neurones were clearly attenuated by concomitant application of ketanserin, a highly specific 5-HT₂ antagonist. Excitatory responses to dl-homocysteic acid were not affected by ketanserin. Previous administration of zimelidine, a selective 5-HT uptake inhibitor, caused a significant enhancement of the excitatory effects of 5-HT while similar application of gepirone did not affect the size of the excitatory responses to 5-HT. These results give electrophysiological support to the idea that 5-HT neurotransmission operating through 5-HT₂ receptors may exert a phasic control on functional processes in the MT. It is possible that 5-HT₂ mechanisms in this region may mediate at least part of the therapeutic effects of 5-HT uptake inhibitors in panic disorders.     

Effects of adrenalectomy and type I or type II glucocorticoid receptor activation on 5-HT1A and 5-HT2 receptor binding and 5-HT transporter mRNA expression in rat brain

We evaluated the effects of adrenalectomy (ADX) and replacement with glucocorticoid receptor agonists on serotonin (5-HT) 5-HT_1A and 5-HT₂ receptor binding in rat brain. 5-HT_1A receptor binding was increased in the CA2–CA4 and the dentate gyrus of the hippocampus 1 week after ADX. This effect was prevented by the systemic administration of aldosterone (10 μg/μl/h) but not by RU28362 (10 μg/μl/h). No significant effect was observed on 5-HT₂ receptor binding in rat cortex. The expression of 5-HT transporter mRNA was unchanged in the raphe nucleus as measured by in situ hybridization.     

5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons

Serotonin (5-HT) has been shown to phase shift circadian rhythms in mammals and to affect responses of the circadian system to light, but it is not clear which receptors are involved in these actions. We found that drugs which act as 5-HT_1A receptor agonists suppressed photic responses of hamster SCN cells, but these drugs also exhibit high affinity for the recently cloned 5-HT₇ receptor. We therefore studied the effects of 5-HT agonists and antagonists with differential affinities for 5-HT₇ and 5-HT_1A receptors on responses of hamster SCN cells to retinal illumination. We confirmed that the 5-HT receptor agonists 5-HT, 8-OH-DPAT and 5-CT, dose-dependently reduced photic activation of SCN cells. These effects could be blocked by co-application of antagonists with high affinities for 5-HT₇ receptors: ritanserin or clozapine. The 5-HT_1A/B/D antagonist, cyanopindolol, which is inactive at 5-HT₇ receptors, did not antagonize the actions of 8-OH-DPAT. Selective 5-HT_1A antagonists, WAY100635 and p-MPPI, had weak or no antagonist effects on the responses to 8-OH-DPAT in the SCN, but they effectively antagonized the actions of 8-OH-DPAT in the hippocampus. In the cerebellar cortex where few 5-HT₇ receptors are present, ritanserin failed to antagonize the effects of 8-OH-DPAT. Our results indicate that the 5-HT₇ receptor subtype plays a major role in mediating the effects of 5-HT on photic responses of SCN cells in the hamster.     

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [H]WAY-100635 and [C]WAY-100635

The distribution of 5-HT_1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT_1A receptor antagonist [³H]WAY-100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities of [³H]WAY-100635 binding, reflecting low densities of 5-HT_1A receptors. The labeling was very low in basal ganglia, such as nucleus caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT_1A receptors with [³H]WAY-100635 was antagonized by the addition of the 5-HT_1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 μM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [³H]WAY-100635 has a K_d of approximately 2.5 nM. The selective labeling of 5-HT_1A receptors with [³H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT_1A receptor subtype in vitro. [¹¹C]WAY-100635 is used for the characterization of 5-HT_1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t_1/2=20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [¹¹C]WAY-100635 gave images qualitatively similar to those of [³H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 μM), blocked all binding of [¹¹C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT_1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT_1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [¹¹C]WAY-100635. These data provide a strong basis for expecting [¹¹C]WAY-100635 to behave as a highly selective radioligand in vivo.     

5-HT1A and 5-HT2 receptors differentially regulate the excitability of 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro

