Keith Frank Martin

Determination of in vivo amino acid neurotransmitters by high-performance liquid chromatography with o-phthalaldehyde-sulphite derivatisation

The measurement of amino acid neurotransmitters by high-performance liquid chromatography (HPLC) has emerged as a reliable and sensitive method. This paper describes a method which employs electrochemical (EC) detection of amino acid derivatives formed by a reaction with o-phthalaldehyde (OPA) in the presence of sulphite ions. This is discussed in relation to the problems of previously reported methods based on OPA derivatisation. Precise separation of the following 7 amino acid standards is achieved using isocratic elution: serine, glycine, taurine, glutamate, arginine, alanine and GABA, in order of increasing retention time. Total elution time is 25 min. Derivatisation proceeds at room temperature and the derivatives are stable for up to 5 h. This technique has the sensitivity to determine the concentrations of amino acid neurotransmitters in cerebrospinal fluid (CSF) and an in vivo microdialysis method is discussed for the detection of basal and potassium-stimulated levels of gamma-aminobutyric acid and glutamate from rat hippocampus.

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.

Opposing roles for 5-HT1B and 5-HT3 receptors in the control of 5-HT release in rat hippocampus in vivo

1 Intracerebral microdialysis was used to determine whether 5‐hydroxytryptamine (5‐HT) release in the ventral hippocampus of rats anaesthetized with chloral hydrate was modulated by 5‐HT3 receptors. 2 It was confirmed that 5‐methoxy‐3‐(1,2,3,6‐tetrahydro‐4‐pyridinyl)‐1H‐indole (RU 24969), a selective 5‐HT1B receptor agonist, decreased 5‐HT release in a dose‐ and concentration‐related manner when administered i.p. (1 and 5 mg kg−1) or via the dialysis probe (0.1 and 1 μm) respectively. The effect of RU 24969 infusion (1 μm) was attenuated by concurrent infusion of metitepine (10 μm) into the hippocampus. 3 When infused into the hippocampus for 15 min, the selective 5‐HT3 receptor agonist, 2‐methyl‐5‐hydroxytryptamine (2‐methyl‐5‐HT; 0.1–10 μm) increased dialysate 5‐HT levels in a concentration‐related manner; an effect which was abolished by concurrent infusion of 3‐tropanyl‐3,5‐dichlorobenzoate (1 μm, MDL 72222), a selective 5‐HT3 antagonist. 4 MDL 72222 had no effects on hippocampal 5‐HT release when administered via the dialysis probe (1 or 10 μm). 5 The data show that 5‐HT3 and 5‐HT1B receptors have opposing roles in the control of 5‐HT release in the hippocampus, with 5‐HT3 receptors facilitating and 5‐HT1B receptors inhibiting 5‐HT efflux, respectively. They also indicate that the facilitatory 5‐HT3 receptors are not tonically activated.

Measurement of extracellular basal levels of serotonin in vivo using nafion-coated carbon fibre electrodes combined with differential pulse voltammetry

Carbon fibre electrodes combined with differential pulse voltammetry have been used for a number of years to monitor changes in the extracellular concentrations of ascorbic acid, dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid. However, the primary objective of in vivo electrochemists has been to monitor changes in the extracellular concentrations of the neurotransmitter amines; dopamine and serotonin rather than their metabolites. In this paper we describe a new chemically- and electrically-pretreated Nafion-coated carbon fibre electrode which can be used to monitor basal levels of serotonin in the extracellular fluid in the frontal cortex and the dorsal raphe nucleus of rat. These electrodes combined with differential pulse voltammetry detect dopamine (Peak A at -70 mV) and serotonin (Peak B at +240 V) oxidation peaks in vitro but not the oxidation of ascorbic acid, dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid or uric acid, at concentrations up to 10 microM. These electrodes were able to detect serotonin concentration as large as 1 nM in vitro. When used in vivo the oxidation peaks obtained in the frontal cortex and dorsal raphe indicate the basal concentrations of serotonin to be 5 nM and 10 nM respectively. Pharmacological interventions in rats implanted with normal carbon fibre electrodes or with Nafion carbon fibre electrodes further demonstrate that the new Nafion electrodes measure serotonin in vivo. The Nafion-coated electrodes therefore may be a useful tool for the study of serotoninergic systems in vivo with the added advantage that they cause minimal damage due to their small tip size (30 micron).

