Antinociceptive effect of ketanserin in mice: involvement of supraspinal 5-HT2 receptors in nociceptive transmission

The involvement of 5-HT₂ receptors in pain transmission was investigated in mice. Subcutaneous administration of the selective 5-HT₂ receptor antagonist ketanserin produced dose-dependent antinociception in the hot-plate and acetic acid-induced writhing tests withED₅₀ values (95% confidence limit) of 1.51 (1.13–1.89) and 0.62 (0.10–1.40) mg/kg, respectively, but was without any significant effect on the tail-flick test. Pretreatment with the catecholamine depletors 6-hydroxydopamine (2.5 μg, i.c.v.) orα-methyl-p-tyrosine (200 mg/kg, s.c.), or the serotonin synthesis inhibitorp-chlorophenylalanine methylester (200 mg/kg, s.c.), resulted in a significant decrease in the antinociceptive effect of ketanserin. Likewise, intrathecal (i.t.) administration of 1 μg/mouse of idazoxan (anα₂-antagonist), methysergide (mixed 5-HT₁, and 5-HT₂ antagonist) or ketanserin also reversed the antinociceptive effect of s.c. administered ketanserin. The results of this work indicate that 5-HT₂ receptors located supraspinally may inhibit descending nociceptive neurotransmission. In addition, these studies suggest that 5-HT₂ receptors located at the spinal level modulate nociception.     

Intracellular recording of light responses from visually identified ganglion cells in the rabbit retina

In this report electrophysiological recordings were made from fluorescently labeled ganglion cells in the rabbit retina. Using a retinal strip preparation, cells in the ganglion cell layer were stained following a brief application of the fluorescent dye acridine orange to the bathing solution. Through an epifluorescence microscope the tip of a recording microelectrode could be positioned near a cell of interest. Extracellular recordings from ganglion cells showed that good recovery of light responses was obtained following a brief exposure of the retina to fluorescent light (400–440 nm excitation). The rate of recovery, however, depended upon the prevailing background light level. Large acridine orange-stained cell bodies in the peripheral retina were impaled under visual control by micropipette electrodes filled with either Lucifer Yellow or the fluorescent dye pyranine. When stained intracellularly, all possessed an axon identifying them as ganglion cells. The majority (~ 80%) of the cells recorded intracellularly were identified physiologically as either ON-center or OFF-center brisk ganglion cells. The other cells encountered were ON-OFF directionally selective ganglion cells.     

Morphine, 5-HT2 and 5-HT3 receptor antagonists reduce c-fos expression in the trigeminal nuclear complex following noxious chemical stimulation of the rat nasal mucosa

Noxious chemical stimulation of the rat nasal mucosa induces the expression of the immediate early gene c-fos in trigeminal brainstem neurons. In the present study, we applied the irritant mustard oil (1%) into the left nostril of urethane anesthetized rats. Immunohistochemical methods were used to evaluate the expression of Fos protein in the trigeminal subnuclei interpolaris and caudalis and to test the effects of putative analgesics that might depress synaptic transmission in neurons related to nociception. For this purpose, morphine (3 mg/kg and 10 mg/kg), the 5-HT₂ antagonist ketanserin (0.5 mg/kg and 5 mg/kg) and the 5-HT₃ antagonist ICS 205–930 (0.1 mg/kg and 1 mg/kg) were administered intravenously prior to noxious stimulation. Pretreatment with any of the three compounds reduced Fos-like immunoreactivity. The effect of morphine was reversible with naloxone. The reduction of the expression of Fos-like immunoreactivity by exogenous morphine speaks in favour of an opiodergic link in the modulation of orofacial pain in the trigeminal nuclei. The effects of the 5-HT receptor antagonists are most likely mediated via 5-HT₂ and 5-HT₃ receptors located on primary afferent fibres.     

