Clozapine blocks D-amphetamine-induced excitation of dopamine neurons in the ventral tegmental area.

Current antipsychotic drugs are thought to inhibit central dopamine (DA) transmission by blocking DA receptors. Here, we provide evidence that the atypical antipsychotic drug clozapine may produce part of its effect by inhibiting a subset of excitatory inputs to DA neurons. Thus, in chloral hydrate-anesthetized rats, systemic administration of D-amphetamine produced two opposing effects on DA neurons in the ventral tegmental area. Under control conditions, D-amphetamine inhibited the firing of the cell through D2-like receptors. When D2-like receptors were blocked by raclopride, D-amphetamine excited DA neurons, instead of producing no effect. The excitation, expressed as an increase in firing rate and a slow oscillation in firing pattern, was suppressed by the adrenergic alpha1 receptor antagonist prazosin, suggesting an involvement of alpha1 receptors. In rats pretreated with the typical antipsychotic drug haloperidol, D-amphetamine also excited DA neurons. However, when given after clozapine, D-amphetamine produced no significant effects. The failure of D-amphetamine to produce an excitation is not due to an incomplete blockade of D2-like receptors by clozapine because co-treatment with clozapine and raclopride also failed to enable the excitatory effect of D-amphetamine. The suggestion that clozapine inhibits the excitatory effect of D-amphetamine is further supported by the finding that clozapine, given after D-amphetamine, reliably reversed D-amphetamine-induced excitation in raclopride-treated rats. Thus, different from raclopride and haloperidol, clozapine may inhibit DA transmission through two additive mechanisms: blockade of DA receptors and inhibition of an amphetamine-sensitive, excitatory pathway that innervates DA neurons.     

Bombesin potently stimulates water intake in the pigeon

Bombesin, injected intracerebroventricularly, evoked a potent dipsogenic response in the pigeon.The effect was dose-dependent and apparently specific since no other behavioural alteration was ever observed. The findings suggest that endogenous bombesin-like peptides may be involved in the control of water intake in the pigeon.     

Dissociation of haloperidol, clozapine, and olanzapine effects on electrical activity of mesocortical dopamine neurons and dopamine release in the prefrontal cortex.

The aim of the present study was to compare the effects of the typical antipsychotic haloperidol and the atypical antipsychotics clozapine and olanzapine on both extracellular dopamine (DA) levels in the medial prefrontal cortex (mPFC) as well as electrical activity of mesoprefrontal DA (mPFC-DA) neurons. Extracellular single unit recordings and microdialysis experiments were carried out in different groups of chloral hydrate anesthetised rats under identical experimental conditions. Intravenous administration of haloperidol, clozapine, and olanzapine increased the firing rate and burst activity of antidromically-identified mPFC-DA neurons; maximal increase in firing rate of approximately 140, 155, and 70 %, was produced by haloperidol, clozapine, and olanzapine at doses of 0.2, 2.5, and 1 mg/kg, i.v., respectively. Intravenous administration of the same doses increased extracellular DA levels in mPFC by 20%, 190%, and 70%, respectively. Moreover, while haloperidol and olanzapine increased extracellular levels of the deaminated DA metabolite DOPAC, by 60% and 40%, respectively, clozapine was totally ineffective. The D1 receptor antagonist SCH 23390 modified neither DA output nor neuronal firing. To determine whether the effect of the three antipsychotics on DA release might depend on a direct action on the mPFC, rats were perfused locally via inverse dialysis in the mPFC at concentrations ranging from 10(-6) to 10(-4)M. While clozapine and olanzapine increased extracellular DA concentrations by up to 400% of basal level, haloperidol was totally ineffective. The results obtained from this study indicate that the rank potency of the three antipsychotics in stimulating the firing rate of DA neurons projecting to mPFC, correlates with their affinity for D2 receptors and doses used clinically. On the other hand, their stimulating effect on DA release does not correlate with their effect on neuronal firing but depends on a direct action on the mPFC.     

