Identification and distribution of 5-HT3 recognition sites within the human brainstem

The present studies demonstrate the presence of specific [3H]GR65630 binding sites within the human brainstem using the techniques of in vitro receptor autoradiography and ligand binding to homogenates. Autoradiography revealed the greatest accumulation of specific binding in the area postrema and subpostrema (AP/ASP). A lower level of specific binding was identified in the nucleus tractus solitarius (excluding area subpostrema). No specific binding was evident in the remainder of the hindbrain at this level. Discrete dissection followed by ligand binding to homogenates revealed that the specific binding of [3H]GR65630 (defined by the presence of 30 microM metoclopramide) was differentially distributed with highest levels in the AP/ASP (112.1 fmol/mg protein) and lower levels in the dorsal vagal complex (nucleus tractus solitarius--excluding the area subpostrema--dorsal motor nucleus of the vagus and hypoglossal nucleus) (DVC) and olivary nucleus (ON) (22.9 and 3.9 fmol/mg, respectively). No specific binding was detectable in the reticular formation (RF) located ventral to the dorsal vagal complex. The specific [3H]GR65630 binding site was pharmacologically similar to the 5-HT3 receptor since the potent and selective 5-HT3 receptor antagonists ICS 205-930 and zacopride (100 nM) and the agonist 5-HT (10 microM) inhibited binding to the same extent as metoclopramide in each of the individual areas (90, 60 and 20% in the AP/ASP, DVC and ON, respectively). The 5-HT1-like and 5-HT2 receptor antagonist methysergide (10 microM) failed to compete for the binding site. 5-HT3 receptor recognition sites within the AP/ASP and the DVC may be functionally involved in the ability of 5-HT3 receptor antagonists to control emesis.     

Reversal of the antinociceptive effects of intrathecally administered serotonin in the rat by a selective 5-HT3 receptor antagonist

The ability of the highly selective 5-HT3 receptor antagonist ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) to block the increase in tail flick (TFL) and hot plate latencies (HPL) produced by intrathecally (i.t.) administered serotonin (5-HT) was examined in pargyline pretreated rats. ICS 205-930 (0.1 microgram, i.t.) blocked the ability of 5-HT (200 micrograms) to increase TFL and HPL. Significant hyperalgesia, as measured by a decrease in TFL and HPL compared to saline controls, also resulted from either the coadministration of ICS 205-930 (10 micrograms) and 5-HT (200 micrograms) or from ICS 205-930 (100 micrograms) alone. These data suggest an important role for 5-HT3 receptors in modulating spinal nociceptive responses.     

Electrophysiological characterization of functionally distinct 5-hydroxytryptamine receptors on guinea-pig submucous plexus

Intracellular recordings were made from neurons of the guinea-pig submucous plexus and the actions of 5-hydroxytryptamine on the postsynaptic membrane and on evoked synaptic potentials were examined. 5-Hydroxytryptamine produced two types of direct postsynaptic responses: (1) A depolarization associated with a fall in input resistance was observed in all cells. Voltage-clamp and ion substitutions showed that this depolarization resulted primarily from an inward sodium current. This response could be as brief as 30 ms; it showed desensitization and was selectively abolished by 0.2-2 microM ICS 205-930. (2) A depolarization (or inward current) associated with a decreased conductance was observed in about 50% of neurons, usually after the first response was blocked by ICS 205-930. This response was due to a decreased potassium conductance; the minimum time course of this response was 8-10 s. It did not show desensitization and was not sensitive to blockade by currently available antagonists of 5-hydroxytryptamine, nicotinic and/or muscarinic receptors. Higher concentrations of 5-hydroxytryptamine were required to produce the sodium conductance increase than the potassium conductance decrease; 2-methyl-5-hydroxytryptamine was equally effective in producing these responses. 5-Hydroxytryptamine also caused a barrage of "spontaneous" nicotinic excitatory post-synaptic potentials which were sensitive to tetrodotoxin. This response desensitized, was blocked by ICS 205-930 and is presumed to reflect excitation of other cholinergic cell bodies in the plexus by the sodium conductance increase mechanism described. The evoked nicotinic excitatory postsynaptic potential and the adrenergic inhibitory postsynaptic potential were decreased by 5-hydroxytryptamine; a portion of this inhibition showed desensitization and was blocked by ICS 205-930 as well as by the muscarinic receptor antagonists, atropine and pirenzepine. The ICS 205-930-insensitive portion of this inhibition could not be attributed to activation of 5-hydroxytryptamine-1 or 5-hydroxytryptamine-2 receptors. Thus, the following conclusions are drawn: 5-hydroxytryptamine excites submucous plexus neurons by activating two distinct 5-hydroxytryptamine receptors. Activation of the 5-hydroxytryptamine-3 receptor (sensitive to ICS 205-930) produces a depolarization mediated by an increased sodium conductance. The same effect occurring in other cholinergic cell bodies initiates action potentials which are responsible for the 5-hydroxytryptamine-induced release of acetylcholine.(ABSTRACT TRUNCATED AT 400 WORDS)     