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

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

Expression of 5-HT2A, 5-HT2B and 5-HT2C receptors in the mouse embryo

Expression patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors during mouse embryogenesis were investigated using highly specific monoclonal antibodies. Differential and overlapping spatio-temporal patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor immunoreactivity were observed during active phases of morphogenesis of a variety of embryonic tissues, including neuroepithelia of brain and spinal cord, notochord, somites, cranial neural crest, craniofacial mesenchyme and epithelia, heart myocardium and endocardial cushions, tooth germs, whisker follicles, cartilage and striated muscle. The functional significance of these receptors was tested by exposing headfold stage mouse embryos to different subtype-selective 5-HT(2) receptor antagonists for 2 days in whole embryo culture. The most potent was the pan 5-HT(2) receptor antagonist ritanserin, which has high affinity for the 5-HT(2B) receptor. Ritanserin caused 100% malformed embryos at a dose of 1 microM. The 5-HT(2A/2C) receptor antagonist mianserin also caused a significant number of malformed embryos, but only when used at a 10 fold higher dose (10 microM). Ketanserin, which primarily targets 5-HT(2A) receptors, did not cause a significant number of malformed embryos at any dose tested. Together with previous evidence that 5-HT acts as an important morphoregulatory signal during mouse embryogenesis, present evidence for the early and continued expression of functional 5-HT(2) receptors throughout gestation raises the possibility that psychotropic drugs taken during pregnancy could interfere with developmental actions of 5-HT during prenatal development of neural and non-neural tissues.     

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.     

Cationic modulation of 5-HT2 and 5-HT3 receptors in rat sensory neurons: the role of K, Ca and Mg

The effects of changes in external K⁺, Ca²⁺, and Mg²⁺ concentrations on 5-HT₂- and 5-HT₃ receptor-mediated depolarizations of the resting membrane potential in rat dorsal root ganglion (DRG) cells was studied. In cells exhibiting a 5-HT₂-mediated response, 5-HT and α-methyl 5-HT depolarized the resting membrane potential (RMP) and increased the slope of the current–voltage (I/V) relationship. The equilibrium potential (E_r) for the depolarization was linearly related to the logarithm of the [K⁺]_o, indicating the depolarization resulted from a decrease in resting K⁺ conductance. In a subpopulation of large-diameter acutely dissociated DRG neurons recorded from using the whole-cell patch-clamp configuration, 5-HT produced an inward shift in the current required to hold cells at −60 mV. This inward shift in holding current was associated with a reduction in membrane conductance and reversed near E_k. This data suggests that the 5-HT₂ receptor-mediated depolarization and increase in R_in seen in intact DRG preparation is produced by blockade of an outward K⁺ leak current. Increases in [K⁺]_o reduced the increase in R_in and depolarization induced by 5-HT with 50% inhibition of the depolarization occurring at 8.3 mM of [K⁺]_o. Half-normal Ca²⁺ (1.2 mM) produced a downward shift of the 5-HT concentration–response curve, reducing the maximal response by 40%, with minimal effect on the half-maximal response. Mg²⁺ ions did not affect this 5-HT response. In cells exhibiting a 5-HT₃ receptor response, 5-HT and 2-methyl-5-HT produced depolarization with decreased R_in. The E_r for this depolarizing response (−30.2±1.8 mV) became less negative (−11.5 mV) in 10 mM [K⁺]_o with minimal effect on the amplitude of the depolarization. In Na⁺-free superfusate, the 5-HT-induced depolarization was converted to hyperpolarization. This indicated the 5-HT₃ response increased a mixed Na⁺/K⁺ conductance. Elevated Ca²⁺ or Mg²⁺ markedly reduced the 5-HT₃ response. Incubation with 3.5 mM Ca²⁺ shifted the 5-HT concentration–response curve downward and to the right, decreasing the maximal response by 49% and increasing the EC₅₀ by 10-fold. Elevated Mg²⁺ produced similar effects. In cells where both 5-HT₂- and 5-HT₃-mediated responses could be demonstrated, the elevation of K⁺ or the reduction of Ca²⁺ converted a 5-HT₂ response to a 5-HT₃ response. The above data suggest that elevation of [K⁺]_o or reduction of [Ca²⁺]_o produced by rapid firing rates of sensory neurons will favor the expression of 5-HT₃ responses over 5-HT₂ responses.     

