Michael R. Prow

[3H]Nisoxetine—A radioligand for noradrenaline reuptake sites: Correlation with inhibition of [3H]noradrenaline uptake and effect of DSP-4 lesioning and antidepressant treatments

Nisoxetine is a potent and selective inhibitor of noradrenaline uptake into noradrenergic neurones. [3H]Nisoxetine binding to rat frontal cortical membranes was of high affinity. The binding data of both competition and saturation studies fitted a single site binding model. [3H]Nisoxetine binding was potently inhibited by the selective noradrenaline uptake inhibitors desipramine and protriptyline. In addition, a very good correlation was obtained between the ability of 25 monoamine reuptake inhibitors and related compounds both to inhibit [3H]nisoxetine binding and to inhibit [3H]noradrenaline uptake in rat frontal cortex. DSP-4 (10-100 mg/kg, i.p.) dose-dependently depleted cortical noradrenaline concentrations (51-100%), with no significant effects on 5-HT and dopamine. These depletions, which were used as a marker of loss of noradrenergic nerve terminals, were associated with a dose-dependent decrease in the number of [3H]nisoxetine binding sites (20-97%) with no change in binding affinity. Furthermore, a good correlation was obtained between cortical noradrenaline concentrations and the number of [3H]nisoxetine binding sites. These data support the view that [3H]nisoxetine binds to a single population of homogeneous sites associated with the noradrenaline transporter complex. Using this ligand, the effects of repeated administration of both antidepressant drugs with a range of pharmacological actions and of electroconvulsive shock on noradrenaline reuptake sites were examined. The number and affinity of [3H]nisoxetine binding sites were unaltered by all treatments. It is unlikely, therefore, that antidepressant therapy would produce adaptive changes in noradrenaline uptake sites.

Characterization of 8-OH-DPAT-induced hypothermia in mice as a 5-HT1A autoreceptor response and its evaluation as a model to selectively identify antidepressants.

1 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) dose‐dependently induced hypothermia in mice. 2 The 5‐HT1A receptor partial agonists, buspirone, gepirone and ipsapirone, also dose‐dependently induced hypothermia. 3 The 8‐OH‐DPAT temperature response was antagonized by the 5‐HT1 receptor antagonists quipazine (2 mg kg−1, i.p.), (±)‐propranolol (10 mg kg−1, i.p.). (±)‐pindolol (5 mg kg−1, i.p.), spiroxatrine (0.5 mg kg−1, i.p.) and metitepine (0.05 mg kg−1, i.p.), but not by 5‐HT2 (ketanserin) or 5‐HT3 (MDL 72222, GR 38032F) receptor antagonists. 4 The response was also antagonized by the dopamine D2 receptor antagonists, haloperidol and BRL 34778. No other catecholamine or muscarinic receptors were involved in mediating the response. 5 Destruction of 5‐hydroxytryptamine (5‐HT)‐containing neurones with the neurotoxin, 5,7‐dihydroxytryptamine (75 μg, i.c.v.), abolished the response to 8‐OH‐DPAT indicating that the 5‐HT1A receptors involved were located on 5‐HT neurones. 6 Chronic antidepressant treatment down‐regulated this 8‐OH‐DPAT response. In addition, chronic administration of anxiolytics and neuroleptics was also effective in this respect. Down‐regulation was also observed following repeated administration of 8‐OH‐DPAT (0.5 mg kg−1, s.c.), (±)‐pindolol (10 mg kg−1, i.p.) and ketanserin (0.5 mg kg−1, i.p.). 7 In conclusion, these data confirm that 8‐OH‐DPAT‐induced hypothermia is mediated by 5‐HT1A autoreceptors. They also indicate that the response involves D2 receptors. The present study also shows that a wide range of antidepressant drugs down‐regulate this response although this property is not restricted to antidepressant treatments. Therefore, care should be exercised when interpreting data from this paradigm.

