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.     

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.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7–12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in 43% of the neurons (53/123); however, in a minority population (6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I_5-HT) in neurons voltage-clamped near the resting potential (-60 mV); the effect was mimicked by the 5-HT₃ receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT₃ receptors. Further, I_5-HT was selectively inhibited by the 5-HT₃ receptor-specific antagonist MDL72222 (1–10 μM), but was unaffected by either 5-HT₁/5-HT₂ receptor antagonist, spiperone, or by 5-HT₂ receptor-specific antagonist, ketanserin (50–100 μM). I_5-HT displayed moderate inward rectification and had a mean reversal potential (±S.E.M.) of −4.3±6.6 mV (n=6). Application of 5-HT (dose range: 0.1–100 μM) produced a dose–response curve that was fitted by the Hill equation with EC₅₀=3.4 μM and Hill coefficient=1.6 (n=8). The activation phase of I_5-HT (10 μM 5-HT at −60 mV) was well fitted by a single exponential with mean (±S.E.M.) time constant of 45±30 ms (n=6). The desensitization phase of I_5-HT was best fitted by a single exponential with mean (±S.E.M.) time constant of 660±167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (±S.E.M) of 2.7±1.5 pS (n=4) at −60 mV. In the minority (6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT₂ receptor antagonist, ketanserin (50 μM). Taken together, these results suggest that 5-HT₃ receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.     

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.     

Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)₇ receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT₇ receptors induced by a treatment with agonists (0.1, 1, and 10 μM, 0.5 to 3.5 h). Maximum responses, but not the EC₅₀ values, in the concentration–response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor α-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT_1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT₇ receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.     

Actions of 5-HT on human neocortical neurones in vitro

Using intracellular recordings, we have studied the action of 5-hydroxytryptamine (5-HT) on slices of human temporal, occipital and frontal cortex maintained in vitro. The recordings were usually made 1.2 to 1.5 mm down from the pial surface, in or around layer III. The action of 5-HT (30–50 μM) was studied on 21 cells (from 12 individuals) which had electrophysiological characteristics of glutamatergic pyramidal neurones. 5-HT depolarised the majority (11) of these cells with a median response of 5 mV. It produced a hyperpolarising response in five neurones (median=−4 mV) and a combined hyperpolarising/depolarising response in two others. No response was detected in three cells. The depolarising response was probably mediated by reducing a resting potassium conductance. Ketanserin (0.1 and 1.0 μM) and spiperone (1 μM) reduced the response indicating that it was likely mediated by 5-HT_2A receptors. The hyperpolarising response was associated with the opening of ion channels and was blocked by the selective 5-HT_1A receptor antagonist WAY-100635 (100 nM). 5-HT inhibited spontaneous synaptic potentials. This effect was reduced by ketanserin (1 μM) but not by WAY-100635 (100 nM). It is concluded that human neocortical neurones in vitro can be depolarised via 5-HT_2A receptors and hyperpolarised via 5-HT_1A receptors.     

Quantitative autoradiography of 5-HT1D and 5-HT1E binding sites labelled by [H]5-HT, in frontal cortex and the hippocampal region of the human brain

In human cortex and hippocampus area, [³H]5-HT (5 nM) labels 5-HT_1A, 5-HT_1D and 5-HT_1E sites. After masking 5-HT_1A receptors by 0.1 μM 8-OH-DPAT, the binding displaced by 0.1 μM 5-CT presumably represented 5-HT_1D sites and the remaining binding 5-HT_1E sites. In frontal cortex, 5-HT_1A receptors represented the main binding in layers II and VI and a lower fraction on other layers. 5-HT_1D and 5-HT_1E sites, were more homogeneously distributed in layers II to VI (21–34% of specific [³H]5-HT binding). 5-HT_1E sites were of similar affinities (K_D close to 6–8 nM) in the cortical layers II to VI. In CA1 field of hippocampus, (pyramidal layer, stratum radiatum, molecular layer), CA2 and dentate gyrus, 5-HT_1A receptors represented the major fraction, 5-HT_1D sites a significant fraction and 5-HT_1E a minor fraction of the specific [³H]5-HT binding. In CA3–CA4 fields, 5-HT_1A receptors were less densely present, 5-HT_1D sites were predominant and 5-HT_1E sites represented a significant fraction (27%). The highest densities of 5-HT_1E sites have been measured in subiculum, where 5-HT_1A, 5-HT_1D, and 5-HT_1E binding sites were equally represented and in entorhinal cortex where 5-HT_1E sites represented the major binding in layer III. They were also present in layers II and IV (29 and 24%) and, to a lesser extent, in layers V and VI. 5-HT_1A sites were predominant in layer VI, II and V and were less abundant in other layers. 5-HT_1D were homogeneously present in layers II, III, IV and were present in low amounts in other layers. No 5-HT_1E were detected in choroid plexus, where [³H]5-HT was dramatically reduced by mesulergine (5-HT_2C receptors). No significant displacement of [³H]5-HT by mesulergine was measured in other structures.     

