Effect of Atomoxetine on Hyperactivity in an Animal Model of Attention-Deficit/Hyperactivity Disorder (ADHD)

Background

Hyperactivity related behaviors as well as inattention and impulsivity are regarded as the nuclear symptoms of attention-deficit/hyperactivity disorder (ADHD).

Purpose

To investigate the therapeutic effects of atomoxetine on the motor activity in relation to the expression of the dopamine (DA) D2 receptor based on the hypothesis that DA system hypofunction causes ADHD symptoms, which would correlate with extensive D2 receptor overproduction and a lack of DA synthesis in specific brain regions: prefrontal cortex (PFC), striatum, and hypothalamus.

Methods

Young male spontaneously hypertensive rats (SHR), animal models of ADHD, were randomly divided into four groups according to the daily dosage of atomoxetine and treated for 21 consecutive days. The animals were assessed using an open-field test, and the DA D2 receptor expression was examined.

Results

The motor activity improved continuously in the group treated with atomoxetine at a dose of 1 mg/Kg/day than in the groups treated with atomoxetine at a dose of 0.25 mg/Kg/day or 0.5 mg/Kg/day. With respect to DA D₂ receptor immunohistochemistry, we observed significantly increased DA D₂ receptor expression in the PFC, striatum, and hypothalamus of the SHRs as compared to the WKY rats. Treatment with atomoxetine significantly decreased DA D₂ expression in the PFC, striatum, and hypothalamus of the SHRs, in a dose-dependent manner.

Conclusion

Hyperactivity in young SHRs can be improved by treatment with atomoxetine via the DA D2 pathway.     

Differential Interactions of Desipramine with Amphetamine and Methamphetamine: Evidence that Amphetamine Releases Dopamine from Noradrenergic Neurons in the Medial Prefrontal Cortex

Amphetamine is more effective than methamphetamine at raising dopamine levels in the prefrontal cortex. The current study tested the hypothesis that norepinephrine transporters are involved in this difference. Using microdialysis, dopamine, norepinephrine, and serotonin were measured in the rat prefrontal cortex after administration of methamphetamine or amphetamine, with and without perfusion of desipramine. Amphetamine raised norepinephrine levels more than methamphetamine did. Desipramine raised dopamine and serotonin levels but did not alter metabolite levels. Desipramine attenuated the increase in dopamine by amphetamine while increasing the dopamine released by methamphetamine. These data suggest that methamphetamine and amphetamine differ in altering prefrontal cortical dopamine levels and in interacting with norepinephrine transporters. It is proposed that amphetamine releases dopamine in the prefrontal cortex primarily through norepinephrine transporters, whereas methamphetamine interacts minimally with norepinephrine transporters.     

Huperzine A Reverses Cholinergic and Monoaminergic Dysfunction Induced by Bilateral Nucleus Basalis Magnocellularis Injection of β-Amyloid Peptide (1–40) in Rats

(1) Huperzine A, a promising therapeutic agent for Alzheimer’s disease (AD), was tested for its effects on cholinergic and monoaminergic dysfunction induced by injecting β-amyloid peptide-(1–40) into nucleus basalis magnocellularis of the rat. (2) Bilateral injection of 10 μg β-amyloid peptide-(1–40) into nucleus basalis magnocellularis produced local deposits of amyloid plaque and functional abnormalities detected by microdialysis. In medial prefrontal cortex, reductions in the basal levels and stimulated release of acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine were observed. However, oral huperzine A (0.18 mg/kg, once daily for 21 consecutive days) markedly reduced morphologic abnormalities at the injection site in rats infused with β-amyloid peptide-(1–40). Likewise, this treatment ameliorated the β-amyloid peptide-(1–40)-induced deficits in extracellular acetylcholine, dopamine, and norepinephrine (though not 5-hydroxytryptamine) in medial prefrontal cortex, and lessened the reduction in nicotine or methoctramine-stimulated release of acetylcholine and K⁺-evoked releases of acetylcholine and dopamine. (3) The present results provide the first direct evidence that huperzine A acts to oppose neurotoxic effects of β-amyloid peptide on cholinergic, dopaminergic, and noradrenergic systems of the rat forebrain.     