5-Hydroxytryptamine-induced sensitization and activation of peripheral fibres in the neonatal rat are mediated via different 5-hydroxytryptamine-receptors

The effects of 5-hydroxytryptamine on peripheral nociceptive fibres were studied in an in vitro preparation of the neonatal rat spinal cord with attached tail. The activation of peripheral fibres in the tail by noxious stimuli (bradykinin, capsaicin, heat) was recorded as a depolarization of a ventral root in the lumbar region of the spinal cord (L₃–L₅). Responses evoked by brief applications of submaximal or threshold concentrations of bradykinin or capsaicin to the tail were enhanced by 5-hydroxytryptamine and the 5-hydroxytryptamine_1C/5-hydroxytryptamine₂-receptor agonist -methyl-5-hydroxytryptamine but not by the 5-hydroxytryptamine₃-receptor agonist 2-methyl-5-hydroxytryptamine or the 5-hydroxy-tryptamine₁-receptor agonist 5-car☐amidotryptamine. Sensitization induced by 5-hydroxytryptamine and -methyl-5-hydroxytryptamine was blocked by the selective 5-hydroxytryptamine₂-receptor antagonist ketanserin. Neither the 5-hydroxytryptamine₃/5-hydroxytryptamine₄-receptor antagonist ICS 205–930 nor the 5-hydroxytryptamine₁/5-hydroxytryptamine₂-receptor antagonist methiothepin blocked the 5-hydroxytryptamine-induced sensitization. The responses evoked by submaximal thermal stimuli were also enhanced following the sensitization of peripheral nociceptors with 5-hydroxytryptamine or -methyl-5-hydroxytryptamine. The -methyl-5-hydroxytryptamine-induced enhancement of thermal responses was reduced by ketanserin. 5-Hydroxytryptamine did not evoke a ventral root response unless peripheral fibres were sensitized with threshold concentrations of bradykinin or capsaicin. This effect was mimicked under the same conditions by 5-car☐amidotryptamine but not by -methyl-5-hydroxytryptamine or 2-methyl-5-hydroxytryptamine. The excitatory effect of 5-hydroxytryptamine was blocked by methiothepin but not by ICS 205–930 or ketanserin. Neither 5-hydroxytryptamine-induced sensitization nor 5-hydroxytryptamine-evoked activation of peripheral fibres was blocked by indomethacin.

These data indicate that two types of receptor are involved in the peripheral actions of 5-hydroxytryptamine in nociception. 5-Hydroxytryptamine-induced sensitization involves a 5-hydroxytryptamine₂-receptor, whereas 5-hydroxytryptamine-evoked excitation involves a 5-hydroxytryptamine₁-like-receptor.     