Mediation of the antidepressant-like effect of 8-OH-DPAT in mice by postsynaptic 5-HT1A receptors.

1 The 5‐hydroxytryptamine (5‐HT)1A agonist 8‐hydroxy‐2‐(dipropylamino)tetralin (8‐OH‐DPAT) has been evaluated in a mouse model for detecting potential antidepressants (Porsolt test). The effects of various receptor antagonists, lesions of brain monoaminergic neurones and chronic drug treatments on this 8‐OH‐DPAT‐induced response have also been determined. 2 8‐OH‐DPAT (0.3–10.0 mg kg−1, s.c.) dose‐dependently increased the mobility of mice in the Porsolt test. Other selective 5‐HT1A receptor ligands (0.3–30 mg kg−1, s.c.) either mimicked the 8‐OH‐DPAT response (ipsapirone, at 10 and 30 mg kg−1, s.c.) or were inactive (buspirone and gepirone). However, each of these compounds (≤ 100 mg kg−1, p.o.) inhibited the response to 8‐OH‐DPAT (3 mg kg−1, s.c.) when given concurrently. 3 The putative 5‐HT1A antagonists, spiroxatrine (1–30 mg kg−1, p.o.), (±)‐pindolol (30 mg kg−1, p.o.) and methiothepin (3–10 mg kg−1, p.o.), each attenuated the 8‐OH‐DPAT (3 mg kg−1, s.c.)‐induced increase in mobility. 4 The dopamine D1 receptor antagonist, SCH 23390 (3–10 mg kg−1, p.o.), weakly reversed the 8‐OH‐DPAT response. Antagonists at 5‐HT1C/5‐HT2 receptors (ketanserin; 0.1–3.0 mg kg−1, p.o.), 5‐HT3 receptors (ondansetron; 0.03–10 mg kg−1, p.o.), α1‐adrenoceptors (prazosin; 1–3 mg kg−1, p.o.), α2‐adrenoceptors (idazoxan; 3–30 mg kg−1, p.o.), β1‐adrenoceptors (metoprolol; 1–30 mg kg−1, p.o.), β2‐adrenoceptors (ICI 118,551; 1–30 mg kg−1, p.o.), dopamine D2 receptors (sulpiride; 10–300 mg kg−1, p.o.) and opiate receptors (naloxone; 3–100 mg kg−1, p.o.) had no effect on the 8‐OH‐DPAT response. 5 Selective destruction of 5‐HT neurones with 5,7‐dihydroxytryptamine or inhibition of 5‐HT synthesis with p‐chlorophenylalanine did not change the 8‐OH‐DPAT response in the Porsolt test. This response was also unaltered by pretreatment with the noradrenergic neurotoxin, DSP‐4. 6 Administration of 8‐OH‐DPAT (3 mg kg−1, s.c.) twice‐daily for 10 days attenuated the hypothermia, but not the increased mobility, induced by 8‐OH‐DPAT (3 mg kg−1, s.c.). Similarly, repeated administration of amitriptyline (3–30 mg kg−1), desipramine (3–30 mg kg−1) or dothiepin (10–100 mg kg−1) also attenuated the former, but not the latter, response. 7 We conclude that 8‐OH‐DPAT produces an antidepressant‐like effect in the Porsolt test which is mediated via postsynaptic 5‐HT1A receptors.

