The role of dopamine and serotonin in the prolongation of post-decapitation convulsions in mice.

L-DOPA (320 mg/kg, i.p.) increased the duration of the clonic phase of post-decapitation convulsions (PDC) by 60% in mice pretreated with the peripheral decarboxylase inhibitor, Ro 4-4602 (50 mg/kg, i.p.). Assays of brains at the time of decapitation showed a 300% increase in dopamine (DM), an 80% reduction in serotonin (5-HT) and no change in norepinephrine (NE) levels. The effect of L-DOPA on PDC was not blocked by haloperidol (0.5 – 5.0 mg/kg), a blocker of DM receptors, nor by diethyldithiocarbamate (400 mg/kg) an inhibitor of NE synthesis. Parachlorophenylalanine (300 mg/kg × 3 days) produced an 80% reduction in 5-HT and a prolongation of PDC similar to that observed after L-DOPA. Prolongation of PDC was also seen after the 5-HT antagonists methysergide (5 mg/kg) and cinanserin (10 mg/kg), but not after cyproheptadine (10 mg/kg). The 5-HT precursor, 5-hydroxytryptophan (100 mg/kg), produced no change in PDC when used alone but inhibited L-DOPA's prolongation of PDC. The results suggest that L-DOPA acts by depleting 5-HT in bulbospinal pathways and thus enhancing reflex activity in the spinal cord.     

Characterisation of Inhibitory 5-Hydroxytryptamine Receptors That Modulate Dopamine Release in the Striatum

Abstract: The effect of a series of indoleamines on the potassium-evoked tritium release of previously accumulated [³H]dopamine from rat striatal slices has been investigated. The indoleamines 5-hydroxytryptamine, 5-methoxy-tryptamine, 5-methoxy- N, N' -dimethyltryptamine and tryptamine (10⁻⁷ to 10⁻³ M) all reduced potassium-evoked release of tritium, to a maximum of 50%. The uptake of [³H]dopamine was unaffected by these compounds. A series of 5-hydroxytryptamine antagonists were examined for their ability to reduce the inhibition of potassium-evoked tritium release induced by 5-methoxytryptamine. The relative order of antagonist potency obtained was methysergide > metergoline > methiothepin > cinanserin > cyproheptadine > mianserin, and was consistent with an action on 5-hydroxytryptamine receptors. It is concluded that there are inhibitory 5-hydroxytryptamine receptors located on the terminals of dopaminergic neurones in the striatum.     

Pharmacological studies on serotonin-mediated behaviour

1. Administration to rats of a monoamine oxidase inhibitor (for example tranylcypromine: Tcp) followed by L-tryptophan increases the rate of synthesis and release of 5-hydroxytryptamine (5-HT) and results in a series of behavioural changes, some of which can be recorded on activity meters or scored. Various putative 5-HT agonists and the releasing drugs, fenfluramine and p-chloroamphetamine, also produce these changes. 2. A supersensitive behavioural response was produced by specific lesioning or p-chlorophenylalanine pretreatment and lesioning and sectioning experiments suggested several of the behaviours to be either hind-brain or spinally mediated. 3. A role for dopamine and GABA in the behaviour was demonstrated, but depletion of brain noradrenaline by specific lesioning or administration of disulfiram did not influence the behavioural changes. 4. The behaviour produced by administration of Tcp/L-tryptophan or 5-methoxy N,N-dimethyl tryptamine was inhibited by the suggested 5-HT antagonists, methysergide, methergoline and (--)-propranolol, but not by cinanserin, mianserin and cyproheptadine, other putative antagonists. In contrast, all the antagonists inhibited the behaviour when it was produced by injection of the agonist, quipazine. 5. The possible reasons for these differences is discussed in the light of the receptor binding characteristics of the drugs and the possible existence of different 5-HT receptor populations.     

Effects of Methiothepin and Lysergic Acid Diethylamide on Serotonin Release In Vitro and Serotonin Synthesis In Vivo: Possible Relation to Serotonin Autoreceptor Function

