Ewen MacDonald

Gene targeting — homing in on α2-adrenoceptor-subtype function

Abstract The α 2 -adrenoceptor was subdivided into three subtypes: α 2A -, α 2B - and α 2C -adrenoceptors almost ten years ago. Since then, the search has been on to discover and develop subtype-selective agonists and antagonists, but as yet no major breakthrough has been made. In the past year, several strains of genetically engineered mice have become available, either overexpressing, totally lacking or expressing heavily modified α 2 -adrenoceptor subtypes. Ewen MacDonald, Brian Kobilka and Mika Scheinin describe how these mice may be utilized to elucidate the physiological functions of the receptor subtypes and the properties of future subtype-selective drugs.

Enhanced BDNF Signaling is Associated with an Antidepressant-like Behavioral Response and Changes in Brain Monoamines

Summary1.Neurotrophins and serotonin have both been implicated in the pathophysiology of depression and in the mechanisms of antidepressant treatments.2.Brain-derived neurotrophic factor (BDNF) influences the growth and plasticity of serotonergic (5-HT) neurons via the activation of trkB receptor.3.Transgenic mice overexpressing the full-length trkB receptor (TrkB.TK+) and showing increased trkB activity in brain, and their wild type (WT) littermates, were injected with the antidepressant fluoxetine or saline, and analyzed behaviorally in the forced swimming test paradigm and biochemically for the concentrations of brain monoamines and their metabolites.4.The TrkB.TK+ mice displayed increased latency to immobility in the forced swim test, suggesting resistance to behavioral despair.5.Fluoxetine increased the latency to immobility in wild-type mice to a similar level as seen in the trkB.TK+ mice after saline treatment, but had no further behavioral effect in the swimming behavior of the trkB.TK+ mice.6.Only minor differences in the levels of brain monoamines and their metabolites were observed between the transgenic and wild-type mice.7.These data, together with other recent observations, suggest that trkB activation may play a critical role in the behavioral responses to antidepressant drugs in mice.

Behavioural and neurochemical effects of antipamezole, a novel α2-adrenoceptor antagonist

Abstract The effects of antipamezole (MPV-1248), a novel selective and specific α 2 -adrenoceptor antagonist, were studied on monoamine metabolism in rat brain and CSF. In addition, the ability of the drug to antagonize the behavioural and neurochemical effects of two α 2 -adrenoceptor agonists, detomidine and medetomidine, was assessed. Atipamezole, 0.03–3.0 mg/kg, had no gross behavioral effects on the rats. Above 3 mg/kg, the rats showed increased vocalization and some hostility, rapid breathing and piloerection. The drug caused dose-dependent, rapid and relatively long-lasting increase in the central turnover of noradrenaline (NA) as reflected by increases in the levels of the major metabolites of NA in brain and CSF and an increase in the depleting effect of α-methyl-para-tyrosine on brain NA levels. An increase in the turnover of serotonin (5-HT) in brain was indicated by a decrease in the concentration of 5-HT and a corresponding increase in the level of its metabolite, 5-hydroxyindoleacetic acid. Atipamezole was able to antagonize the sedative, hypothermic and neurochemical effects of two potent α 2 -agonists, detomidine and medetomidine. These results give support for the characterization of atipamezole as a potent antagonist at central α 2 -adrenoceptors with a rapid onset of action.

Medetomidine — a novel α2-adrenoceptor agonist: A review of its pharmacodynamic effects

Abstract 1. 1. The pharmacodynamic effects of medetomidine, a novel α2-adrenoceptor agonist, are reviewed. 2. 2. In receptor binding experiments, and in isolated organ preparations medetomidine shows high specificity and selectivity to α2-adrenoceptors. Its α 2 α 2 selectivity ratio is 1620 compared to 220 of clonidine. It is a highly potent full agonist at α2-adrenoceptors, a fact that also distinguishes it from clonidine. 3. 3. Medetomidine induces a dose-dependent decrease in the central release and turnover of norepinephrine (NE) measured as changes in metabolite concentrations or using pharmacological intervention techniques. 4. 4. The selectivity, specificity and potency of medetomidine is further supported by various in vivo experiments showing dose-dependent hypotensive, bradycardic, sedative, anxiolytic mydriatic, hypothermic and analgesic effects. 5. 5. The pharmacological, neurochemical and behavioral effects of medetomidine can be inhibited by prior, simultaneous or subsequent administration of selective and specific α2-antagonists. 6. 6. In humans medetomidine is well-tolerated and pharmacodynamic effects including e.g. dose-dependent decrease of vigilance, blood pressure, heart rate, salivary secretion and plasma NE are compatible with an agonistic action at α2-adrenoceptors.

