Peter H. Hutson

Low doses of the putative serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) elicit feeding in the rat

The effects of the putative serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on food intake in non-deprived male rats were investigated. Low doses of 8-OH-DPAT (15–60 μg/kg) significantly increased food intake, without affecting drinking, grooming, rearing or locomotion. Microstructural analysis of the elicited feeding behaviour revealed that the rate of eating after 8-OH-DPAT treatment was very similar to that previously reported following 16 h food deprivation. Higher drug doses (250–4,000 μg/kg) also elicited feeding and caused locomotor stimulation and serotonin-related stereotyped behaviour (i.e. forepaw padding, headweaving, wet dog shakes, flat body posture). When feeding and stereotypy were observed concurrently, response competition was evident and feeding behaviour was fragmented into numerous short eating bouts. As drug-induced stereotypy declined with time, this fragmented pattern of eating was succeeded by long bouts of eating which were similar to those observed at doses of 15–60 μg/kg 8-OH-DPAT. The induction of feeding by a serotonin agonist appears paradoxical, since drugs which enhance brain serotonergic activity usually inhibit feeding.

Neurochemical and behavioural evidence for mediation of the hyperphagic action of 8-OH-DPAT by 5-HT cell body autoreceptors.

Administration of 60 micrograms/kg s.c. of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), a dose previously shown to cause hyperphagia in satiated rats (but not to cause the 5-HT behavioural syndrome) decreased 5-HIAA and 5-HIAA/5-HT ratio in several brain regions, the most marked effects being in pons + medulla oblongata, a region containing 5-HT cell bodies and ascending 5-HT axons. Micro-infusion of 8-OH-DPAT (250 and 500 ng) into the dorsal or medial raphe nuclei significantly increased food intake and feeding duration but did not produce the 5-HT behavioural syndrome. Results suggest that 8-OH-DPAT induced hyperphagia is mediated via a agonist action on somatodendritic 5-HT autoreceptors.

Hippocampal 5-HT synthesis and release in vivo is decreased by infusion of 8-OHDPAT into the nucleus raphe dorsalis

Infusion of the serotonin-1A (5-HT1A) agonist, 8-hydroxy-2-di(n-propylamino)tetralin (8-OHDPAT) (0.5, 1.0 and 2.0 micrograms) into the dorsal raphe, decreased extracellular 5-HT as measured by dialysis in the ventral hippocampus and also decreased 5-HT synthesis in both the hippocampus and the rest of the brain as measured by 5-hydroxytryptophan (5-HTP) accumulation following decarboxylase inhibition by NSD 1015.

Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression

The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression.

Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of “ischaemia” (glucose‐free Krebs‐bicarbonate medium gassed with N2/CO2 on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20‐fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia‐induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 mM) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia‐induced release do not involve an initial release of K or an influx of calcium.

Anxiogenic properties of an inverse agonist selective for α3 subunit‐containing GABAA receptors

1 α3IA (6‐(4‐pyridyl)‐5‐(4‐methoxyphenyl)‐3‐carbomethoxy‐1‐methyl‐1H‐pyridin‐2‐one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype. 2 α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15‐1788 in vivo binding assay. 3 At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. α1‐containing receptors), α3IA (30 mg kg−1 i.p.), like the nonselective partial inverse agonist N‐methyl‐β‐carboline‐3‐carboxamide (FG 7142), not only caused behavioural disruption in an operant, chain‐pulling assay but was also anxiogenic in the elevated plus maze, an anxiogenic‐like effect that could be blocked with the benzodiazepine antagonist Ro 15‐1788 (flumazenil). 4 Neurochemically, α3IA (30 mg kg−1 i.p.) as well as FG 7142 (15 mg kg−1 i.p.) increased the concentration of the dopamine metabolite 3,4‐dihydroxyphenylacetic acid in rat medial prefrontal cortex by 74 and 68%, respectively, relative to vehicle‐treated animals, a response that mimicked that seen following immobilisation stress. 5 Taken together, these data demonstrate that an inverse agonist selective for GABAA receptors containing an α3 subunit is anxiogenic, and suggest that since α3‐containing GABAA receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic.

