Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex

The aim of the present study was to examine the role of 5-HT₃ receptors in spontaneous and K⁺-evoked acetylcholine (ACh) release from rat entorhinal cortex and striatal slices. The 5-HT3 receptor antagonists ondansetron and granisetron (0.01–10 μM) produced a concentration-dependent increase in both spontaneous and K⁺-evoked [³H]ACh release in the two brain regions studied. The release of ACh was Ca²⁺-dependent and tetrodotoxin-sensitive. 5-HT₃ receptor agonists, such as 2-methyl-5-HT and 1-phenylbiguanide, at concentrations up to 1 μM, did not show any intrinsic effect on [³H]ACh release in both rat brain regions. However, 2-methyl-5-HT, 1 μM, fully blocked the ondansetron-induced enhancement in both basal and K⁺-evoked ACh release, suggesting that 5-HT₃ through 5-HT₃ receptor activation, tonically inhibits ACh release. The possible implication of interposed inhibitory systems in ACh release after 5-HT₃ receptor blockade was subsequently analyzed. While the effect of ondansetron was not modified by haloperidol or naloxone, the GABA_A receptor antagonist bicuculline produced a marked potentiation of ACh release in the entorhinal cortex but not in the striatum. The results suggest that in this cortical area 5-HT activates 5-HT₃ receptors located on GABAergic neurons which in turn inhibit cholinergic function.     

Evidence for 5-HT4 receptor involvement in the enhancement of acetylcholine release by p-chloroamphetamine in rat frontal cortex

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 μM), 5-HT_1A/1B/β-adrenoceptor antagonists (−)-pindolol (8 mg/kg, i.p.) and (−)-propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (1 mg/kg, i.p.; 10 μM) and 5-HT₃ antagonist ondansetron (1 mg/kg, i.p.; 10 μM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT₄ receptors.     

Modulation of neurotransmitter release induced by brain-derived neurotrophic factor in rat brain striatal slices in vitro

The atypical antipsychotic drugs (APDs) clozapine, olanzapine, risperidone, and ziprasidone preferentially increase dopamine (DA) release in rat medial prefrontal cortex (mPFC). These effects have been shown to depend upon potent 5-HT(2A) relative to weak D(2) antagonism, and 5-HT(1A) agonism as well. Atypical APDs also increase acetylcholine (ACh) release in the mPFC, but not the nucleus accumbens (NAC) or striatum (STR), whereas typical APDs such as haloperidol, S(-)-sulpiride and thioridazine do not produce either effect in the mPFC. This study examined the role of 5-HT(1A) agonism, 5-HT(2A) and D(2) antagonism, and the combination thereof, in the ability of clozapine to increase ACh release in rat mPFC. R(+)-8-OH-DPAT (0.2 mg/kg), a 5-HT(1A) agonist, WAY100635 (0.2-0.5 mg/kg), a 5-HT(1A) antagonist, and DOI (0.6-2.5 mg/kg), a 5-HT(2A/2C) agonist, increased ACh release in the mPFC, whereas M100907 (0.03-1 mg/kg), a 5-HT(2A) antagonist, did not. DOI (2.5 mg/kg) and M100907 (0.1 mg/kg) had no effect on ACh release in the NAC or STR. WAY100635 and M100907 inhibited the ability of R(+)-8-OH-DPAT and DOI, respectively, to increase ACh release in the mPFC. WAY100635, which inhibits clozapine-induced DA release in the mPFC, failed to inhibit clozapine (20 mg/kg)-induced ACh release in that region. Similarly, the combination of M100907 and haloperidol (0.1 mg/kg), which enhances DA release in the mPFC, failed to increase ACh release in that region. These results suggest that 5-HT(1A) agonism and 5-HT(2A) antagonism, as well as DA release, contribute minimally to the ability of clozapine, and perhaps other atypical APDs, to increase ACh release in the mPFC.     