Effects of the 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide in the rat kindling model of epilepsy

The present study was conducted to identify serotonin (5-HT) receptor subtypes involved in the development of amygdala (AM) kindling. We used 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT_1A agonist, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT₂ agonist, both of which were injected subcutaneously 15 min prior to each daily electrical stimulation to the rat AM. Treatment with 8-OH-DPAT (1 mg/kg) slightly suppressed behavioral and electrographic seizure development during the course of kindling. In contrast, DOI (1 mg/kg) strongly facilitated kindling development and reduced the number of stimulations needed to produce generalized seizures. These facilitatory effects of DOI were completely blocked by pretreatment with a 5-HT₂ antagonist ketanserin. The present results suggest that the activation of 5-HT_1A receptors can retard the development of AM kindling, whereas 5-HT₂ receptors play a facilitatory role in this developmental seizure process.     

Phosphoinositide hydrolysis linked 5-HT2 receptors in fibroblasts from choroid plexus

A serotonin (5-HT)-mediated phosphoinositide hydrolysis response was characterized in fibroblasts cultured from rabbit choroid plexus. 5-HT elicited a maximum 8-fold increase in [³H]inositol-phosphate ([³H]IP) formation, while the partial agonists, (+)-lysergic acid diethylamide and (−)-1-(4-bromo-2,5-dimethyoxyphenyl)-2-aminopropane caused 2- and 5-fold increases, respectively. Mianserin, ketanserin, and spiperone were equipotent at blocking the 5-HT-mediated response. Thus, agonist and antagonist profiles indicate interactions with 5-HT₂ receptors.     

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.     

Selective abolition of OFF responses in kainic acid-lesioned chicken retina

When ganglion cell responses were recorded from optic axons in the superficial layers of the chicken optic tectum, the responses recorded are predominantly ON-OFF transient, with some ON transient, and rare OFF transient responses. Several weeks after excitotoxic lesion of the retina with 40 nmol of kainic acid injected intravitreally, only ON transient responses could be recorded from the contralateral optic tectum. ON response latency and threshold were not affected. At low light intensities responses in the kainic acid-lesioned retinas showed a sustained component which was not detected in control retinas, but at high light intensities, the sustained component disappeared and the responses were extremely transient. The disappearance of the OFF responses seems to be due to elimination of the OFF component of the responses of cells which are normally ON-OFF transient, rather than the silencing of these cells, leaving only the normally ON transient cells. Morphological evidence suggests that approximately two thirds of the bipolar cells and most amacrine cells are destroyed by the kainic acid lesion (Ingham and Morgan, Neuroscience, 9 (1983) 165-181), and pharmacological logic (Morgan, Prog. Retinal Res., 2 (1983) 247-266) suggests that the missing bipolar cells should be OFF bipolar cells. These results therefore suggest that ON-OFF transient cells receive direct input from bipolar cells, which determines their basic response type. These results also suggest that amacrine cells have little if any role to play in the generation of the basic centre responses of these ON-OFF transient ganglion cells, and that while amacrine cells may have a role in the generation of transient responses in the inner plexiform layer, transient responses can be generated without the intervention of amacrine cells, particularly at high intensities.     