A comparison of the effects on central 5-HT function of sibutramine hydrochloride and other weight-modifying agents

1 Effects on 5‐HT function of sibutramine and its active metabolites, BTS 54 354 and BTS 54 505, were compared with fluoxetine, (+)‐fenfluramine and (+)‐amphetamine. 2 In vitro sibutramine weakly inhibited [3H]‐5‐HT uptake into brain synaptosomes. BTS 54 354, BTS 54 505 and fluoxetine were powerful [3H]‐5‐HT uptake inhibitors, whereas (+)‐fenfluramine and (+)‐amphetamine were very much weaker. Conversely, whilst sibutramine, its metabolites and fluoxetine did not release [3H]‐5‐HT from brain slices at 10−5M, (+)‐fenfluramine and (+)‐amphetamine concentration‐dependently increased [3H]‐5‐HT release. 3 Sibutramine and fluoxetine had no effect on 5‐hydroxytryptophan (5‐HTP) accumulation in either frontal cortex or hypothalamus at doses <10 mg kg−1. In contrast, (+)‐amphetamine (3 mg kg−1) reduced 5‐HTP in hypothalamus, whilst (+)‐fenfluramine (1 mg kg−1) decreased 5‐HTP in both regions. 4 Sibutramine (10 mg kg−1 i.p.) and fluoxetine (10 mg kg−1 i.p.) produced slow, prolonged increases of extracellular 5‐HT in the anterior hypothalamus. In contrast, (+)‐fenfluramine (3 mg kg−1 i.p.) and (+)‐amphetamine (4 mg kg−1 i.p.) induced rapid, short‐lasting increases in extracellular 5‐HT. 5 Only (+)‐fenfluramine (10 mg kg−1) altered 5‐HT2A receptors in rat frontal cortex when given for 14 days, producing a 61% reduction in receptor number and a 18% decrease in radioligand affinity. 6 These results show that sibutramine powerfully enhances central 5‐HT function via its secondary and primary amine metabolites; this effect, like that of fluoxetine, is almost certainly mediated through 5‐HT uptake inhibition. By contrast, (+)‐fenfluramine enhances 5‐HT function predominantly by increasing 5‐HT release. (+)‐Amphetamine, though weaker than (+)‐fenfluramine, also enhances 5‐HT function by release.

Quantification of presynaptic α2-adrenoceptors in rat brain after short-term DSP-4 lesioning

Abstract The relative numbers of pre- and postsynaptic α 2 -adrenoceptors were determined in various rat brain regions after short-term DSP-4 ( N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine) lesioning. For these studies, rats pretreated with zimeldine (10 mg/kg i.p.) were injected with DSP-4 (100 mg/kg i.p.) and were killed either 3 or 15 days later. At the 3 day time-point, DSP-4 treatment produced marked reductions in the noradrenaline content of the cortex (93%), hippocampus (89%), hypothalamus (83%) and cerebellum (92%) with no change in the levels of dopamine or 5-HT. This treatment also decreased the number of α 2 -adrenoceptors labelled with [ 3 H]idazoxan in the cortex (20%), hippocampus (18%), cerebellum (24%) and hypothalamus (39%). Fifteen days after DSP-4 lesioning, the marked reductions of noradrenaline were sustained in the cortex, hippocampus and cerebellum, but there was a considerable reversal of the effect of DSP-4 in the hypothalamus. At this time-point, the decrease in α 2 -adrenoceptors was attenuated in cortex (4%) and cerebellum (0%) and their number was increased in hippocampus (8%) and hypothalamus (7%). Together, the data argue that presynaptic α 2 -adrenoceptors comprise approximately 20% of the total α 2 -adrenoceptors population in the cortex, hippocampus and cerebellum, but about 40% of it in the hypothalamus. Furthermore, they also demonstrate that although the number of presynaptic α 2 -adrenoceptors in rat brain can be determined by the reduction of radioligand-receptor binding shortly after DSP-4 lesioning, this effect is rapidly masked by receptor proliferation in response to noradrenergic denervation.

A comparison of the effects of sibutramine hydrochloride, bupropion and methamphetamine on dopaminergic function: evidence that dopamine is not a pharmacological target for sibutramine.