We have used intracellular recording techniques to examine the effects of 5-hydroxytryptamine (5-HT, serotonin) on 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro. Bath-applied 5-HT (30–300 μM) had two opposing effects on the membrane excitability of these cells, reflecting the activation of distinct 5-HT receptor subtypes. As demonstrated previously in the rat, 5-HT evoked a hyperpolarization and inhibition of 5-HT neurones, which appeared to involve the activation of an inwardly rectifying K⁺ conductance. This hyperpolarizing response was blocked by the 5-HT_1A receptor-selective antagonist WAY-100635 (30–100 nM). In the presence of WAY-100635, 5-HT induced a previously unreported depolarizing, excitatory response of these cells, which was often associated with an increase in the apparent input resistance of the neurone, likely due to the suppression of a K⁺ conductance. Like the hyperpolarizing response to 5-HT, this depolarization could be recorded in the presence of the Na⁺ channel blocker tetrodotoxin. In addition, the response was not significantly attenuated by the α₁-adrenoceptor antagonist prazosin (500 nM), indicating that it is not due to the release of noradrenaline, or to the direct activation of α₁-adrenoceptors by 5-HT. The 5-HT₃ receptor antagonist granisetron (1 μM) and the 5-HT₄ receptor antagonist SB 204070 (100 nM) failed to reduce the depolarizing response to 5-HT; however, ketanserin (100 nM), mesulergine (100 nM) and lysergic acid diethylamide (1 μM) significantly reduced or abolished the depolarization, indicating that this effect of 5-HT is mediated by 5-HT₂ receptors.     

5-HT2 receptor antagonists and migraine therapy

5-Hydroxytryptamine (5-HT; serotonin) has been implicated in the pathophysiology of migraine, and several drugs with potent 5-HT₂ receptor blocking activity (methysergide, pizotifen, cyproheptadine and mianserin) have been recognized as being clinically effective in migraine prophylaxis, although the selective 5-HT₂ receptor antagonist ketanserin (the principal agent used to identify 5-HT₂ receptor-mediated actions) seems to be ineffective in migraine. Pizotifen is the most widely used 5-HT₂ receptor antagonist in migraine prophylaxis, because of its superior efficacy compared with cyproheptadine, and because the incidence and severity of adverse effects with pizotifen is lower compared with methysergide and mianserin. These agents have additional antagonistic effects at histamine H₁, muscarinic cholinergic, ₁-adrenergic, ₂-adrenergic and dopamine receptors, but drugs which are selective for these non-5-HT receptors appear to be of no benefit in migraine. Actions mediated by 5-HT₂ receptors which could be of relevance to migraine comprise cranial vasoconstriction, increased cranial capillary permeability and platelet aggregation, and some central nervous system effects and neuroendocrine functions. Although pizotifen, cyproheptadine and mianserin are considered to be relatively specific for 5-HT₂ receptors, these agents and methysergide all share a high affinity for 5-HT_1C binding sites; ketanserin, however, has little affinity for these sites, thus activation of 5-HT_1C receptors may be an important step in the pathogenesis of migraine. It is not yet known which 5-HT_1C receptor-mediated actions of 5-HT are relevant to migraine, but some behavioural actions and cranial vasodilatation via release of endothelium-derived relaxing factor may be involved. If 5-HT_1C rather than 5-HT₂ receptor-mediated actions are important, then other 5-HT₂ receptor antagonists with a high affinity at 5-HT_1C binding sites, such as LY 53857, metergoline, mesulergine, ritanserin and SCH 23390, may also prove to be effective in migraine. The efficacy of methysergide may also depend on other 5-HT₁-like receptor-mediated actions such as cranial vasoconstriction, and inhibition of cranial vascular neurogenic inflammation. The efficacy of these agents implies that 5-HT is causally involved in at least some of the underlying pathophysiology of migraine.     

Differential effects of 5-HT1A receptor deletion upon basal and fluoxetine-evoked 5-HT concentrations as revealed by in vivo microdialysis

An involvement of serotonin (5-HT) 1A receptors in the etiology of psychiatric disorders has been suggested. Hypo-responsiveness of the 5-HT_1A receptor is linked to anxiety and constitutive deletion of the 5-HT_1A receptor produces anxiety-like behaviors in the mouse. Evidence that 5-HT_1A receptor inactivation increases the therapeutic effects of antidepressants has also been presented. The present studies used in vivo microdialysis and homologous recombination techniques to examine the contribution of 5-HT_1A autoreceptors to these effects. Basal and fluoxetine-evoked extracellular concentrations of 5-HT were quantified in the striatum, a projection area of dorsal raphe neurons (DRN), of wild-type (WT) and 5-HT_1A receptor knock out (KO) mice. The density of 5-HT transporters was also determined. Basal 5-HT concentrations did not differ in WT and KO mice. Fluoxetine (10 mg/kg) increased 5-HT concentrations in both genotypes. This increase was, however, 2-fold greater in KO mice. In contrast, no differences in K⁺-evoked 5-HT concentrations were seen. Similarly, neither basal nor stimulation-evoked DA differed across genotype. Autoradiography revealed no differences between genotype in the density of 5-HT transporters or post-synaptic 5-HT_2A receptors, an index of 5-HT neuronal activity. These experiments demonstrate that, under basal and KCl stimulated conditions, adaptive mechanisms in the 5-HT system compensate for the lack of 5-HT_1A autoreceptor regulation of DRN. Furthermore, they suggest that the absence of release-regulating 5-HT_1A autoreceptors in the DRN can not account for the anxiety phenotype of KO mice. The enhanced response to fluoxetine in KO mice is consistent with pharmacological studies and suggests that adaptive mechanisms that occur in response to 5-HT_1A receptor deletion are insufficient to oppose increases in 5-HT concentrations produced by acute inhibition of the 5-HT transporter.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