The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain

Spontaneously hypertensive rats (SHR) are considered to represent a genetic animal model for attention-deficit hyperactivity disorder (ADHD). In the present studies, we compared the locomotor activity, learning and memory functions of juvenile male SHR, with age- and gender-matched genetic control Wistar-Kyoto rats (WKY). In addition, we investigated potential differences in brain morphology by magnetic resonance imaging (MRI). In other complimentary studies of the central nervous system, we used real-time PCR to examine the levels of several dopaminergic-related genes, including those coding for the five major subtypes of dopamine receptor (D1, D2, D3, D4 and D5), those coding for enzymes responsible for synthesizing tyrosine hydroxylase and dopamine-beta-hydroxylase, and those coding for the dopamine transporter. Our data revealed that SHR were more active than WKY in the open field (OF) test. Also, SHR appeared less attentive, exhibiting inhibition deficit, but in the absence of memory deficits relative to spatial learning. The MRI studies revealed that SHR had a significantly smaller vermis cerebelli and caudate-putamen (CPu), and there was also a significantly lower level of dopamine D4 receptor gene expression and protein synthesis in the prefrontal cortex (PFC) of SHR. However, there were no significant differences between the expression of other dopaminergic-related genes in the midbrain, prefrontal cortex, temporal cortex, striatum, or amygdala of SHR and WKY. The data are similar to the situation seen in ADHD patients, relative to normal volunteers, and it is possible that the hypo-dopaminergic state involves a down regulation of dopamine D4 receptors, rather than a general down-regulation of catecholamine synthesis. In conclusion, the molecular and behavioural data that we obtained provide new information that may be relevant to understanding ADHD in man.     

Spatial Memory in Spontaneously Hypertensive Rats (SHR)

The spontaneously hypertensive rat (SHR) is an established animal model of ADHD. It has been suggested that ADHD symptoms arise from deficits in executive functions such as working memory, attentional control and decision making. Both ADHD patients and SHRs show deficits in spatial working memory. However, the data on spatial working memory deficits in SHRs are not consistent. It has been suggested that the reported cognitive deficits of SHRs may be related to the SHRs’ locomotor activity. We have used a holeboard (COGITAT) to study both cognition and activity in order to evaluate the influence of the activity on the cognitive performance of SHRs. In comparison to Wistar-Kyoto (WKY) rats, SHRs did not have any impairment in spatial working memory and reference memory. When the rats’ locomotor activity was taken into account, the SHRs’ working memory and reference memory were significantly better than in WKY rats. The locomotor activity appears to be a confounding factor in spatial memory tasks and should therefore be controlled for in future studies. In the SHR model of ADHD, we were unable to demonstrate an impairment of working memory which has been reported in patients with ADHD.     

Regulation of Extracellular Dopamine by the Norepinephrine Transporter

Abstract: There is growing evidence of an interaction between dopamine and norepinephrine. To test the hypothesis that norepinephrine terminals are involved in the uptake and removal of dopamine from the extracellular space, the norepinephrine uptake blocker desmethylimipramine (DMI) was infused locally while the extracellular concentrations of dopamine were simultaneously monitored. DMI increased the extracellular concentrations of dopamine in the medial prefrontal cortex and nucleus accumbens shell but had no effect in the striatum. The combined systemic administration of haloperidol and the local infusion of DMI produced an augmented increase in extracellular dopamine in the cortex compared with the increase produced by either drug alone. This synergistic increase in dopamine overflow is likely due to the combination of impulse-mediated dopamine release produced by haloperidol and blockade of the norepinephrine transporter. No such synergistic effects were observed in the nucleus accumbens and striatum. Local perfusion of the α₂-antagonist idazoxan also increased the extracellular concentrations of dopamine in the cortex. Although the stimulation of extracellular dopamine by idazoxan and DMI could be due to the increased extracellular concentrations of norepinephrine produced by these drugs, an increase in dopamine also was observed in lesioned rats that were depleted of norepinephrine and challenged with haloperidol. This contrasted with the lack of an effect of haloperidol on cortical dopamine in unlesioned controls. These results suggest that norepinephrine terminals regulate extracellular dopamine concentrations in the medial prefrontal cortex and to a lesser extent in the nucleus accumbens shell through the uptake of dopamine by the norepinephrine transporter.     