Pharmacology of the GABAB receptor in amphibian retina

Amacrine and ganglion cells in the amphibian retina contain GABA_B, as well as GABA_A, receptors. Baclofen, a GABA_B agonist, hyperpolarizes the dark membrane potential of these third order neurons and makes their light responses more transient. GABA_B receptors in the retina have a similar agonist profile to GABA_B receptors described at other sites in the brain. Namely, preferential activation by the R-enantiomer of baclofen, and agonist sensitivity in the order 3-aminopropylphosphinic acid > baclofen > 3-aminopropylphosphonic acid. The GABA_B receptor was not activated by 4-aminobutylphosphonic acid. Several antagonists, such as phaclofen, saclofen, and 2-hydroxysaclofen, were ineffective in the amphibian retina. However, CGP35348 blocked the action of applied baclofen and produced effects on the light response that were opposite to those of baclofen. Applied agonists and antagonists support the hypothesis that GABA_Breceptors serve to regulate the balance of sustained and transient signals to the inner retina.     

Reduction in opioid and non-opioid forms of swim analgesia by 5-HT2 receptor antagonists

Acute exposure to continuous (CCWS) or intermittent (ICWS) cold-water swims elicits non-opioid and opioid forms of analgesia respectively. Intrathecal administration of methysergide blocks ICWS, but not CCWS analgesia. The present study evaluated the role of serotonin (5-HT) receptor subtypes in the mediation of CCWS and ICWS analgesia on the tail-flick and jump tests following administration of methysergide, a non-specific 5-HT antagonist and pirenpirone and ketanserin, two 5-HT₂ receptor subtype antagonists. Systemic methysergide was more effective in reducing CCWS analgesia (50–58%, 0.1–1.0 mg/kg) than ICWS analgesia (21%, 5 mg/kg) on both pain tests. Systemic pirenpirone (0.04–0.2 mg/kg) and ketanserin (1–5 mg/kg) were also more effective in reducing CCWS analgesia (43–57%) on both tests than ICWS analgesia (pirenpirone: 0.4 mg/kg, 34%; ketanserin: 5 mg/kg, 21%) on the tail-flick test. Indeed, both 5-HT₂ receptor antagonists potentiated ICWS analgesia on the jump test. While serotonin antagonist effects upon hypothermia could not account for CCWS analgesia effects, similar potentiations in ICWS analgesia and hypothermia were observed following pirenpirone and ketanserin. Finally, both 5-HT₂ receptor antagonists differentially reduced CCWS hypothermia and potentiated ICWS hypothermia. These data suggest differential serotonergic modulation of the two forms of swim analgesia with opioid-mediated ICWS analgesia acting through spinal 5-HT₁ receptors and non-opioid-mediated CCWS analgesia acting through supraspinal 5-HT₂ receptors.     

Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myo-inositol reduces serotonin (5-HT2) receptor induced homologous and heterologous desensitization

The effect of myo-inositol was examined on 5-HT₂ receptor mediated facilitation of NMDA depolarization of rat neocortical neurons in vitro. Myo-inositol (1–10 mM) potentiated the 5-HT facilitation, the potentiation increasing linearly with log 5-HT concentration. Myo-inositol also eliminated 5-HT induced heterologous desensitization of muscarinic andα₁-adrenergic receptor mediated facilitation. Our findings suggest that 5-HT induced homologous and heterologous desensitization results in part from depleting phosphoinositide substrate.     

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.     

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

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

Immunocytochemical staining of cholinergic amacrine cells in rabbit retina

Cholinergic neurons of rabbit retina were labelled with an antibody against choline acetyltransferase, the synthesizing enzyme for acetylcholine. Two populations of cells are immunoreactive. Type a cell bodies lie in the inner nuclear layer (INL), their dendrites branching narrowly in sublamina a of the inner plexiform layer (IPL), while type b cell bodies lie in the ganglion cell layer (GCL) with dendrites branching in sublamina b of the IPL. The irregular networks of clustered immunoreactive dendrites are similar, but not identical, in the two sublaminae. Type b cells are more numerous than type a cells in central retina. No axons were stained. It appears that the immunoreactive neurons are normally placed and displaced starburst/cholinergic amacrine cells.