Quinine potently blocks single K+ channels activated by dopamine D-2 receptors in rat corpus striatum neurons

In single channel recordings from acutely dissociated neurons of the rat corpus striatum, a membrane K+ channel which is activated by dopamine D-2 receptors was blocked by nanomolar concentrations of quinine. An intermittent partial blockade was observed at 4-10 nM quinine, with a voltage dependence consistent with quinine binding to the channel near the extracellular surface of the membrane. A nearly complete blockade of channel current was observed at 100 nM quinine and above. Such concentrations are known to be too low to block various other ion channels, and may be attained in human brain at antimalarial dosages of quinine. Blockade of this channel by quinine may provide a useful experimental probe of dopaminergic function, as an alternative to D-2 receptor binding site blockade by neuroleptics.     

The autophagic inhibitor 3-methyladenine potently stimulates PKA-dependent lipolysis in adipocytes

BACKGROUND AND PURPOSE

The class III PI3K inhibitor, 3-methyladenine (3-MA), is commonly used to selectively block autophagy. Recent findings suggest a strong relationship between autophagy and lipid turnover. Here, we explore the effect of 3-MA on adipocyte lipolysis.

EXPERIMENTAL APPROACH

Assays were performed in 3T3-L1 cells. Cells were treated with 3-MA and wortmannin, a pan PI3K and autophagy inhibitor. Pharmacological and genetic manipulation of endogenous autophagic and lipolytic pathways was used to ascertain the contribution of 3-MA to the observed effects on lipolysis.

KEY RESULTS

3T3-L1 cells that were exposed to 3-MA showed a consistent increase in lipolysis, approximately 50% over basal levels. The effect of 3-MA was not secondary to autophagic inhibition as treatment of 3T3-L1 cells with wortmannin yielded no such changes. Dosing and time course experiments showed that 3-MA''s ability to activate lipolysis was more sensitive than its inhibitory effect on autophagy. Knockdown of adipose triglyceride lipase (ATGL) negated the stimulatory effect of 3-MA by >90%, indicating that 3-MA enhanced ATGL-dependent hydrolysis of triacylglycerols. Additionally, the lipolytic effect of 3-MA was dependent on the activation of PKA and 3-MA induced a rapid and potent elevation of intracellular cAMP levels in adipocytes.

CONCLUSIONS AND IMPLICATIONS

Cumulatively, we show that 3-MA potently modulated a cellular mechanism and its underlying signalling pathways not associated with autophagy. Furthermore, we describe a novel stimulatory effect on a major signalling pathway. Our findings provide valuable information to studies employing 3-MA as a specific inhibitor for PI3K and autophagy.     

The ability of the mesocortical dopamine system to operate in distinct temporal modes

Background This review discusses evidence that cells in the mesocortical dopamine (DA) system influence information processing in target areas across three distinct temporal domains. Discussions Phasic bursting of midbrain DA neurons may provide temporally precise information about the mismatch between expected and actual rewards (prediction errors) that has been hypothesized to serve as a learning signal in efferent regions. However, because DA acts as a relatively slow modulator of cortical neurotransmission, it is unclear whether DA can indeed act to precisely transmit prediction errors to prefrontal cortex (PFC). In light of recent physiological and anatomical evidence, we propose that corelease of glutamate from DA and/or non-DA neurons in the VTA could serve to transmit this temporally precise signal. In contrast, DA acts in a protracted manner to provide spatially and temporally diffuse modulation of PFC pyramidal neurons and interneurons. This modulation occurs first via a relatively rapid depolarization of fast-spiking interneurons that acts on the order of seconds. This is followed by a more protracted modulation of a variety of other ionic currents on timescales of minutes to hours, which may bias the manner in which cortical networks process information. However, the prolonged actions of DA may be curtailed by counteracting influences, which likely include opposing actions at D1 and D2-like receptors that have been shown to be time- and concentration-dependent. In this way, the mesocortical DA system optimizes the characteristics of glutamate, GABA, and DA neurotransmission both within the midbrain and cortex to communicate temporally precise information and to modulate network activity patterns on prolonged timescales.     