Specific [3H]Ro 5-4864 binding to rat spinal cord membranes: evidence for peripheral type benzodiazepine recognition sites

The binding of [3H]Ro 5-4864 and [3H]methylclonazepam to membranes prepared from adult rat spinal cord has been studied in vitro. Scatchard analysis of saturation isotherms suggested that both 3H-labeled ligands bind to single binding sites ([3H]Ro 5-4864 Kd = 2 nM, [3H]methylclonazepam Kd = 3.5 nM), although [3H]Ro 5-4864 bound to 3 times the number of sites labeled by [3H]methylclonazepam (respective Bmax values were 15 vs 5.3 pmol/g tissue). Displacement experiments with clonazepam, flunitrazepam and Ro 5-4864 indicated that [3H]Ro 5-4864 and [3H]methylclonazepam binding had the expected pharmacologic specificity for peripheral and central benzodiazepine recognition sites respectively (i.e. [3H]methylclonazepam binding was sensitive to clonazepam but not Ro 5-4864 whereas [3H]Ro 5-4864 binding was potently inhibited by Ro 5-4864 but not clonazepam. Flunitrazepam had similar affinities for both sites). Thus, in addition to central type benzodiazepine receptors, the rat spinal cord contains comparatively high concentrations of peripheral benzodiazepine recognition sites.     

Autoradiographic distribution of [3H]acetylcholine binding sites in the cervical spinal cord of man and some other species

The distribution of [3H]acetylcholine ([3H]ACh) and [3H]ACh co-incubated with 1-mM nicotine (muscarinic receptor), and [3H]ACh co-incubated with 1.5 microM atropine (nicotinic receptor) binding sites were studied in man and compared to monkey, cat and rat using quantitative in vitro autoradiography. The highest density of total [3H]ACh binding sites was found in laminae II-III, IX (motor neuron areas) and X close to the central canal. The distribution pattern of the muscarinic cholinergic binding sites was similar to that of the total cholinergic binding. In general the number of nicotinic binding sites in the spinal cord was relatively small. The largest number of such binding sites was found in laminae II-III of the dorsal horn and in laminae X around the central canal. It is evident that the spinal cord has a 2-3 times higher number of muscarinic than of nicotinic cholinergic receptors.     

Postnatal development of [3H]flunitrazepam and [3H]strychnine binding sites in rat spinal cord localized by quantitative autoradiography

The development of inhibitory receptors in rat spinal cord was investigated by autoradiography using [3H]flunitrazepam as a ligand for benzodiazepine receptors and [3H]strychnine as a ligand for glycine receptors. The development of benzodiazepine receptors follows a similar pattern at all levels of the spinal cord. The density of [3H]flunitrazepam binding sites is already high at birth, increases 2-fold by days 3-7 and thereafter declines to levels already present at birth. In contrast, [3H]strychnine binding sites are weakly expressed at birth and increase up to 7-fold between days 4 and 21. A craniocaudal gradient in the development of glycine receptors is not apparent. However, maturation of [3H]strychnine binding in the ventral horn precedes that in the dorsal horn for 3-4 days. In summary, the developmental expression of these two inhibitory receptors in the spinal cord appears to be regulated differently.     

Stereospecific interaction of bicuculline with specific [3H]strychnine binding to rat spinal cord synaptosomal membranes

The gamma-aminobutyric acid (GABA) antagonist (+/-)-bicuculline inhibits specific [3H]strychnine binding to postsynaptic glycine receptor sites in rat spinal cord synaptosomal membranes with an inhibition constant of about 5 microM, which is fairly similar to its inhibition constant reported for the GABA receptor. This effect is highly stereospecific, since the affinity of (-)-bicuculline for the specific [3H]strychnine binding sites is more than ten times less than that of the pharmacologically active (+)-bicuculline. Besides an unspecific effect at the glycine receptor, the results could suggest that the glycine and the GABA receptors are located close together in spinal cord membranes, so that the antagonist states of both receptors may be able to interfere with each other in some mechanistic way.     