Abstract: An in vitro system characterizing the presyn- aptic serotonin (5-HT) autoreceptor which controls the release of 5-HT from rat brain slices is described. Using this system, methiothepin (1–10 μ M) demonstrated 5-HT autoreceptor antagonist activity -by enhancing 5-HT release, while several recognized postsynaptic 5-HT receptor antagonists were inactive: mianserin, cinanserin, cyproheptadine, methysergide. The activity of methiothepin was highest in hypothalamic slices and lowest in striatal slices and was inhibited by the autoreceptor agonists lysergic acid diethylamide (LSD) and 5-methoxy- tryptamine (5-MT). The reversal of the methiothepin-enhanced 5-HT release from hypothalamic slices by LSD was not influenced by 0.3 μ M tetrodotoxin. The peripheral administration of LSD to rats has been shown to reduce 5-HT synthesis and release by a mechanism thought to involve, in part, an autoreceptor-mediated reduction in impulse flow of 5-HT neurons. In the present experiments, intraperitoneal injection of methiothepin antagonized the LSD-induced reduction in hypothalamic 5-HT synthesis (5-hydroxytryptophan accumulation) while exerting no influence by itself. Conversely, compounds which were not active as 5-HT autoreceptor antagonists in vitro (i.e., cyproheptadine, methysergide, cinanserin) did not influence the effect of LSD on 5-HT synthesis. Further, the reduction in 5-hydroxytryptophan (5-HTP) accumulation by LSD showed regional differences in inhibition by methiothepin (hypothalamus > cortex > striatum) which paralleled the autoreceptor antagonist activity of methiothepin in vitro. These data suggest that similar autoreceptor mechanisms control 5-HT release and synthesis in terminal 5-HT projection areas and that the reduction in 5-HT accumulation by LSD and the antagonism by methiothepin may represent a useful biochemical measure of 5-HT autoreceptor activity in vivo.     

5-HT1a receptors positively coupled to C-AMP formation in the rabbit retina

Serotonin, 8-OH-DPAT, buspirone, ipsapirone, RU-24969, dopamine and isoproterenol all stimulate C-AMP production in rabbit retinal homogenates. The dopamine and isoproterenol responses are specifically antagonized by haloperidol and propranolol, respectively. In contrast the effects produced by the other substances are specifically nullified by spiroxatrine, a known 5-HT_1a antagonist. Since 5-HT₂ antagonists (methysergide, kentanserin), 5-HT_1b antagonist (propranolol), 5-HT₄ antagonist (ICS-205930), 5-HT₃ antagonist (MDL-72222, ICS-205930) or the primarily ₂-antagonist mianserin, had no influence on the stimulation of C-AMP caused by serotonin or 8-OH-DPAT, it is concluded that 5-HT_1a receptors, positively coupled to C-AMP production exist in the retina.     

Serotonergic mechanism in the control of β-endorphin and acth release in male rats

The role of the serotonergic mechanism in the regulation of β-endorphin (β-EP) and adrenocorticotropin (ACTH)-like immunoreactivity in plasma was investigated. Increases in β-EP and ACTH-LI produced by quipazine maleate (QPZ), a serotonergic agonist, 1 hr after injection could be completely prevented by the serotonin (5-HT) antagonist, cinanserin (CIN), which when injected alone, decreased basal plasma concentrations of both β-EP-LI and ACTH-LI. Concurrent injections of L-5-HTP with the 5-HT reuptake inhibitor, fluoxetine, produced an additive increase in plasma β-EP-LI 1 hr after injection. Injection of the 5-HT antagonist, cyproheptadine, significantly decreased plasma β-EP-LI. Stress by immobilization for 30 min or exposing the rats to 40° ± 1°C for 30 min produced an approximate 4-fold increase in plasma β-EP-LI and ACTH-LI, which was potentiated by I.P. injections of fluoxetine. Furthermore, the stress induced increases in plasma concentrations of β-EP-LI and ACTH-LI were significantly reduced by the serotonin antagonists metergoline and cinanserin. These results suggest that 5-HT is a potent stimulator of both β-EP and ACTH release and the increase in plasma concentrations of ACTH and β-EP induced by stress are probably mediated, at least in part, by central serotonergic mechanisms.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

A pharmacologic analysis of the action of platelet-activating factor in the induction of hindpaw edema in the rat

Platelet-activating factor (PAF), a phospholipid product of neutrophils, alveolar macrophages, monocytes, and platelets and an important mediator of inflammatory reactions, was studied for its ability to evoke hindpaw edema in the rat. PAF caused edema, peaking at 1 hr and gradually declining over the next 2 hr. The H1 and H2 antihistamines, mepyramine and cimetidine, the serotonin/histamine antagonist, cyproheptadine, and the serotonin antagonist, methysergide, were ineffective in reducing PAF-induced paw edema. Indomethacin, acetylsalicylic acid, and dexamethasone did not inhibit the peak edematous response but significant reduction was noted with only dexamethasone at 3 hr. Prazosin and propranolol did not prevent PAF-induced edema, whereas, yohimbine, phentolamine, rauwolscine, verapamil and theophylline partially inhibited edema. Clonidine and guanfacine did not induce edema when injected into the rat hindpaw. These results suggest that PAF elicits edema at vascular sites of the rat hindpaw which are partially dependent on extracellular Ca2+ movement, are not due to alpha-1 or alpha-2-adrenoreceptor stimulation, histamine, serotonin, or prostaglandin activity, and demonstrates variable sensitivities to agents blocking Ca2+ entry. Inhibition of specific PAF-sensitive receptors await the discovery of specific PAF antagonists.     