Behavioural and neurochemical effects of medetomidine, a novel veterinary sedative

The effects of the novel veterinary sedative, medetomidine, were studied in rats. In addition to a dose-dependent sedation, which at high doses (greater than 100 micrograms/kg) included loss of the righting reflex and hypothermia, there was a concurrent decrease in the turnover rate of biogenic amines in the brain. Noradrenaline turnover was dose dependently decreased as judged by (i) the decrease in the brain concentration of its metabolite, MHPG-SO4, (ii) a decrease in the ability of alpha-methyl-p-tyrosine methyl ester to deplete brain noradrenaline stores and (iii) a dose-dependent decrease in the level of unconjugated MHPG in the CSF of freely moving rats. Brain dopamine turnover was also inhibited at higher doses as judged by the alpha-methyl-p-tyrosine method and by a decrease in the concentration of HVA in the rat brain 4 h after medetomidine. Serotonin turnover as estimated by the ratio of biogenic amine to its metabolite was also significantly depressed. These changes in brain biogenic amine turnover were inhibited by prior or simultaneous administration of alpha 2-adrenoceptor antagonists, either yohimbine or the more specific, novel alpha 2-antagonist, atipamezole.

Behavioural and neurochemical effects of atipamezole, a novel α2-adreneceptor antagonist

The effects of atipamezole (MPV-1248), a novel selective and specific α2-adrenoceptor antagonist, were studied on monoamine metabolism in rat brain and CSF. In addition, the ability of the drug to antagonize the behavioural and neurochemical effects of two α2-adrenoceptor agonists, detomidine and medetomidine, was assessed. Atipamezole, 0.03–3.0 mg/kg, had no gross behavioural effects on the rats. Above 3 mg/kg, the rats showed increased vocalization and some hostility, rapid breathing and piloerection. The drug caused dose-dependent, rapid and relatively long-lasting increase in the central turnover of noradrenaline (NA) as reflected by increases in the levels of the major metabolites of NA in brain and CSF and an increase in the depleting effect of α-methyl-para-tyrosine on brain NA levels. An increase in the turnover of serotonin (5-HT) in brain was indicated by a decrease in the concentrations of 5-HT and a corresponding increase in the level of its metabolite, 5-hydroxyindoleacetic acid. Atipamezole was able to antagonize the sedative, hypothermic and neurochemical effects of two potent α2-agonists, detomidine and medetomidine. These results give support for the characterization of atipamezole as a potent antagonist at central α2-adrenoceptors with a rapid onset of action.

Central α1-adrenoceptors: Their role in the modulation of attention and memory formation

Adrenoceptors presently are classified into three main subclasses: alpha1-, alpha2-, and beta-receptors, each with three (perhaps more) subtypes. All three alpha1-adrenoceptor subtypes are present in rat brain. The purpose of this review is to assess the role of alpha1-adrenoceptors in the modulation of synaptic transmission and plasticity, as well as their ability to modulate higher cerebral functions, such as attentional and memory processes. However, since there are no truly subtype-specific agonists or antagonists available at present, it is virtually impossible to allocate a particular central effect to one or other of the subtypes. The activation of alpha1-adrenoceptors reduces the firing probability and glutamate release in the cornu ammonis of the hippocampus. Alpha1-Adrenoceptors may flexibly modulate weak and strong activation of the pyramidal neurones in the neocortex. Alpha1-Adrenoceptors play only a minor role in the modulation of long-term potentiation in the hippocampus, and may influence many brain functions also via non-neuronal mechanisms. since glial cells can express alpha1-adrenoceptors. At the behavioural level, the activation of alpha1-adrenoceptors promotes vigilance and influences working memory and behavioural activation, while having only a minor role in the modulation of long-term memory.