Selective irreversible blockade of 5-hydroxytryptamine1A and 5-hydroxytryptamine1C receptor binding sites in the rat brain by 8-MeO-2'-chloro-PAT: a quantitative autoradiographic study

The possible irreversible blockade of 5-hydroxytryptamine1 receptor subtypes 5-hydroxytryptamine1A, 5-hydroxytryptamine1B/5-hydroxytryptamine1D and 5-hydroxytryptamine1C by the chloramine 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetralin (8-MeO-2'-chloro-PAT) was investigated in rat brain sections by quantitative autoradiography using [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT), [3H]5-hydroxytryptamine, [125I]BH-8-MeO-N-PAT and [125I]cyanopindolol as radio-ligands. A marked reduction (-50% to -75%) of [3H]8-OH-DPAT and [125I]BH-8-MeO-N-PAT specific binding to 5-hydroxytryptamine1A sites in the hippocampus (CA1 area) and the dorsal raphe nucleus, and of [3H]5-hydroxytryptamine specific binding to 5-hydroxytryptamine1C sites in the choroid plexus was found in sections exposed to 1 microM 8-MeO-2'-chloro-PAT and then washed extensively. In contrast the specific binding of [3H]5-hydroxytryptamine to 5-hydroxytryptamine1B/5-hydroxytryptamine1D sites and of [125I]cyanopindolol to 5-hydroxytryptamine1B sites in the substantia nigra and dorsal subiculum remained unaltered by this treatment. Similarly [125I]cyanopindolol binding to beta-adrenergic receptors was not affected by 8-MeO-2'-chloro-PAT. Prior occupancy of 5-hydroxytryptamine1A sites by 10 microM 5-hydroxytryptamine or 8-OH-DPAT, and of 5-hydroxytryptamine1C sites by 10 microM 5-hydroxytryptamine prevented any subsequent blockade by 8-MeO-2'-chloro-PAT. These data indicate that 8-MeO-2'-chloro-PAT should be a useful alkylating agent for achieving selective irreversible blockade of central 5-hydroxytryptamine1A and 5-hydroxytryptamine1C receptors in vivo in the rat.     

Effects of spinal cord lesions and rhizotomies on cholinergic and opiate receptor binding sites in rat spinal cord

Changes in the distribution of [3H]quinuclidinylbenzilate ([3H]QNB), [3H] alpha-bungaro-toxin ([3H]alpha-Btx) and [3H]etorphine binding sites were studied autoradiographically, and cholinacetyltransferase (ChAT) activity radioenzymatically, in the C6-7 segments of rats 1-20 days after combined dorsal and ventral C3-8 rhizotomies and spinal cord lesions at C3. After dorsal and ventral rhizotomies the number of [3H]QNB, [3H]alpha-Btx and [3H]etorphine binding sites were reduced ipsilaterally in the dorsal horn and those of [3H]QNB and [3H]alpha-Btx in the ventral horn. In the ventral horn ChAT activity was significantly reduced. After a unilateral spinal cord lesion at C3, ChAT activity was reduced in the ipsilateral ventral horn at C6-7 caudal to the lesions, whereas no change in receptor binding sites was observed.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [alpha-125I]bungarotoxin and [3H]nicotine

The topographical distribution of [alpha-125I]bungarotoxin [125I]BTX, [3H]nicotine ([3H]Nic), [3H]acetylcholine ([3H]ACh) (in the presence of atropine) binding in rat tel- and diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [3H]ACh and [3H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [3H]Nic were plotted against binding data for [3H]ACh in various brain nuclei, a significant correlation was obtained. Considering [125I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [125I]BTX binding in various brain nuclei and the degree of binding observed with [3H]Nic or [3H]ACh. The present results underline the view that the high-affinity [3H]Nic and [3H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [125I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Autoradiographic distribution of phencyclidine receptors in the rat brain using [3H]1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP)

The distribution of phencyclidine (PCP) receptors in the rat brain was determined by autoradiography using 1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP). [3H]TCP appeared to bind to PCP receptors as only PCP-like drugs and sigma-opioids inhibited the binding of [3H]TCP. The areas of the rat brain with the highest density of radiolabeled binding sites were the superficial layers of cerebral cortex, hippocampus and dentate gyrus. Moderate densities of binding sites were found in the medial geniculate nuclei, caudate nucleus, nucleus accumbens, interpeduncular nucleus, superior colliculus, periaqueductal gray and cerebellum. Low densities of binding sites were observed in spinal cord, most of the brainstem, the substantia nigra and most of the hypothalamus.     