Crucial role of the 5-HT2C receptor, but not of the 5-HT2A receptor, in the down regulation of stimulated dopamine release produced by pressure exposure in freely moving rats

Helium pressure of more than 2 MPa is a well known factor underlying pressure-dependent central neuroexcitatory disorders, referred to as the high-pressure neurological syndrome. This includes an increase in both serotonin (5-HT) and dopamine (DA) release. The relationship between the increase in 5-HT transmission produced by helium pressure and its effect on DA release has been clarified in a recent study, which have first demonstrated that the helium pressure-induced increase in DA release was dependent on some 5-HT receptor activation. In the present study, we examined in freely moving rats the role of 5-HT_2A and 5-HT_2C receptors in the increase in DA release induced by 8 MPa helium pressure. We used the 5-HT_2A receptor antagonist ketanserin and the 5-HT_2C receptor agonist m-CPP which have been demonstrated to reduce DA function. Because neither ketanserin is an ideal 5-HT_2A receptor antagonist nor m-CPP an ideal 5-HT_2C receptor agonist, additional experiments were made at normal pressure to check up on the selectivity of ketanserin and m-CPP for 5-HT_2A and 5-HT_2C receptors, respectively. Administration of m-CPP reduced both DA basal level and the helium pressure-induced increase in DA release, whereas administration of ketanserin only showed a little effect on the increase in DA release produced by high helium pressure. These results suggest that the 5-HT_2C receptor, but not the 5-HT_2A receptor, would play a crucial role in the helium pressure-induced increase in DA release. This further suggests that helium pressure may simultaneously induce an increase in 5-HT transmission at the level of 5-HT_2A receptors and a decrease in 5-HT transmission at the level of 5-HT_2C 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.     

Intranigral GR-113808, a selective 5-HT4 receptor antagonist, attenuates morphine-stimulated dopamine release in the rat striatum

GR-113808, a potent and selective 5-HT₄ receptor antagonist, was infused through a microdialysis probe into the striatum and nucleus accumbens of awake rats, and basal and morphine-stimulated extracellular concentrations of dopamine (DA) were measured in these regions. At 1 and 10 μM GR-113808 did not effect the extracellular concentrations of DA in either region and 100 μM significantly reduced dialysate DA only in the striatum. A subcutaneous dose of 5 mg/kg morphine significantly raised extracellular concentrations of DA in the striatum and nucleus accumbens from 60 to 120 min after injection and the effect was not modified by 10 μM GR-113808 infused through the probe 20 min before and for 60 min after morphine. Bilateral injections of GR-113808 (1, 2.5 and 10 μg/0.5 μl) in the substantia nigra pars compacta did not affect dialysate DA in the striatum, except for a significant increase 120 min after the injection of 10 μg but the highest dose of GR-113808 prevented the increase of striatal DA caused by 5 mg/kg morphine s.c. The results suggest that 5-HT₄ receptors in the substantia nigra modulate the activity of the dopaminergic nigrostriatal system only when the neurons are activated.     

DOI, a 5-HT2A/2C receptor agonist, attenuates clozapine-induced cortical dopamine release

(±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI, 1.25, 2.5 and 5 mg/kg), a serotonin (5-HT)_2A/2C agonist, produced an inverted U-shaped increase in DA release in rat medial prefrontal cortex (mPFC) with a significant effect only at 2.5 mg/kg. This effect was completely abolished by M100907 (0.1 mg/kg), a 5-HT_2A antagonist, and WAY100635 (0.2 mg/kg), a 5-HT_1A antagonist, neither of which when given alone affected dopamine release. DOI (2.5 mg/kg), but not the 5-HT_2C agonist Ro 60-0175 (3 mg/kg), attenuated clozapine (20 mg/kg)-induced mPFC dopamine release. These results suggest that 5-HT_2A receptor stimulation increases basal cortical dopamine release via 5-HT_1A receptor stimulation, and inhibits clozapine-induced cortical dopamine release by diminishing 5-HT_2A receptor blockade.     

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.     