Sibutramine hydrochloride, a novel monoamine reuptake inhibitor antidepressant, has been studied to determine whether it alters dopaminergic function in the brain. Its effects have been compared with bupropion, a dopamine reuptake inhibitor, and methamphetamine, a dopamine reuptake inhibitor and releasing agent. Sibutramine (0.1–3 mg/kg PO) and methamphetamine (0.3–30 mg/kg PO) both prevented reserpine (0.75 mg/kg IV) ptosis in rats with ED50 values of 0.6 mg/kg and 4.2 mg/kg, respectively. Bupropion (10–100 mg/kg PO) was ineffective against reserpine ptosis. The efflux of [3H]-dopamine from preloaded rat striatal slices was not altered by 10−7–10−5 M concentrations of sibutramine, BTS 54 354, BTS 54 505 (secondary and primary amine metabolites, respectively) or bupropion. In contrast, methamphetamine (10−8–10−4 M) caused a significant concentration-dependent increase in [3H]-dopamine release. Sibutramine (3 mg/kg IP or 6 mg/kg PO) and bupropion (10 mg/kg IP or 430 mg/kg PO) did not alter 3-methoxy-tyramine (3-MT) levels in rat striatum. Striatal 3-MT concentrations were, however, dose-dependently increased by methamphetamine (0.3–10 mg/kg IP or 0.42–4.2 mg/kg PO). Sibutramine (6 mg/kg PO) did not induce circling in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopaminergic neuronal tract. Bupropion (10–100 mg/kg PO) did not induce circling at the lowest dose, but caused increasing ipsilateral rotation at higher doses. Methamphetamine (0.42 or 4.2 mg/kg PO) induced ipsilateral circling with marked effects at the higher dose. In a two-choice lever pressing model using rats trained to discriminated-amphetamine (0.5 mg/kg IP) from saline, sibutramine (0.3–3 mg/kg IP) generalised to the saline lever. Bupropion (3–30 mg/kg IP) generalised tod-amphetamine at the highest dose, while methamphetamine (0.1–5 mg/kg IP) generalised to this lever at doses as low as 0.3 mg/kg. Overall, the rank order of potency for enhancing central dopaminergic function is methamphetamine > bupropion >> sibutramine. The data therefore indicate that dopamine is unlikely to be an important pharmacological target for reuptake inhibition by sibutramine.

Clonidine-induced hypoactivity and mydriasis in mice are respectively mediated via pre- and postsynaptic α2-adrenoceptors in the brain

Since brain alpha 2-adrenoceptors occur both pre- and postsynaptically, experiments were carried out to determine the synaptic locations of those receptors mediating clonidine-induced hypoactivity and mydriasis. Intraperitoneal (i.p.) injection of clonidine (1-3000 micrograms/kg) to mice dose dependently induced these two responses and also decreased brain concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG). The ED50 values were: 120 micrograms/kg for hypoactivity (95% confidence limits 103-140 micrograms/kg), 54 micrograms/kg for mydriasis (95% confidence limits 40-74 micrograms/kg) and 18 micrograms/kg for MHPG reduction (95% confidence limits 8-36 micrograms/kg) suggesting that these responses could all be presynaptically mediated. However, methamphetamine which increases noradrenaline turnover was found to dose dependently produce mydriasis, but not hypoactivity, after peripheral (0.1-5 mg/kg i.p.) or central (0.5-10 micrograms i.c.v.) injection. The mydriasis produced by methamphetamine (0.5 mg/kg i.p.) was abolished by i.c.v. injection of 1 micrograms idazoxan or yohimbine, but not 2.5 micrograms prazosin or pindolol, showing this effect was mediated by central alpha 2-adrenoceptors. Methamphetamine (1-10 micrograms i.c.v.) potentiated the mydriasis induced by clonidine (50 micrograms/kg i.p.) suggesting this was a postsynaptic alpha 2-adrenoceptor response. By contrast, methamphetamine (1-10 micrograms i.c.v.) dose dependently reversed clonidine (100 micrograms/kg i.p.) hypoactivity indicating this response was mediated by presynaptic alpha 2-adrenoceptors. These hypotheses were confirmed by destruction of noradrenergic neurones using DSP-4 (100 mg/kg i.p. x 2). This treatment prevented the mydriasis response to methamphetamine (0.5 mg/kg i.p.), but not clonidine (100 micrograms/kg i.p.) and markedly attenuated clonidine (100 micrograms/kg i.p.) hypoactivity.