Autoradiographic localization of binding sites for [3H]histamine and H1- and H2-antagonists on cultured neurones and glial cells

By means of autoradiography we have studied the cellular localization of binding of [3H]histamine and H1- and H2-antagonists in explant cultures of rat cerebellum, brain stem and spinal cord. In brain stem and spinal cord cultures, a relatively great number of neurones revealed binding sites for [3H]histamine and to a lesser extent also for the H1-antagonist [3H]pyrilamine and for the H2-antagonist [3H]tiotidine. In contrast, only a small number of labelled neurones was found in cerebellar cultures. The intensity of labelling was usually much stronger for [3H]histamine than for its antagonists, suggesting that binding sites for histamine might reflect both H1- and H2-receptors. Glial cells also showed binding sites for [3H]histamine and the H1- and H2-antagonists, the number of labelled astrocytes by these radioligands was, however, smaller than that observed with [3H]noradrenaline and alpha- and beta-adrenergic antagonists. It is suggested that in addition to alpha- and beta-adrenoceptors, glial cells also possess receptors for histamine.     

Characterization of two distinct 5-HT receptors coupled to adenylate cyclase activation and ion current generation in NCB-20 cells.

The level of cyclic AMP in NCB-20 cells was increased by serotonin (5-HT), 5-methoxytryptamine and 2-methyl-5-HT with EC50 of 0.5 +/- 0.1, 1.0 +/- 0.1, 10 +/- 0.1 microM, respectively. The 5-HT-mediated increase of cyclic AMP content was completely blocked by metergoline but unaffected by 5-HT3 antagonists, ICS 205-930, MDL 72222, quipazine and 5-HT2 antagonist, ketanserin. Putative 5-HT1A agonists (8-OH-DPAT, ipsapirone, and buspirone) and 5-HT1B agonists (TFMPP and m-CPP) affected neither basal nor forskolin-dependent cyclic AMP accumulation. Receptor binding studies suggest that NCB-20 cells are devoid of 5-HT1A and 5-HT1B receptor sites. Application of 5-HT onto NCB-20 cells resulted in membrane depolarization by an evoked inward current which displayed rapid desensitization. 5-HT-mediated current had a reversal potential around 0 mV and was potently and reversibly inhibited by ICS 205-930. Our data suggest that in NCB-20 cells the 5-HT3 receptor is involved in the generation of inward currents, while the 5-HT receptor coupled to adenylate cyclase does not seem to correspond to any of the known receptor subtypes.     

Quantitative autoradiography of [3H]norharman [( 3H]beta-carboline) binding sites in the rat brain

The anatomical distribution of [3H]norharman binding sites was determined by quantitative autoradiography in rat brain slices. They are enriched in hypothalamic, thalamic, accumbens and amygdaloid nuclei as well as in hippocampal, neocortical and olfactory-related structures. The distribution pattern differs from that of other previously described receptors or binding sites (e.g. monoamine oxidase, benzodiazepine, tryptamine, 5-hydroxytryptamine receptors (5-HT1A, 5-HT1B, 5-HT1C, 5HT2], which suggests that a unique class of [3H]norharman binding sites exists in the rat brain. The findings are consistent with previous experiments which showed high affinity binding sites for [3H]norharman in rat brain membranes (KD 1.552 nM; autoradiography KD 5.5 nM). A correspondence in the displacing activity of drugs was found for both methods (crude membrane fraction: harman much greater than tryptamine much greater than 5-hydroxytryptamine greater than N-methyl-beta-carboline-3-carboxamide (FG 7142) = diazepam; autoradiography: harman much greater than tryptamine much greater than FG 7142 greater than 5-hydroxytryptamine greater than diazepam). Provided that the binding sites represent functional receptors, the present anatomical findings may explain the biological effects of norharman, e. g. pro-conflict behaviour (limbic-hypothalamic structures), tonic-clonic convulsions (limbic-cortical structures) and alterations of locomotor activity (accumbens nucleus).     