Cholinergic depletion in nucleus accumbens impairs mesocortical dopamine activation and cognitive function in rats

In rats, selective depletion of the cholinergic interneurons in the ventral striatum (nucleus accumbens or N.Acc.) results in heightened behavioural sensitivity to amphetamine and impaired sensorimotor gating processes, suggesting a hyper-responsiveness to dopamine (DA) activity in the N.Acc. We hypothesized that local cholinergic depletion may also trigger distal functional alterations, particularly in prefrontal cortex (PFC). Adult male Sprague-Dawley rats were injected bilaterally in the N.Acc. with an immunotoxin targeting choline acetyltransferase. Two weeks later, cognitive function was assessed using the delayed alternation paradigm in the T-maze. The rats were then implanted with voltammetric recording electrodes in the ventromedial PFC to measure in vivo extracellular DA release in response to mild tail pinch stress. The PFC was also examined for density of tyrosine hydroxylase (TH)-labelled varicosities. In another cohort of control and lesioned rats, we measured post mortem tissue content of DA.Depletion of cholinergic neurons (restricted to N.Acc.) significantly impaired delayed alternation performance across delay intervals. While (basal) post mortem indices of PFC DA function were unaffected by N.Acc. lesions, in vivo mesocortical DA activation was markedly reduced; this deficit correlated significantly with cognitive impairments. TH-labelled varicosities however, were unaffected in cortical layer V relative to controls. These data suggest that selective depletion of cholinergic interneurons in N.Acc. triggers widespread functional impairments in mesocorticolimbic DA function and cognition. The possible relevance of these findings is also discussed in relation to schizophrenia, where reduced density of cholinergic neurons in ventral striatum has been reported.     

Clozapine concentrations in brain regions: Relationship to dopamine metabolite increase

Levels of clozapine in rat striatum and tuberculum olfactorium were quantitated by a gas chromatographic technique. The relationship of the increase in 3,4-dihydroxyphenylacetic acid (DOPAC) in these regions produced by clozapine to the concentration of clozapine was explored. One hour after 10, 20 or 40 mg/kg clozapine i.p. the concentration of drug increased in proportion to the dose and at each dose was similar in striatum and T.O. The percent increase in DOPAC in both areas was related to the clozapine concentration in a typical dose-response manner and was greater in the striatum than the T.O. A relatively high concentration of clozapine (40 μM) was required to produce a half-maximal elevation of DOPAC. Striatal clozapine levels were similar in acutely and chronically treated animals. The concentrations of clozapine in straitum and T.O. reflect the dose injected and do not account for its atypical properties.     

Clozapine antagonism of D-1 and D-2 dopamine receptor-mediated behaviors

When tested in rats supersensitive to dopamine agonists, the atypical neuroleptic clozapine displayed pharmacological properties expected of both a D-1 and D-2 receptor antagonist. The locomotor response induced by the D-1 receptor agonist SKF-38393 in neonatal-6-hydroxydopamine (6-OHDA)-lesioned rats was reversed in a dose-related fashion, although a complete blockade of this behavior was not observed indicative for only a partial antagonism of D-1 receptor function. Clozapine also blocked the self mutilation resulting from L-dihydroxyphenylalanine (L-DOPA) administration to neonatal-6-OHDA-lesioned rats, an effect previously linked to D-1 receptor activation. At higher doses, clozapine blocked the locomotor activity elicited by the D-2 agonist LY-171555 in adult-6-OHDA-lesioned rats. Therefore, the action of clozapine on D-1 as well as D-2 receptor-mediated behaviors contributes to its pharmacological effects. The ability of clozapine to stop self-mutilatory behavior in neonatal-6-OHDA-lesioned rats suggests that this drug might be an effective treatment for self-injurious behavior associated with the Lesch-Nyhan syndrome and mental retardation.     