Influence of serotonergic and adrenergic antagonists on TRH-induced prolactin release in the monkey.

The influence of methysergide, cyproheptadine and SQ 10,631 (serotonergic receptor blockers) at the dose of 35 μg/kg, 50 μg/kg and 5 mg/kg, respectively, and propranolol, phentolamine and phenoxybenzamine (adrenergic receptor blockers) at the dose of 1 mg/kg on TRH-induced prolactin release was studied in sexually mature female monkeys. The serotonergic antagonists had no effect on TRH-induced prolactin release. Both β and α adrenergic antagonist gave a similar potentiation of the TRH-induced prolactin response but only phenoxybenzamine plus TRH was statistically different (P < 0.05) from TRH alone. The effect of the adrenergic receptor blockers is believed to be due to actions on dopamine receptors.     

A comparative study of several serotonin receptor antagonists on basal and stimulus induced release of insulin and glucagon in the intact rat.

Several commonly used serotonin receptor antagonists were studied for their ability to influence basal plasma insulin and glucagon (using 30K antibody) levels as well as the response of these hormones to a glucose or arginine challenge administered systematically to overnight fasted rats. Cyproheptadine, in contrast to other antagonists employed, induced large increases of insulin, glucagon and glucose, although this hyperinsulinemia was of a smaller magnitude when compared with hormone levels observed during an equivalent hyperglycemia resulting from glucose administration. The pancreatic response to a glucose load (increased insulin and decreased glucagon release) and an arginine load (increased insulin and glucagon release) were prevented by cyproheptadine pretreatment. Basal insulin levels were bot consistently altered by methysergide or cinanserin and were slightly elevated by metergoline. Basal glucagon levels were unaffected by these drugs. These three agents potentiated the insulinotropic effect of an arginine load whereas only metergoline exerted a similar effect on the response to glucose loading. Glucagon release in response to these stimuli was not significantly altered by drug pretreatment.     

Serotonin sensitive adenylate cyclase in horse brain synaptosomal membranes.

In different membranal preparations isolated from horse brain stritum we have shown the existence of an adenylate cyclase system sensitive to serotonin (5-HT). Activation of the adenylate cyclase was determined by measuring cAMP using a radioimmunoassay. This serotoninergic sensitive enzyme is characterized by a high apparent affinity constant (in the nanomolar range), located on synaptosomal membranes. It is inhibited by antiserotoninergic drugs (cyproheptadine, cinanserin, methysergide, LSD), and synergistically activated by GTP. This serotoninergic activation is clearly additive to the activation induced by dopamine. It appears different from the adenylate cyclase system previously described in the literature which is also activated by 5-HT, but which has a low apparent affinity constant (in the micromolar range); the latter is apparently located in non-synaptosomal membranes, and its activation by 5-HT is non-additive to the activation induced by dopamine.The serotoninergic sensitive adenylate cyclase reported in this study, might be related to the serotoninergic binding system which we have previously described which has similar affinity constant, a similar subcellular distribution and which is inhibited in the same concentration ranges by antiserotoninergic drugs. These two systems might represent a synaptosomal serotoninergic receptor complex.     

Species-specific effects on hemolymph glucose control by serotonin, dopamine, and L-enkephalin and their inhibitors in Squilla mantis and Astacus leptodactylus (crustacea)

Hemolymph glucose level is controlled by crustacean Hyperglycemic Hormone (cHH) released from the eyestalk neuroendocrine centers under conditions of both physiological and environmental stress. Biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue. We investigated the effect of serotonin, dopamine, and Leucine-enkephalin in vivo--injected into the stomatopod Squilla mantis and the decapod Astacus leptodactylus--whether increasing or depressing glycemia. Serotonin had a marked effect in elevating glucose level compared with initial values in both species. 5-HT1-like receptors are more involved in mediating serotonin action as co-injected cyproheptadine was a more effective antagonist than ketanserin (5-HT2-like receptor inhibitor). Dopamine injection in intact animals produced a decrease below initial levels of hemolymph glucose. This effect was significantly antagonized by domperidone. No significant effect of both amines occurred in eyestalkless animals. L-enkephalin shows a differential effect: in S. mantis it induced hypoglycemia while in A. leptodactylus it caused an increase of glucose level. Co-injected antagonist naloxone affected the direction of the response. Serotonin appears to provide a major control on glucose mobilization, whereas dopamine and L-enkephalin act as modulators whose plasticity in use or action varies among species.     