Monitoring the Effect of a Tryptophan Load on Brain Indole Metabolism in Freely Moving Rats by Simultaneous Cerebrospinal Fluid Sampling and Brain Dialysis

Abstract: Rats were given L‐tryptophan, 50 mg/kg i.p., and its concentration in the CNS was monitored in individual freely moving animals using repeated sampling of cisternal CSF and concurrent striatal dialysis. The 5‐hydroxytryptamine metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) was also measured. Results were compared with changes of central tryptophan and 5‐HIAA concentrations in brains of rats killed at various times after administration of L‐tryptophan, 50 mg/kg i.p. Tryptophan changes in CSF were proportionate to those in whole brain and followed essentially identical time courses. Results for the striatal dialysate and whole striatum also paralleled each other. Similarly, results for 5‐HIAA showed proportionality between CSF and brain and between dialysate and striatum. The data obtained were used to determine pharmacokinetic data for individual rats, i.e., areas under curves for both tryptophan and 5‐HIAA and half‐lives for the decline of tryptophan. Kinetic parameters varied considerably from rat to rat. However, mean half‐lives for tryptophan in CSF, brain, dialysate, and striatum were all comparable. Results in general show the value of repeated CSF sampling and intracerebral dialysis for concurrent monitoring of changes of indole metabolism in the whole brain and a specific brain region, respectively. The methods should be suitable for the continuous monitoring of changes of central transmitter metabolism in parallel with observation of behavior following environmental or dietary changes or drug administration. They also should be of use in the investigation of drug kinetics in the CNS.

Evidence that the hyperphagic response to 8-OH-DPAT is mediated by 5-HT1A receptors

The 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) at a dose of 1 mg/kg s.c. increased food intake in free feeding rats. 8-OH-DPAT-induced feeding was blocked by metergoline which has comparable affinity for 5-HT1A, 5-HT1B, 5-HT1C and 5-HT2 receptors. This is consistent with the hyperphagia being mediated by an action at 5-HT receptors. Evidence against the involvement of 5-HT2 or 5-HT3 receptors was provided by the lack of effect of methysergide, ketanserin, MDL 72222 and ICS 205930 on the feeding response. Blockade of the hyperphagia by (-)- but not (+)pendolol which stereoselectively interacts with 5-HT1 receptors indicated an involvement of this receptor type. The lack of effect of ketanserin suggests that the 5-HT1C site is not involved as it has high affinity for both 5-HT2 and 5-HT1C receptors. Blockade of the hyperphagia by spiperone suggests mediation by 5-HT1A rather than 5-HT1B receptors. Although spiperone also blocks dopamine and alpha 2-adrenoreceptors, involvement of these sites is unlikely as neither the DA antagonist haloperidol nor the alpha 2-adrenoceptor antagonist idazoxan blocked 8-OH-DPAT-induced feeding. These results indicate that 8-OH-DPAT-induced feeding is mediated by 5-HT1A receptors.

Para-chlorophenylalanine prevents feeding induced by the serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT)

The effects of para-chlorophenylalanine pre-treatment (PCPA, 150 mg/kg IP daily for 3 days) on feeding and stereotyped behaviour elicited by the serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) in rats were investigated. PCPA depleted brain serotonin and 5-hydroxyindoleacetic acid concentrations by 90% and increased feding during a 2-h day-time test. 8-OH-DPAT (60–4000 μg/kg SC) increased food intake in control animals but decreased in in PCPA-treated animals during the 2-h test. PCPA treatment had no effect on 8-OH-DPAT-induced locomotion or serotonin-related stereotyped behaviour (i.e. forepaw treading, headweaving, wet dog shakes, etc). Since PCPA prevents the operation of pre-synaptic serotonergic mechanisms, the failure of 8-OH-DPAT to increase food intake in PCPA-treated rats suggests that 8-OH-DPAT-induced hyperphagia is autoreceptor mediated.

Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes

The modulation of [3H]ACh release by nicotinic compounds was studied in superfused rat hippocampal synaptosomes loaded with [3H]choline. (−)-Nicotine (0.1–10 μM) evoked a dose-dependent increase in [3H]ACh release; higher concentrations were less effective. Nicotine-evoked release was Ca2+-dependent, and blocked by the nicotinic antagonists dihydro-β-erythroidine, mecamylamine, and pempidine. The α7-selective antagonist methyllycaconitine did not inhibit nicotine-evoked release when tested at 1 μM, although at 10 μM some attenuation of the response was observed. Six agonists tested were equally efficacious in stimulating [3H]ACh release, as judged by the maximum responses, and gave the following EC50 values: (±)-epibatidine 0.12 μM; (+)-anatoxin-a 0.14 μM; (−)-nicotine 0.99 μM; (−)-cytisine 1.06 μM; ABT-418 2.6 μM; isoarecolone 43 μM. Each agonist generated a “bell-shaped” dose response curve, suggesting desensitisation at higher concentrations. This is supported by analysis of repetitive stimulation with (−)-nicotine and (−)-cytisine: S2/S1 ratios declined sharply with increasing concentration, whereas subsequent KCl-evoked release remained constant. These results are discussed in terms of possible nicotinic receptor subtypes that might be present on hippocampal nerve terminals.