5-HT3 receptors control dopamine release in the nucleus accumbens of freely moving rats

ICS 205-930, a selective and potent 5-HT3 receptor antagonist applied either systemically, or locally into the ventral tegmental area, antagonized the stimulation of dopamine release in the nucleus accumbens, induced by the subcutaneous administration of morphine. These findings, obtained by the use of brain microdialysis in awake freely-moving rats, demonstrate in vivo a functional role of 5-HT3 receptors in the brain. Since stimulation of dopamine release in the nucleus accumbens is a prerequisite for the expression of the rewarding properties of morphine, its suppression by ICS 205-930 suggests a possible application of 5-HT3 receptor antagonists in the treatment of addiction.     

The distribution of serotonin binding sites in the hippocampal region of the rat brain. An autoradiographic study

The distribution of serotonin binding sites was studied in the rat hippocampal region by using contact-film autoradiography after in vitro incubations of brain sections with 5-[3H]hydroxytryptamine, [3H]spiperone, and [3H]ketanserin, respectively. Biochemical studies of the 5-[3H]hydroxytryptamine binding to sections cut through the hippocampal region showed that at saturating concentrations of 5-[3H]hydroxytryptamine (2-2.5 nM) the specific binding was at least 50% of the total. The 5-[3H]hydroxytryptamine binding sites were found to be heterogeneously distributed within the hippocampal region with the highest densities present in the following parts: layers I and II and layers IV through VI of the entorhinal area, the radial layer of the subiculum and subfield CA1 of the Ammon's horn and the molecular layer of the area dentata. Moderate to low densities of binding was observed in layer III of the entorhinal area, the pre- and parasubiculum, the stratum pyramidale of the Ammon's horn, and the granular cell layer of the area dentata. Removal of the 5-hydroxytryptamine nerve terminals by systemic injections of the 5-hydroxytryptamine neurotoxin parachloroamphetamine resulted in no detectable reductions of 5-[3H]hydroxytryptamine binding in any brain region. Lesions of hippocampal cell bodies by intrahippocampal injections of ibotenic acid prevented the binding of 5-[3H]hydroxytryptamine within the area of the cell loss. Comparisons between the distribution of 5-hydroxytryptamine immunoreactive nerve terminals and the 5-[3H]hydroxytryptamine binding sites showed that in some areas of sparse 5-hydroxytryptamine innervation the 5-[3H]hydroxytryptamine binding was close to background (e.g. the pyramidal cell layer, the stratum lucidum) whereas in areas with little 5-[3H]hydroxytryptamine binding (e.g. layer III of the lateral entorhinal area, the presubiculum) a very dense 5-hydroxytryptamine innervation was found. The hippocampal 5-[3H]hydroxytryptamine binding was displaced neither by ketanserin (1 microM) nor by spiperone (1 microM), two drugs that bind to cortical 5-hydroxytryptamine2 receptors in the rat brain. Furthermore, the pattern of hippocampal [3H]spiperone binding differed considerably from that of 5-[3H]hydroxytryptamine. The [3H]ketanserin binding in the hippocampal region did not exceed background levels, except in the hilus of area dentata in the ventral hippocampus and entorhinal layer VI at the same level, where moderate binding was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonergic fibers induce a long-lasting inhibition of monosynaptic reflex in the neonatal rat spinal cord.