Hyperthermia induced by morphine administration to the VTA of the rat brain: an effect dissociable from morphine-induced reward and hyperactivity

Morphine action at opiate receptors in the ventral tegmental area (VTA) of the rat brain has been implicated in the production of increased locomotor activity and in morphine's rewarding properties. In the present experiments, bilateral administration of morphine (18 micrograms tapped into the tips of 28 gauge cannulae) into the VTA resulted in an increase in body temperature in rats. This effect was both reversed and blocked by a systemic injection of the opiate receptor blocker, naloxone, suggesting that it was due to morphine action at opiate receptors. The neuroleptic, pimozide, injected systemically four hours prior to morphine administration completely blocked the increased locomotor activity but had no effect on the hyperthermia. These data demonstrate that the hyperthermia was not brought about by the increased physical activity. Furthermore, these results suggest that while morphine-induced reward and increased locomotor activity may be mediated by an interaction of morphine and the ascending mesolimbic dopamine system, the hyperthermia is not. In an additional experiment, the effect of systemic injections of the central neurotransmitter receptor antagonists, scopolamine, phenoxybenzamine, and methergoline, on the hyperthermia induced by morphine in the VTA was investigated. Only the serotonin antagonist, methergoline, attenuated the hyperthermia.     

Effects of biogenic amines and related agonists and antagonists on the isolated heart of the common cuttlefish Sepia officinalis L

1. Effects of noradrenaline and the related compounds adrenaline, dopamine, octopamine, tyramine, clonidine and isoprenaline were studied in isolated heart preparations from the cuttlefish Sepia officinalis L. 2. All analogues produced a positive inotropic affect, with noradrenaline being the most potent substance. The chronotropic effects of the tested compounds differed widely. 3. The action of substances of the phenylethanolamine group were not antagonized by propranolol but were partly antagonized by phentolamine. 4. Serotonin and its analogues also produced cardio-excitation. These effects were blocked by cyproheptadine but not by methysergide. 5. These results indicate the presence of two different receptors in the Sepia myocardium: one type reacting with noradrenaline most effectively and a second type being stimulated by serotonin.     

Histamine and serotonin suppression of lymphocyte response to phytohemagglutinin and allogeneic cells

Histamine added to murine spleen cells suppressed in vitro proliferation of lymphocytes induced by PHA or allogeneic spleen cells. Another vasoactive amine, serotonin (5-hydroxytryptamine), exerted a similar inhibitory activity on PHA- or allogeneic cell-induced lymphocyte proliferation. Anti-H2 histamine antagonists, cimetidine, metiamide, and ranitidine, blocked the histamine and serotonin suppressive effect. Cyproheptadine, an anti-H1 histamine and anti-serotonin antagonist, and methysergide, an anti-serotonin antagonist, also blocked histamine and serotonin inhibitory activities. These data suggest the presence, on lymphocytes, of receptors for serotonin which might be related to histamine receptors.     

Serotonin (5-HT2) receptor mediated enhancement of cortical unit activity.

Simultaneous single-unit and intracortical activity were recorded from neocortical neurons in urethane-anaesthetized rats to investigate the role of serotonin (5-HT) in modifying cortical excitability. Units, at a depth of 775-1100 microns from the pial surface, discharged in a burst-pause pattern that was correlated with slow wave activity. Application of noxious somatic stimulation resulted in cortical desynchronization and altered the pattern of unit activity such that firing was continuous, i.e., the pauses were eliminated. Intravenous administration of the mixed 5-HT1C/5-HT2 antagonists (cinanserin, cyproheptadine, ketanserin, and ritanserin) prevented both desynchronization and the change in unit activity induced by noxious stimulation within 2.5-15 min of the injection. The basic pattern of burst-pause activity remained intact, but the number of spikes per burst was typically reduced, whereas interburst intervals were increased. Iontophoretic application of these antagonists onto cortical neurons resulted in actions similar to those observed following systemic administration. Intravenous and iontophoretic application of m-trifluomethylphenylpiperazine (5-HT1C agonist, 5-HT2 antagonist) resulted in actions indistinguishable from those observed with the above antagonists, from which we conclude 5-HT2 and not 5-HT1C receptors mediate the alteration in unit activity observed with noxious stimulation. The results are discussed with respect to an interaction between N-methyl-D-aspartate and 5-HT2 receptors leading to the enhanced unit activity observed with noxious stimulation.     