5-HT1A/1B, 5-HT6, and 5-HT7 serotonergic receptors recruitment in tonic-clonic seizure-induced antinociception: Role of dorsal raphe nucleus

Pharmacological studies have been focused on the involvement of different neural pathways in the organization of antinociception that follows tonic-clonic seizures, including 5-hydroxytryptamine (5-HT)-, norepinephrine-, acetylcholine- and endogenous opioid peptide-mediated mechanisms, giving rise to more in-depth comprehension of this interesting post-ictal antinociceptive phenomenon. The present work investigated the involvement of 5-HT_1A/1B, 5-HT₆, and 5-HT₇ serotonergic receptors through peripheral pretreatment with methiothepin at doses of 0.5, 1.0, 2.0 and 3.0 mg/kg in the organization of the post-ictal antinociception elicited by pharmacologically (with pentylenetetrazole at 64 mg/kg)-induced tonic-clonic seizures. Methiothepin at 1.0 mg/kg blocked the post-ictal antinociception recorded after the end of seizures, whereas doses of 2.0 and 3.0 mg/kg potentiated the post-ictal antinociception. The nociceptive thresholds were kept higher than those of the control group. However, when the same 5-hydroxytryptamine receptors antagonist was microinjected (at 1.0, 3.0 and 5.0 μg/0.2 μL) in the dorsal raphe nucleus, a mesencephalic structure rich in serotonergic neurons and 5-HT receptors, the post-ictal hypo-analgesia was consistently antagonized. The present findings suggest a dual effect of methiothepin, characterized by a disinhibitory effect on the post-ictal antinociception when peripherally administered (possibly due to an antagonism of pre-synaptic 5-HT_1A serotonergic autoreceptors in the pain endogenous inhibitory system) and an inhibitory effect (possibly due to a DRN post-synaptic 5-HT_1B, 5-HT₆, and 5-HT₇ serotonergic receptors blockade) when centrally administered. The present data also suggest that serotonin-mediated mechanisms of the dorsal raphe nucleus exert a key-role in the modulation of the post-ictal antinociception.     

5-HT1B and 5-HT1D receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase

5-Hydroxytryptamine (5-HT) is implicated in migraine and agonist directed aganist 5-HT_1B and 5-HT_1D receptors are commonly used as effective therapies. The antimigraine mechanisms involve the inhibition of intracranial sensory neuropeptide release. In order to determine which 5-HT₁ receptor subtypes are involved we have by immunocytochemistry examined the distribution of 5-HT_1B and 5-HT_1D receptors in the human trigeminal ganglia, and addressed which of them colocalize with calcitonin gene-related peptide (CGRP), substance P (SP) or nitric oxide synthase (NOS). We detected that 5-HT_1D receptor immunoreactivity (i.r.) was predominantly expressed in medium-sized cells (86% of positive cells, 30–60 μm). About 9% of the 5-HT_1D receptor i.r. cells were large in size (>60 μm) and 5% were small in size (<30 μm). In a similar pattern, 5-HT_1B receptor i.r. was mainly expressed in medium-sized cells (81% in 30–60 μm, 15% in >60 μm and 4% in <30 μm). Double immunostaining was used to determine whether the 5-HT_1B or 5-HT_1D receptor immunoreactive cells co-localized with either CGRP, SP or NOS. Thus, 89% of the CGRP i.r. cells expressed 5-HT_1D receptor i.r. and 65% of the CGRP positive cells were 5-HT_1B receptor positive. Most of the 5-HT_1D (95%) and the 5-HT_1B (94%) receptor i.r. cells showed SP immunostaining and 83% of 5-HT_1D receptor and 86% of 5-HT_1B receptor i.r. cells contained NOS. In conclusion, both 5-HT_1B and 5-HT_1D receptors are expressed in the human trigeminal ganglion and they are mainly localized in medium-sized cells and they seem to colocalize with CGRP, SP and NOS.     

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex

The anatomic relationship between serotonergic (5-HT) axons and 5-HT₂ receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT₂ receptors, localized by the ligand N1-methyl-2-¹²⁵I-LSD (¹²⁵I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT₂ receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT₂ receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT₂ receptors in layer V_a of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT₂ receptors and fine axons in other areas of the forebrain, suggesting that 5-HT₂ receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT₂ receptors may mediate the effects of dorsal but not median raphe projections.