α2-Adrenergic drug effects on brain monoamines, locomotion, and body temperature are largely abolished in mice lacking the α2A-adrenoceptor subtype

α2-ARs regulate brain monoaminergic function by inhibiting neuronal firing and release of monoamine neurotransmitters, noradrenaline (NA), serotonin (5-HT) and dopamine (DA). Both α2A- and α2C-AR inhibit monoamine release in vitro in brain slices, but the in vivo roles of individual α2-AR subtypes in modulating monoamine metabolism have not been characterised. Metabolism of brain monoamine neurotransmitters, locomotor activity and body temperature were investigated in mice with targeted inactivation of the gene encoding α2A-AR (α2A-knockout, α2A-KO) and wild-type (WT) mice after treatment with the α2-AR agonist dexmedetomidine and the antagonist atipamezole. Dexmedetomidine caused profound hypothermia (up to 14.7° C mean reduction in rectal temperature) and locomotor inhibition in WT mice, and inhibited the turnover of NA, 5-HT and DA, but increased NA turnover in α2A-KO mice. α2-AR agonist-induced hypothermia and locomotor inhibition were attenuated, but not totally abolished, in α2A-KO mice. These results suggest that α2A-ARs are principally responsible for the α2-AR mediated inhibition of brain monoamine metabolism, but other α2-ARs, possibly α2C-ARs, are also involved, especially in the striatum. However, secondary effects of the physiological alterations caused by drug administration, especially hypothermia, may have contributed to the observed neurochemical changes in WT mice.

Locomotor activity and evoked dopamine release are reduced in mice overexpressing A30P-mutated human α-synuclein

We have generated a transgenic mouse line overexpressing mutated human A30P alpha-synuclein under the control of the prion-related protein promoter. Immunohistology revealed mutated human A30P alpha-synuclein protein in numerous brain areas, but no gross morphological changes, Lewy bodies, or loss of dopaminergic cell bodies. The transgenic mice displayed decreased locomotion, impaired motor coordination, and balance. In vivo voltammetry showed that A30P mice responded to longer stimulation of the ascending dopaminergic pathways with less dopamine release in striatum and had a slower rate of dopamine decline after repeated stimulations or after alpha-methyl-p-tyrosine-HCl treatment. However, dopamine re-uptake or transporter levels were similar in transgenic and control mice. Our data provide evidence that overexpression of mutated human A30P alpha-synuclein in mice leads to a reduced size of the dopamine storage pool. This is in agreement with the previously postulated involvement of alpha-synuclein in the turnover of transmitter vesicles and may explain the observed motor deficits in A30P mice.

Evaluation of the effects of a specific α2-adrenoceptor antagonist, atipamezole, on α1- and α2-adrenoceptor subtype binding, brain neurochemistry and behaviour in comparison with yohimbine

In the present study we evaluated the α1- and α2-adrenoceptor subtype binding, central α2-adrenoceptor antagonist potency, as well as effects on brain neurochemistry and behavioural pharmacology of two α2-adrenoceptor antagonists, atipamezole and yohimbine. Atipamezole had higher selectivity for α2- vs. α1-adrenoceptors than yohimbine regardless of the subtypes studied. Both compounds had comparable affinity for the α2A-, α2C- and α2B-adrenoceptors, but yohimbine had significantly lower affinity for the α2D-subtype. This may account for the fact that significantly higher doses of yohimbine than atipamezole were needed for reversal of α2-agonist (medetomidine) -induced effects in rats (mydriasis) and mice (sedation and hypothermia). The effect on central monoaminergic activity was estimated by measuring the concentrations of transmitters and their main metabolites in whole brain homogenate. At equally effective α2-antagonising doses in the rat mydriasis model, both drugs stimulated central noradrenaline turnover (as reflected by increase in metabolite levels) to the same extent. Atipamezole increased dopaminergic activity only slightly, whereas yohimbine elevated central dopamine but decreased central 5-hydroxytryptamine turnover rates. In behavioural tests, atipamezole (0.1–10 mg/kg) did not affect motor activity but stimulated food rewarded operant (FR-10) responding (0.03–3 mg/kg) whereas yohimbine both stimulated (1 mg/kg) and decreased (≥ 3 mg/kg) behaviour in a narrow dose range in these tests. In the staircase test, both antagonists increased neophobia, but in the two compartment test only yohimbine (≥ 3 mg/kg) decreased exploratory behaviour. The dissimilar effects of the antagonists on neurochemistry and behaviour are thought to be caused by non α2-adrenoceptor properties of yohimbine. In conclusion, the α2-antagonist atipamezole blocked all α2-adrenoceptor subtypes at low doses, stimulated central noradrenergic activity and had only slight effects on behaviour under familiar conditions, but increased neophobia. The low affinity for the α2D-adrenoceptor combined with its unspecific effects complicates the use of yohimbine as pharmacological tool to study α2-adrenoceptor physiology and pharmacology.