pH-dependent modulation of agonist interactions with [3H]-ketanserin-labelled S2 serotonin receptors

The effects of varying the pH on the properties of S₂ serotonin receptors labelled by [³H]-ketanserin were examined. Between pH 7.0 and 8.2 the agonist affinities, as determined by competition experiments, increased dramatically. Serotonin, 5-methoxytryptamine, tryptamine, bufotenine and quipazine, demonstrated 15,16,8,6 and 5-fold increases in apparent affinity between pH 7.0 and 8.2. On the other hand the antagonists, ketanserin, cinanserin, and spiperone demonstrated little or no affinity changes between pH 7.0 and 8.2. The largest shift in affinity for an antagonist occurred with spiperone, which displayed a two-fold shift. Although changing pH is a rather non-specific manipulation, the selective affect on agonist interaction with S₂ receptors indicates further investigation of this pH effect may aid in discovering the difference in receptor interactions between serotonin agonists and antagonists.     

Influence of adrenergic antagonists and apomorphine on prolactin release induced by serotonergic antagonists in the monkey.

The influence of adrenergic receptor blockers on the prolactin releasing effect of methysergide and cyproheptadine was examined in sexually mature female monkeys under ketamine anesthesia. Propranolol, a β-adrenergic blocker, at a dose of 1 mg/kg did not alter the prolactin releasing action of 0.1 mg/kg of methysergide but significantly potentiated (P < 0.025) the prolactin releasing action of 0.5 mg/kg of cyproheptadine. Phentolamine and phenoxybenzamine, both α-adrenergic blockers, at 1 mg/kg blunted the prolactin releasing effect of methysergide and cyproheptadine, but the pattern of prolactin blockade was different between the two putative antiserotonergic drugs. The prior administration of apomorphine, 4 mg/kg, a dopamine receptor stimulator, blocked the prolactin releasing effect of methysergide and cyproheptadine. Evidence presented here and from the literature indicate that the prolactin releasing action of methysergide and cyproheptadine is mediated by an antidopaminergic action directly on the pituitary.     

Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail

In the central nervous system of the pond snail Lymnaea stagnalis, serotonergic transmission plays an important role in controlling feeding behavior. Recent electrophysiological studies have claimed that only metabotropic serotonin (5-HT(2)) receptors, and not ionotropic (5-HT(3)) receptors, are used in synapses between serotonergic neurons (the cerebral giant cells, CGCs) and the follower buccal motoneurons (the B1 cells). However, these data are inconsistent with previous results. In the present study, we therefore reexamined the serotonin receptors to identify the receptor subtypes functioning in the synapses between the CGCs and the B1 cells by recording the compound excitatory postsynaptic potential (EPSP) of the B1 cells evoked by a train of stimulation to the CGC in the presence of antagonists: cinanserin for 5-HT(2) and/or MDL72222 for 5-HT(3). The compound EPSP amplitude was partially suppressed by the application of these antagonists. The rise time of the compound EPSP was longer in the presence of MDL72222 than in that of cinanserin. These results suggest that these two subtypes of serotonin receptors are involved in the CGC-B1 synapses, and that these receptors contribute to compound EPSP. That is, the fast component of compound EPSP is mediated by 5-HT(3)-like receptors, and the slow component is generated via 5-HT(2)-like receptors.     

Lack of effect of antagonists on serotonin-induced inhibition in rat hippocampus

Four putative central nervous system 5-hydroxytryptamine antagonists, methysergide, cyproheptadine, metergoline, and ketanserin and also lysergic acid diethylamide were applied by iontophoresis to firing CA1 hippocampal pyramidal cells to test their action on the inhibition produced by 5-hydroxytryptamine. In contrast to a previous report, none of these peripherally active 5-hydroxytryptamine antagonists altered the inhibitory response to submaximal doses of 5-hydroxytryptamine, but they did block after-excitations that followed the inhibitions. All the antagonists and lysergic acid diethylamide produced a depression of firing. When picrotoxin was used to drive the cells, 5-hydroxytryptamine was still able to produce a normal inhibition. The results of this study suggest that CA1 hippocampus is another structure, innervated by serotonergic neurones, where all (peripherally active) serotonin antagonists tested to date are ineffective against 5-hydroxytryptamine induced inhibition.