Decreased GABA release following tonic-clonic seizures is associated with an increase in extracellular glutamate in rat hippocampus in vivo

The effects of maximal electroshock, used as a model of generalized seizures, were studied on extracellular GABA and glutamate levels in the ventral hippocampus of the freely-moving rat, using in vivo microdialysis. Following a maximal electroshock there was a rapid decline in GABA levels (46 +/- 5%) in the 20 min immediately after the seizure and levels remained depressed for a further 60 min. However, although there was a transient small decrease (11 +/- 2%) in glutamate levels in the first 20 min post-ictally, there followed a more prolonged, larger increase in the next 40 min. Maximal electroshock, administered in the absence of extracellular calcium, did not change GABA levels, while glutamate levels were again increased (42 +/- 8%) in the 40-80 min after the shock. Local perfusion with nickel (1 mM) to block T-type calcium channels had no effect on basal GABA or glutamate levels but prevented maximal electroshock-induced changes in both amino acids. Experiments were carried out to test the hypothesis that the post-ictal increased glutamate release was due to the decrease in GABA release. Perfusion with the potent GABA re-uptake inhibitor NNC-711, for 60 min prior to administration of maximal electroshock, increased GABA levels (436 +/- 58%) and abolished the seizure-induced decrease. Basal glutamate levels were not affected by perfusion with NNC-711 but subsequent maximal electroshock also failed to affect levels. Local perfusion with the GABAA receptor antagonist bicuculline (1, 10 and 100 microM) had no effect on basal GABA levels but glutamate levels were increased (46 +/- 5%) after perfusion with 100 microM bicuculline.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of phenytoin and sodium valproate on seizure-induced changes in γ-aminobutyric acid and glutamate release in vivo

The effects of intraperitoneal administration of the anticonvulsants phenytoin and sodium valproate were compared with ethosuximide on maximal electroshock seizure-related changes in rat hippocampal gamma-aminobutyric acid (GABA) and glutamate release in vivo as measured by microdialysis. There were immediate increases in GABA and glutamate in the 5 min post-ictal period, followed by a sustained reduction in GABA levels. Glutamate levels, however, were subsequently reduced until 20 min post-ictal before gradually increasing above basal. All animals displayed tonic hind-limb extension that was blocked by phenytoin (20 mg/kg) and sodium valproate (400 mg/kg) but not ethosuximide (150 mg/kg). Phenytoin attenuated the immediate post-ictal increase observed in glutamate whilst sodium valproate enhanced GABA release and prevented its secondary post-ictal inhibition. Ethosuximide was without effect on the post-ictal changes. These are the first data to show detailed seizure-induced amino acid changes and the in vivo effects of anticonvulsants on them in the seizure model.

Diurnal variation in 5-HT1B autoreceptor function in the anterior hypothalamus in vivo: effect of chronic antidepressant drug treatment

Intracerebral microdialysis was used to examine the function of the terminal 5‐hydroxytryptamine (5‐HT) autoreceptor in the anterior hypothalamus of anaesthetized rats at two points in the light phase of the light–dark cycle. Infusion of the 5‐HT1A/1B agonist 5‐methoxy‐3‐(1,2,3,6‐tetrahydro‐4‐pyridyl)‐1H‐indole (RU24969) 0.1, 1.0 and 10 μM through the microdialysis probe led to a concentration‐dependent decrease (49, 56 and 65% respectively) in 5‐HT output. The effect of RU24969 (1 and 5 μM) was prevented by concurrent infusion of methiothepin (1 and 10 μM) into the anterior hypothalamus via the microdialysis probe. Infusion of methiothepin alone (1.0 and 10 μM) increased (15 and 142% respectively) 5‐HT output. Infusion of RU24969 (5 μM) through the probe at mid‐light and end‐light resulted in a quantitatively greater decrease in 5‐HT output at end‐light compared with mid‐light. Following treatment with either paroxetine hydrochloride (10 mg kg−1 i.p.) or desipramine hydrochloride (10 mg kg−1 i.p.) for 21 days the function of the terminal 5‐HT1B autoreceptor was more markedly attenuated at end‐light. The data show that, as defined by the response to RU24969, the function of the 5‐HT1B receptors that control 5‐HT output in the anterior hypothalamus is attenuated following chronic desipramine or paroxetine treatment in a time‐of‐day‐dependent manner.

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.