Measurement of 3-methoxy-4-hydroxyphenylglycol (MHPG) in mouse brain by h.p.l.c. with electrochemical detection, as an index of noradrenaline utilisation and presynaptic α2-adrenoceptor function

1 A novel method for measurement of 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) in mouse brain by use of high performance liquid chromatography (h.p.l.c.) with electrochemical detection is described. This technique incorporates an ethyl acetate purification procedure and uses 3‐hydroxy‐4‐methoxyphenylglycol (iso‐MHPG) as the internal standard. 2 Inhibition of monoamine oxidase by injection of tranylcypromine (5 and 10 mg kg−1) or pargyline (50 and 100 mg kg−1) markedly decreased brain MHPG concentrations. After injection of the tyrosine hydroxylase inhibitor, α‐methyl‐p‐tyrosine (200 mg kg−1), there were time‐dependent linear decreases in the concentrations of noradrenaline and MHPG in mouse brain. In addition, a very good correlation (r = 0.95, n = 30; P < 0.001) was found between the concentrations of noradrenaline and MHPG present in the brains of the same mice after α‐methyl‐p‐tyrosine treatment. 3 Mouse brain MHPG concentrations were dose‐dependently reduced after administration of the α2‐adrenoceptor agonist, clonidine (1–3000 μg kg−1), and elevated by the antagonists, idazoxan (1 and 5 mg kg−1), and yohimbine (1 and 5 mg kg−1). Intracerebroventricular injection of the α1‐adrenoceptor agonist, phenylephrine (5–50 μg) dose‐dependently increased MHPG levels. The α1‐adrenoceptor antagonist, prazosin, had no effect at the moderate dose of 1 mg kg−1, but increased MHPG concentrations at 5 mg kg−1. The β‐adrenoceptor agonist, clenbuterol (10–1000 μg kg−1) and the antagonist, pindolol (1 and 5 mg kg−1), were both without effect. 4 The decrease in brain MHPG concentrations induced by clonidine (100 μg kg−1) was prevented by prior injection of 1 mg kg−1 of idazoxan or yohimbine, but not by prazosin or pindolol. 5 MHPG levels were decreased after administration of the noradrenaline reuptake inhibitor desipramine (5 and 10 mg kg−1) and the non‐selective monoamine reuptake inhibitors, sibutramine HCl (BTS 54 524; 1 and 3 mg kg−1) and amitryptyline (5 mg kg−1). However, the selective 5‐hydroxytryptamine reuptake inhibitor, zimeldine (5 and 10 mg kg−1), was without effect. Dexamphetamine (1 and 5 mg kg−1) and methamphetamine (1 and 5 mg kg−1) both decreased brain MHPG concentrations in a dose‐related fashion. 6 Overall the data show that MHPG can be used as a functional index of both presynaptic α2‐adrenoceptor activity and noradrenaline turnover and utilisation.

8-OH-DPAT-induced mydriasis in mice: a pharmacological characterisation.

8-Hydroxy(di-n-propylamino)tetralin (8-OH-DPAT; 0.1-50 mg/kg i.p.) evoked a dose-dependent mydriatic response in conscious mice (ED50 = 5.8 mg/kg i.p.) which was maximal after 10 min. 8-OH-DPAT (2 mg/kg i.p.)-induced mydriasis was attenuated by the alpha 2-adrenoceptor antagonists, idazoxan (1 and 3 mg/kg i.p.) and yohimbine (1 and 3 mg/kg i.p.), by the 5-HT1 receptor antagonists, pindolol (10 mg/kg i.p.) and quipazine (2 mg/kg i.p.), and by the selective 5-HT1A receptor antagonist, (-)-N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenyl propionamide ((-)-WAY 100135; 1-10 mg/kg s.c.). These data argue that both central alpha 2-adrenoceptors and 5-HT1A receptors are involved in the mediation of mydriasis induced by 8-OH-DPAT. The synaptic location of these receptors was determined using either N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 100 mg/kg i.p.) or 5,7-dihydroxytryptamine (5,7-DHT; 75 micrograms i.c.v.)+p-chlorophenylalanine (PCPA; 200 mg/kg i.p.); these lesioning procedures respectively produced highly significant losses of whole brain noradrenaline (72% depletion) and 5-HT (78% depletion). The former abolished 8-OH-DPAT (5 mg/kg i.p. (ED50)) mydriasis, whereas the latter was without effect. 8-OH-DPAT (0.5-5 mg/kg i.p.) also dose-dependently increased the noradrenaline metabolite, 3-methoxy-4-hydroxy-phenylglycol (MHPG), in mouse whole brain minus cerebellum. Taken together these results show that 8-OH-DPAT initially stimulates 5-HT1A receptors, and it is likely that this is followed by release of noradrenaline onto postsynaptic alpha 2-adrenoceptors, the latter effect being responsible for the mydriatic response.