Chronic repetitive transcranial magnetic stimulation alters β-adrenergic and 5-HT2 receptor characteristics in rat brain

Repetitive transcranial magnetic stimulation (rTMS) has been shown to affect mood in health and disease. Evidence to date has demonstrated an antidepressant potential for low- and high-frequency rTMS treatment. In animal behavioral models of depression magnetic stimulation of the brain induced similar effects to those of electroconvulsive shock (ECS). In this study the effects of repeated rTMS on rat brain noradrenaline, dopamine, serotonin and their metabolites levels, as well as on β-adrenergic and 5-HT₂ receptor characteristics were studied. After 10 days of treatment, β-adrenergic receptors were significantly up regulated in the frontal cortex, down regulated in the striatum and were unchanged in the hippocampus. 5-HT₂ receptors were down regulated in the frontal cortex and were not changed in the other brain areas. No change in benzodiazepine receptors in the frontal cortex and cerebellum were demonstrated. These findings demonstrate specific and selective alterations induced by repeated rTMS, which are distinct from those induced by other antidepressant treatments. TMS therapeutic effects in humans and behavioral and biochemical effects in animal, suggest that TMS has a unique mechanism of action which requires further investigation.     

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 receptors in the nucleus tractus solitarius: characterisation and role in cardiovascular regulation in the rat

The effects of the local application of drugs acting on 5-HT₂ receptors in the nucleus tractus solitarius (NTS) on the heart rate and blood pressure were investigated in normal and nodose ganglionectomized anaesthetized rats. The unilateral micro-injection of an agonist such as 2,5-dimethoxy-3-bromo-amphetamine (DOB) (0.1–0.5 pmol) or 2,5-dimethoxy-3-nitroamphetamine (DON) (0.1–0.5 pmol) produced a dose-dependent hypotension and bradycardia in both intact and ganglionectomized animals. These cardiovascular effects were similar to those observed after the unilateral micro-injection of low doses (pmol) of 5-HT, and could be prevented by the prior micro-injections of the 5-HT₂ antagonists ketanserin, ritanserin and piremperone. These findings support the hypothesis that 5-HT₂ receptors within the NTS play a role in the reflex regulation of blood pressure. In addition, it was also observed that the micro-injection of subthreshold doses of 5-HT or DOB significantly enhanced the hypotension and bradycardia produced by the unilateral micro-injection of N-methyl-d-aspartate (NMDA). The potentiation of NMDA depressor effects by 5-HT or DOB could be totally prevented by ketanserin or piremperone, suggesting that 5-HT acting upon 5-HT₂ receptors in the NTS may intervene in the reflex control of blood pressure by modulating the glutamatergic transmission.     

5-Hydroxytryptamine7 (5-HT7) receptor immunoreactivity-positive ‘stigmoid body’-like structure in developing rat brains

We examined the expression of 5-hydroxytryptamine(7) (5-HT(7)) receptor protein in developing and adult rats with immunohistochemical technique. In adult male rats, 5-HT(7) receptor immunoreactivity was detected in the septum, striatum, indusium griseum, tenia tecta, thalamus, hippocampus and hypothalamus in the forebrain as well as the pons and cerebellum. In brains of 1, 7, 15 and 21 days old male rats but not of adult ones, 5-HT(7) receptor immunoreactivity-positive dot-like structures were detected. The dot-like structures were visualized in hypothalamus, hippocampus, frontal cortex, brainstem and cerebellum at 1 day old male rats. In 7 days old male rats, the dot-like structures were found in the hypothalamus, medial preoptic area (MPA), bed nucleus of the stria terminalis (BST), amygdaloid nucleus and brainstem reticular formation. In 15 and 21 days old male and female rats, 5-HT(7) receptor immunoreactive dots were most clearly detected in MPA, hypothalamus, raphe pallidus, raphe magnus and brainstem reticular formation. The 5-HT(7) receptor immunoreactivity-positive dot-like structures were shown in the cytoplasm and they were less than 1 microm in diameter in 1 and 7 days old rats and became larger to 1-3 microm in 15 and 21 days old rats. From the distribution and morphologic features, the 5-HT(7) receptor immunoreactivity-positive dot-like structure found in developing rat brains is considered to be identical to a cytoplasmic inclusion named 'stigmoid body'.     

The effect of 5-HT3 receptor antagonists on the morphine-induced excitation of A10 dopamine cells: electrophysiological studies

In extracellular recordings from chloral hydrate anesthetized rats the 5-HT₃ antagonist, BRL 46470A, failed to prevent or reverse the increase in dopamine cell firing rate produced by systemic or iontophoretically applied morphine. A second 5-HT₃ antagonist, tropesitron, was similarly found to be ineffective in antagonizing the effects of systemic morphine. These results suggest that previous microdialysis reports that 5-HT₃ antagonists can prevent the increase in extracellular dopamine levels in the nucleus accumbens produced by morphine are not due to an action of these compounds in suppressing the excitatory effects of morphine on A10 dopamine cell firing rate.     