The transmitter mechanism of a long-lasting descending inhibition of the monosynaptic reflex was investigated in the isolated spinal cord of the neonatal rat. The monosynaptic reflex elicited by dorsal root stimulation was recorded extracellularly from a lumbar ventral root (L3-L5). Electrical stimulation of the upper thoracic part of the hemisected cord caused an inhibition lasting about 40 s of the monosynaptic reflex. This descending inhibition was markedly attenuated by perfusing the spinal cord with reserpine (1 microM) or 5,7-dihydroxytryptamine (10 microM) for 2-6 h. The perfusion with reserpine (1 microM) for 4 h significantly decreased the contents of 5-hydroxytryptamine, dopamine, and norepinephrine of the neonatal rat spinal cord, whereas the perfusion with 5,7-dihydroxytryptamine (10 microM) for 4 h decreased the contents of 5-hydroxytryptamine and dopamine. The descending inhibition was markedly potentiated by a 5-hydroxytryptamine uptake blocker, citalopram (10 nM), and was blocked by a 5-hydroxytryptamine antagonist, ketanserin (10-100 nM). Application of 5-hydroxytryptamine to the spinal cord induced an inhibition of the monosynaptic reflex, a later part of which was blocked by ketanserin. Ketanserin also moderately blocked inhibitions of the monosynaptic reflex caused by norepinephrine and dopamine. Phentolamine (10 microM) abolished the depressant actions of norepinephrine and dopamine, but did not affect that of 5-hydroxytryptamine or the descending inhibition. These results strongly suggest the involvement of 5-hydroxytryptamine, but not dopamine nor norepinephrine, in the descending inhibition. Besides ketanserin, the descending inhibition was blocked by ritanserin, haloperidol, and pipamperone, which have affinities to 5-hydroxytryptamine2 receptors, and also by spiperone and methiothepin, which are antagonists at both 5-hydroxytryptamine1 and 5-hydroxytryptamine2 receptors (all 1 microM). On the other hand, a 5-hydroxytryptamine1C and 5-hydroxytryptamine2 antagonist, mesulergine (1 microM), and 5-hydroxytryptamine3 antagonists, ICS 205-930 and quipazine (both 1 microM), did not depress either the descending inhibition or the 5-hydroxytryptamine-evoked inhibition of the monosynaptic reflex. The results with these antagonists favor the involvement of 5-hydroxytryptamine2 receptors although the results with mesulergine disagree with this notion. 5-Hydroxytryptamine1 agonists, such as 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and 5-carboxyamidotryptamine, and a 5-hydroxytryptamine3 agonist, 2-methyl-5-hydroxytryptamine, induced a long-lasting inhibition of the monosynaptic reflex, which was blocked by ketanserin whereas a 5-hydroxytryptamine2 agonist, S-(+)-alpha-methyl-5-hydroxytryptamine, evoked a biphasic inhibition, in which only the later component was blocked by ketanserin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for presynaptic 5-hydroxytryptamine3 recognition sites on vagal afferent terminals in the brainstem of the ferret

Antagonists acting at the 5-hydroxytryptamine3 receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3H]granisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetron in the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [3H]granisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5-hydroxytryptamine3 receptors are located on vagal afferent terminals in the ferret brainstem.     

Autoradiographic distribution of the D1 agonist [3H]SKF 38393, in the rat brain and spinal cord. Comparison with the distribution of D2 dopamine receptors

The regional distribution of the specific D1 agonist [3H]SKF 38393 (SKF 38393, 2,3,4,5-tetra-hydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) has been studied autoradiographically in the rat CNS. The binding of [3H]SKF 38393 to striatal sections was saturable, stereospecific, reversible, of high affinity (Kd = 9.9 nM) and partly sodium sensitive; it occurred at a single population of sites and possessed the pharmacological characteristics of the dopamine D1 receptor. The highest levels of [3H]SKF 38393 binding sites were found in the caudate-putamen, nucleus accumbens, olfactory tubercle and substantia nigra. Moderately high concentrations of the [3H]ligand were observed in the amygdala, endopyriform nucleus, nucleus olfactorius anterior, lateral septum, primary olfactory cortex, cerebellum (molecular layer) and spinal cord. An intermediate labelling was found in the thalamus, habenula, subthalamic nucleus, hypothalamus, ventral tegmental area, superior colliculus, hippocampus and cerebral cortex. Moderate levels of [3H]SKF 38393 binding were observed in the globus pallidus and arcuate nucleus. The autoradiographic distribution of [3H]SKF 38393 overlapped with that of [3H]N,n-propylnorapomorphine, a radioligand which labels the D2 dopamine receptors, in a number of dopamine-rich brain areas but there were several areas which exhibited a high density of [3H]SKF 38393 binding sites but undetectable concentrations of [3H]N,n-propylnorapomorphine. Moreover, in the spinal cord, the subregional localization of these [3H]ligands clearly differed. Intrastriatal injection of ibotenic acid caused a large decrease in [3H]SKF 38393 and [3H]N,n-propylnorapomorphine binding in the striatum and provoked a reduction of [3H]SKF 38393 but not [3H]N,n-propylnorapomorphine binding in the substantia nigra confirming the view that nigral D1 but not D2 receptors are located on striatonigral fibres.