Effects of cholecystokinin tetrapeptide (CCK4) and anxiolytic drugs on the electrically evoked [H]5-hydroxytryptamine outflow from rat cortical slices

The outflow of [³H]5-hydroxytryptamine ([³H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [³H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK₄ 300–1000 nM, the effect being prevented by the CCK_B receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK₄ seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [³H]5-HT outflow and lowered the CCK₄ threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 μM bacitracin to inhibit CCK metabolism increased [³H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT_1A partial agonist buspirone 300 nM and the GABA_A receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [³H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 μM) and NBQX (10 μM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.     

Functional evidence for the rapid desensitization of 5-HT3 receptors on vagal afferents mediating the Bezold–Jarisch reflex

The aim of this study was to determine whether 5-hydroxytryptamine (5-HT)₃ receptors on cardiopulmonary afferents mediating the Bezold–Jarisch reflex (BJR) desensitize upon repeated exposure to selective agonists. BJR-mediated falls in heart rate, diastolic arterial blood pressure and cardiac output elicited by the 5-HT₃-receptor agonists, phenylbiguanide (100 μg/kg, i.v.) or 2-methyl-5-HT (100 μg/kg, i.v.), progressively diminished upon repeated injection in conscious rats. The BJR responses elicited by 5-HT (40 μg/kg, i.v.) were markedly reduced in rats which had received the above injections of phenylbiguanide or 2-methyl-5-HT whereas the BJR responses elicited by -S-nitrosocysteine (10 μmol/kg, i.v.) were similar before and after the injections of the 5-HT₃ receptor agonists. These findings suggest that tachyphylaxis to 5-HT₃ receptor agonists may be due to the desensitization of 5-HT₃ receptors on cardiopulmonary afferents rather than the impairment of the central or peripheral processing of the BJR.     

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.     

Differential influence of two serotonin 5-HT3 receptor antagonists on spinal serotonin-induced analgesia in rats

We tested the antinociceptive effect of intrathecal (i.t.) administration of 5-HT and the 5-HT₃ receptor agonist, 1-(m-chlorophenyl)-biguanide (mCPBG), in rats submitted to a mechanical noxious stimulus and the influence of the 5-HT₃ receptor selective antagonists, tropisetron and granisetron. Both 5-HT and mCPBG (0.01, 0.1, 1, 10, 20 μg/rat) produced a significant dose-dependent antinociception. The lowest active doses were 0.1 and 1 μg for 5-HT and mCPBG, respectively. The effect, observed with 20 μg, was significantly lower with mCPBG (+33±6%) than with 5-HT (+63±7%). For 5-HT-induced antinociception, the minimal inhibitory doses were 0.001 μg/rat for tropisetron and 10 μg/rat for granisetron. In contrast, the same doses of the two antagonists (from 0.1 μg/rat) similarly inhibited the effect of mCPBG. This study provides evidence that contrary to tropisetron, doses of granisetron able to inhibit the effect of a 5-HT₃ receptor agonist failed to reduce that of 5-HT. This demonstrates a heterogeneity between 5-HT₃ receptor antagonists and questions the true involvement of these receptors in spinal 5-HT-induced antinociception.     

Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca stores in NG108-15 cells

Following mobilization with the inositol 1,4,5-trisphosphate (IP₃)-generating agonist bradykinin, Ca²⁺ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca²⁺ to refill. The process by which this store refills with Ca²⁺ was characterized by recording bradykinin-induced intracellular free Ca²⁺ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca²⁺ ATPase inhibitor, reversibly depleted intracellular Ca²⁺ stores in these cells, but did not recruit detectable Ca²⁺ influx, suggesting that these cells lack substantial capacitative Ca²⁺ entry. The paucity of voltage-sensitive Ca²⁺ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca²⁺ entry in nonexcitable cells might assist refilling IP₃-sensitive Ca²⁺ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca²⁺ entry in nonexcitable cells might inhibit the refilling of the IP₃-sensitive store in NG108-15 cells was explored. The IP₃-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca²⁺ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca²⁺ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca²⁺ stores by a plasmalemmal Ca²⁺ leak, this leak shares a pharmacology similar to the capacitative Ca²⁺ entry pathway described for nonexcitable cells.     

Perfusate serotonin increases extracellular dopamine in the nucleus accumbens as measured by in vivo microdialysis

The effects of local application of serotonin (5-HT) on extracellular levels of dopamine (DA) in the nucleus accumbens (N.AACC) were assessed using in vivo microdialysis. At a perfusate flow rate of 0.3 μl/min the baseline dialysate concentration of DA was 2.1 ± 0.7 nM (mean ± S.E.M.; n = 5) and significantly increased to 142 ± 18%, 220 ± 47% and 332 ± 35% of baseline when 0.1 μM, 0.2 μM and 0.4 μM concentrations of 5-HT were included in the perfusate. Perfusate 5-HT concentrations below 0.1 μM had no effect on dialysate DA. The in vivo dialysis efficiency for 5-HT was found to be 39 ± 12%, and thus the concentrations of 5-HT reaching the extracellular space at the surface of the dialysis membrane were estimated to be 40,80 and 160 nM for the 0.1, 0.2 and 0.4 μM 5-HT perfusates, respectively. The serotonin-induced increase in dialysate DA was attenuated by co-perfusion of 0.4 μM 5-HT with 4 μM concentrations of pindolol (a relatively non-specific 5-HT₁ antagonist; 151 ± 7% vs. 332 ± 35% baseline dialysate DA for 5-HT/antagonist and 5-HT-only perfusates, respectively), LY 53,857 (a specific 5-HT₂ antagonist; 130 ± 17%vs.332 ± 35%) and MDL 7222 (a specific 5-HT₃ antagonist; 143 ± 19%vs.332 ± 35%). While the contributions of the 5-HT₁ receptor subtype are unclear, the inhibitory efficacy of a highly specific 5-HT₂ antagonist and an antagonist specific to 5-HT₂ and 5-HT₃ receptors suggest that both of these receptor subtypes are involved in the serotonin-induced DA increase in the N. ACC.     

Characterization of 5-HT2A receptor desensitization and the effect of cycloheximide on it in C6 cells

Summary. Effect of prolonged pretreatment with serotonin (5-HT) on 5-HT_2A receptor desensitization was examined by the measurement of intracellular calcium ([Ca²⁺]_i) mobilization in C6 cells. 5-HT-induced desensitization of [Ca²⁺]_i mobilization was in a time and dose dependent manner and reached a plateau after 3 hr. After 1 and 3 hr 5-HT pretreatment, 5-HT concentration in the medium little changed. 5-HT pretreatment with cycloheximide, a protein synthesis inhibitor, produced an enhancement of the desensitization for 3 and 6 hr pretreatment. However, 5-HT pretreatment for 3 and 6 hr caused no marked change in the 5-HT_2A receptor mRNA level or Gαq/11 protein in this study, suggesting that 5-HT may decrease 5-HT-induced [Ca²⁺]_i mobilization independent of 5-HT_2A receptor mRNA or G-proteins. Endothelin-1-induced [Ca²⁺]_i mobilization did not alter after 5-HT and/or cycloheximide pretreatment. These results showed that activation of the 5-HT_2A receptor induced homologous desensitization and pretreatment with 5-HT and/or cycloheximide did not change the efficacy of the second messenger pathway from Gq to a [Ca²⁺]_i rise. Received June 6, 2000; accepted October 17, 2000     