The effects of the peptide-coupling agent, EEDQ, on 5-HT2A receptor binding and function in rat frontal cortex.

This ex vivo study in rat frontal cortex determined the influence of 5-HT receptor agonists and antagonists on EEDQ-induced depletion of 5-HT2A binding sites and reduction in their functional coupling to phospholipid hydrolysis. Twenty-four hours after EEDQ (6 mg/kg) administration a marked reduction (66%) of cortical 5-HT2A binding sites with no change in binding affinity was observed. The 5HT2A antagonists ritanserin (1 mg/kg), ketanserin (1 and 5 mg/kg), metergoline (3 mg/kg) or the 5HT2A agonist, DOI (3 and 10 mg/kg) also significantly reduced (by 15-44%) these binding sites 24 h after injection. Thirty minute pretreatment with ritanserin, ketanserin, metergoline or DOI (at the doses above) afforded 49-65% protection against the loss of 5-HT2A binding sites induced by EEDQ (6 mg/kg). DOI (10 mg/kg) pretreatment (-24 h) decreased by 26% the accumulation of [3H]inositol phosphates (IPs) evoked by 5-HT (100 microM), but did not affect that produced by DOI (100 microM). Ketanserin (5 mg/kg, -24 h) decreased 5-HT- and DOI-induced IP formation by 65% and 53%, respectively. The EEDQ (6 mg/kg, -24 h)-evoked reductions (-50%) of 5-HT- and DOI-induced IP formation were not altered by DOI (10 mg/kg) or ketanserin (5 mg/kg) given 30 min before EEDQ. G-protein-stimulated IP accumulation was unaffected by EEDQ (6 mg/kg). Overall, EEDQ reduces 5-HT2A binding sites and function in rat frontal cortex, whereas its effects on binding were attenuated by various 5-HT receptor antagonists and agonists, its effects on function was unaltered by these drugs.

Receptor binding and functional evidence suggest that postsynaptic α2-adrenoceptors in rat brain are of the α2D subtype

This study has determined the subtype(s) of postsynaptic α2-adrenoceptors in rat brain. This question has been addressed by using two separate approaches, i.e. ligand displacement of [3H]2-(2-methoxy)-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX 821002) from membranes prepared from rat cortex after noradrenergic denervation and, secondly, by antagonism of clonidine-induced mydriasis. After rats had been lesioned using N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 100 mg/kg i.p., 30 min after zimeldine 10 mg/kg i.p.), noradrenaline was undetectable in the cortex 3 days later. Displacement of [3H]RX 821002 with a range of agonists and antagonists which distinguish between the known α2-adrenoceptor subtypes (α2A–2D) yielded pKi values which correlated very well with reported values for the α2D-adrenoceptor (r = 0.929; P < 0.001), but not the α2A (r = 0.450; P = 0.192), α2B (r = 0.280, P = 0.434) or α2C (r = 0.283; P = 0.460) subtypes. Similarly, the potencies of various α2-adrenoceptor antagonists to inhibit clonidine (0.03 mg/kg i.p.)-induced mydriasis in conscious rats correlated strongly with their pKi values for α2D-adrenoceptors (r = 0.899; P = 0.015) but not α2A- (r = 0.369; P = 0.472), α2B- (r = −0.224; P = 0.670) or α2C-adrenoceptors (r = 0.253; P = 0.584). These data are, therefore, consistent and argue strongly that postsynaptic α2-adrenoceptors in the rat cortex and Edinger-Westphal nucleus are of the α2D subtype.