Clozapine normalizes prefrontal cortex dopamine transmission in monkeys subchronically exposed to phencyclidine.

The mechanism responsible for the therapeutic effects of the prototypical atypical antipsychotic drug, clozapine, is still not understood; however, there is persuasive evidence from in vivo studies in normal rodents and primates that the ability to elevate dopamine neurotransmission preferentially in the prefrontal cortex is a key component to the beneficial effects of clozapine in schizophrenia. Theoretically, such an effect of clozapine would counteract the deficient dopaminergic innervation of the prefrontal cortex that appears to be part of the pathophysiology of schizophrenia. We have previously shown that following repeated, intermittent administrations of phencyclidine to monkeys there is lowered prefrontal cortical dopamine transmission and impairment of cognitive performance that is dependent on the prefrontal cortex; these biochemical and behavioral changes therefore model certain aspects of schizophrenia. We now investigate the effects of clozapine on the dopamine projections to prefrontal cortex, nucleus accumbens, and striatum in control monkeys and in those withdrawn from repeated phencyclidine treatment, using a dose regimen of clozapine that ameliorates the cognitive deficits described in the primate phencyclidine (PCP) model. In normal monkeys, clozapine elevated dopamine turnover in all prefrontal cortical, but not subcortical, regions analyzed. In the primate PCP model, clozapine normalized dopamine (DA) turnover in the dorsolateral prefrontal cortex, prelimbic cortex, and cingulate cortex. Thus, the present data support the hypothesis that the therapeutic effects of clozapine in this primate model and perhaps in schizophrenia may be related at least in part to the restoration of DA tone in the prefrontal cortex.     

Effect of D2 dopamine agonists on tuberoinfundibular dopamine neurons.

The effects of piribedil and the selective D2 dopamine agonists, quinpirole and quinelorane, on the synthesis and metabolism of dopamine, within tuberoinfundibular neurons, were studied. The synthesis and metabolism of dopamine within these hypothalamic neurons were assessed by measuring the accumulation of DOPA after inhibition of DOPA decarboxylase and the concentration of DOPAC in the median eminence. Quinpirole (0.1-2.5 mg/kg, i.p.) produced a dose-related increase in accumulation of DOPA and concentrations of DOPAC in the median eminence. The increased accumulation of DOPA after administration of quinpirole was evident for at least 4 hr. The accumulation of DOPA in the median eminence also was enhanced after the administration of quinelorane (0.025 mg/kg, i.p.) and piribedil (50 mg/kg, i.p.). The stimulatory effect of quinpirole on accumulation of DOPA in the median eminence was antagonized by haloperidol (1 mg/kg, i.p.) but not by SCH 23390 (0.5 mg/kg, i.p.). Although D2 agonists have been shown to acutely suppress the synthesis and metabolism of dopamine in nigrostriatal and mesocorticolimbic dopamine neurons, it is apparent that activation of D2 receptors enhanced the synthesis and metabolism of dopamine within tuberoinfundibular neurons in the hypothalamus.     

Clozapine increases dopamine release in prefrontal cortex by 5-HT1A receptor activation

Clozapine (1–10 mg/kg s.c.) produces a selective increase in dopamine release in rat prefrontal cortex which is, in large part (~50%), mediated via activation of 5-HT_1A receptors. Clozapine is a moderately potent, partial 5-HT_1A receptor agonist and activation of 5-HT_1A receptors may contribute to its efficacy against negative symptoms and reduced extrapyramidal side effect liability. Agonist affinity for 5-HT_1A receptors could thus be a desirable feature in the design of new antipsychotics.     

Influence of morphine sensitization on the responsiveness of mesolimbic and mesocortical dopamine transmission to appetitive and aversive gustatory stimuli

Rationale  

Repeated treatment with morphine has been shown to sensitize rats to its stimulant effects on motor activity and mesolimbic dopamine (DA) transmission.