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca]i increases and NMDA receptor mRNA levels

We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 μM glutamate/10 μM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca²⁺, respectively, to measure changes in [Ca²⁺]_i. Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca²⁺]_i changes with exposure to 3 or 100 μM glutamate for 5 min, whereas magfura-2 reported larger [Ca²⁺]_i increases with 5-min exposure to 100 μM glutamate than with exposure to 3 μM glutamate, 100 μM kainate or 50 mM K⁺ from 10 DIV onward. This suggests that the magnitude of the [Ca²⁺]_i changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca²⁺. We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca²⁺]_i responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.     

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.     

Involvement of 5-HT7 receptors in serotonergic effects on spike afterpotentials in presumed jaw-closing motoneurons of rats

Intracellular recordings were obtained from rat presumed jaw-closing motoneurons in slice preparations to investigate the involvement of the serotonin₇ (5-HT₇) receptors in serotonergic inhibition of the postspike medium-duration afterhyperpolarization (mAHP) and enhancement of the afterdepolarization (ADP). 5-HT-induced suppression of the mAHP and enhancement of the ADP were mimicked by application of the 5-HT_1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and antagonized by the 5-HT_2/6/7 receptor antagonist clozapine, whereas the 5-HT₂ receptor agonist α-methyl-5-hydroxytryptamine (α-methyl-5-HT) did not affect the mAHP and ADP. 8-OH-DPAT-induced attenuation of the mAHP and enhancement of the ADP were also antagonized by clozapine and another 5-HT_2/6/7 receptor antagonist ritanserin, whereas the 5-HT_1A receptor antagonist pindolol failed to block the 8-OH-DPAT-induced effects on the mAHP and ADP. 8-OH-DPAT-induced suppression of the mAHP and enhancement of the ADP were also antagonized by a protein kinase A (PKA) inhibitor H89, whereas 8-OH-DPAT could inhibit the mAHP and enhance the ADP in the presence of a protein kinase C (PKC) inhibitor chelerythrine. The 8-OH-DPAT-induced suppression of the mAHP was enhanced under raised [Ca²⁺]_o and this enhancement was reduced by chelerythrine. It is suggested that the 5-HT₇ receptors are involved in 5-HT-induced attenuation of the mAHP and enhancement of the ADP through activation of PKA, and the attenuation of mAHP through the 5-HT₇ receptors is enhanced under raised [Ca²⁺]_o by PKC activation.     

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.     

U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells

The aminosteroid U73122 inhibited phospholipase C (PLC)-mediated intracellular Ca²⁺ release in differentiated and undifferentiated NG108-15 cells, as well as rat dorsal root ganglion (DRG) neurons grown in primary culture. 1 μM U73122 blocked bradykinin (BK)-induced increases in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) measured in single cells with indo-1-based dual emission microfluorimetry. A close structural analog, U73343, was without effect. The effects of U73122 were time and concentration-dependent. 1 μM drug produced half maximal inhibition in approximately 3 min. The IC₅₀ for a 20-min exposure was approximately 200 nM. The effects of the compound were irreversible for the duration of experiments as long as 1 h. Treatment with 1 μM U73122, but not U73343 produced a small but significant increase in [Ca²⁺]_i which resulted from Ca²⁺ release from an intracellular store. It is not clear whether this [Ca²⁺]_i increase resulted from inhibition of PLC or an action on the store directly. In differentiated NG108-15 cells U73122 blocked completely depolarization-induced Ca²⁺ influx. In contrast, in DRG neurons U73122 inhibited only slightly voltage-sensitive Ca²⁺ channels. Thus, we caution that U73122 may not be selective at concentrations required for maximal block of PLC and that the selectivity of U73122 is dependent on cell type. Overall, our results are consistent with U73122 inhibiting PLC in neuronal cells and indicate that under the appropriate conditions, this compound is a useful tool for studying inositol 1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ mobilization.     

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.