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.     

Studies on the involvement of the dopaminergic system in the 5-HT2 agonist (DOI)-induced premature responding in a five-choice serial reaction time task

The present experiments investigated whether the enhanced premature (impulsive) responding induced by DOI, [(±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride], a 5-HT_2A/2C receptor agonist, is mediated by activation of the dopaminergic system and if this effect of DOI occurs in the nucleus accumbens. Therefore, the effects of a dopamine (D_1/2) receptor antagonist given alone or combined with DOI were examined on the performance of rats in a five-choice serial reaction time (5-CSRT) task. Secondly, the effects of DOI in nucleus accumbens core and shell were studied, in order to find the target brain area for DOI-induced premature responding. The results indicate that DOI (0.1 mg/kg, subcutaneously) increases the number of premature responses, as found previously. α-Flupenthixol (0.03 mg/kg), a D_1/2 dopamine receptor antagonist, and raclopride (0.015 mg/kg), a D₂ receptor antagonist, attenuated the DOI-induced enhancement in premature responding. SCH 23390 (0.005 mg/kg), a selective D₁ receptor antagonist with little affinity to 5-HT₂ receptors totally blocked the effect of DOI. Those doses of DA antagonists did not significantly decrease premature responding when given alone. On the other hand, higher doses of all of these dopamine antagonists increased the number of omissions and decreased the number of ITI hole responses. In contrast to subcutaneous administration, direct injections of DOI (1, 3, and 10 μg bilaterally) to the nucleus accumbens shell or core had no effect on premature responding. These results suggest that the activation of the dopamine system mediates, at least in part, the effect of a 5-HT₂ agonist on premature responding, but the nucleus accumbens is not the primary site for this action.     

Effects of triiodothyronine and fluoxetine on 5-HT1A and 5-HT1B autoreceptor activity in rat brain: regional differences

The thyroid hormone triiodothyronine (T3) augments and accelerates the effects of antidepressant drugs. Although the majority of studies showing this have used tricyclics, a few studies have shown similar effects with the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. In this study we investigated the effects of fluoxetine (5 mg/kg), T3 (20 μg/kg) and the combination of these drugs, each administered daily for 7 days, on serotonergic function in the rat brain, using in vivo microdialysis. Fluoxetine alone induced a trend towards desensitization of 5-HT_1A autoreceptors as shown by a reduction in the effect of 8-OH-DPAT to lower 5-HT levels in frontal cortex, and desensitized 5-HT_1B autoreceptors in frontal cortex. The combination of fluoxetine and T3 induced desensitization of 5-HT_1B autoreceptors in hypothalamus. Since there is evidence linking hypothalamic function and depression, we suggest that this effect may partly account for the therapeutic efficacy of the combination of an SSRI and T3.     

Cardiovascular effects of 5HT2 and 5HT3 receptor stimulation in the nucleus tractus solitarius of spontaneously hypertensive rats

The effects of local application of 5-HT₂- and 5-HT₃-receptor agonists in the nucleus tractus solitarius (NTS) on the cardiovascular parameters were investigated in anaesthetized spontaneously hypertensive (SH) rats and their genetically normotensive precursors (WKY). Unilateral microinjection of picomolar doses of a 5HT₂ receptor agonist, 2,5-dimethoxy-3-bromo-amphetamine (DOB, 0.025 – 0.5 pmol), produced a dose-dependent hypotension and bradycardia in both SH and WKY rats. These effects could be prevented by prior local microinjection of a 5-HT₂ receptor antagonist, ketanserin (10 pmol). However, for both cardiovascular parameters, DOB was more potent in SH than in WKY rats. Thus, the dose-related responses to DOB were shifted to the left in SH as compared to WKY rats. Bilateral microinjection of the 5-HT₃ receptor agonist, phenylbiguanide (1.7 – 5 nmol), produced an increase in blood pressure and reduced the cardiovagal component of the baroreflex. These effects were not significantly different in SH and WKY rats. These data suggest that 5-HT₂ receptors, but not 5-HT₃ receptors, are supersensitive in the NTS of SH rats.