Comparative neurochemical changes associated with chronic administration of typical and atypical neuroleptics: implications in tardive dyskinesia

An important goal of current neuroleptic research is to develop antipsychotic compounds with the low incidence of extrapyramidal side effects. The therapeutic success and less side-effect of atypical anti-psychotics such as clozapine and risperidone has focused the attention on the role of receptor systems other than dopaminergic system in the pathophysiology of neuroleptics-associated extrapyramidal side effects. The present study compares the effect of chronic administration of typical and atypical antipsychotics on neurochemical profile in rat forebrain. The study was planned to study changes in extracellular levels of norepinephrine, dopamine and serotonin in forebrain region of brain and tried to correlate them with hyperkinetic motor activities (vacuous chewing movements (VCM's), tongue protrusions and facial jerking) in rats, hall mark of chronic extrapyramidal side-effect of neuroleptic therapy tardive dyskinesia. Chronic administration of haloperidol (1 mg/kg) and chlorpromazine (5 mg/kg) resulted in significant increase in orofacial hyperkinetic movements where as clozapine and risperidone showed less significant increase in orofacial hyperkinetic movements as compared to control. There were also significant decrease in the extracellular levels of neurotransmitters dopamine, norepinephrine and serotonin in fore-brain as measured by HPLC/ED after chronic administration of haloperidol and chlorpromazine. Chronic administration of atypical neuroleptics clozapine and risperidone resulted in the decrease in extracellular concentration of dopamine and norepinephrine but the effect was less significant as compared to typical drugs. However, treatment with atypical neuroleptics resulted in 3 fold increase in serotonin levels as compared to forebrain of control rats. Typical and atypical neuroleptics showed varying effects on neurotransmitters, especially serotonin which may account for the difference in their profile of side effects (Tardive dyskinesia).     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Methylphenidate alters NCS-1 expression in rat brain

Methylphenidate has been used as an effective treatment for attention deficit hyperactivity disorder (ADHD). Methylphenidate (MPH) blocks dopamine and norepinephrine transporters causing an increase in extracellular levels. The use of psychomotor stimulants continues to rise due to both the treatment of ADHD and illicit abuse. Methylphenidate sensitization mechanism has still poor knowledge. Neuronal calcium sensor 1 was identified as a dopaminergic receptor interacting protein. When expressed in mammalian cells, neuronal calcium sensor 1 attenuates dopamine-induced D2 receptor internalization by a mechanism that involves a reduction in D2 receptor phosphorylation. Neuronal calcium sensor 1 appears to play a pivotal role in regulating D2 receptor function, it will be important to determine if there are alterations in neuronal calcium sensor 1 in neuropathologies associated with deregulation in dopaminergic signaling. Then, we investigated if methylphenidate could alter neuronal calcium sensor 1 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. These regions were chosen because some are located in brain circuits related with attention deficit hyperactivity disorder. Our results showed changes in neuronal calcium sensor 1 expression in hippocampus, prefrontal cortex and cerebellum mainly in adult rats. The demonstration that methylphenidate induces changes in neuronal calcium sensor 1 levels in rat brain may help to understand sensitization mechanisms as well as methylphenidate therapeutic effects to improve attention deficit hyperactivity disorder symptoms.     

Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Abstract: Monoamine-uptake blockers were applied focally (0.1–1,000 µ M ) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.     

Gene expression of synaptosomal-associated protein 25 (SNAP-25) in the prefrontal cortex of the spontaneously hypertensive rat (SHR)

Dopamine is believed to play an important role in the etiology of attention-deficit/hyperactivity disorder (ADHD). In our previous study, we showed that gene expression of dopamine D4 receptor decreased in the spontaneously hypertensive rat (SHR) in the prefrontal cortex (PFC). In the present study, we explored the potential causes of dysfunction in the dopamine system in ADHD. It is the first time that neuronal activities in both juvenile SHR and WKY rats have been measured by functional MRI (fMRI). Our results showed that in PFC the Blood Oxygenation Level Dependent (BOLD) signal response in SHR was much higher than WKY under stressful situations. We tested the effects of acute and repeated administration of amphetamine on behavioral changes in SHR combined with the expression of the neuronal activity marker, c-fos, in the PFC. Meanwhile dopamine-related gene expression was measured in the PFC after repeated administration of amphetamine. We found that potential neuronal damage occurred through deficit of D2-like receptor protective functions in the PFC of the SHR. We also measured the expression of synaptosomal-associated protein 25 (SNAP-25) in SHR in PFC. The results showed decreased expression of SNAP-25 mRNA in the PFC of SHR; this defect disappeared after repeated injection of D-AMP.     

The Effects of Cholinergic and Dopaminergic Antagonists on Nicotine-Induced Cerebral Neurotransmitter Changes

In a continuing study of nicotine-induced mechanisms in brain areas associated with cognitive processes, the effects of cholinergic and dopaminergic antagonists on nicotine-induced changes in dopamine, norepinephrine, and serotonin were examined. These effects were measured via in vivo microdialysis in the dorsal and ventral hippocampus and in the prefrontal and medial temporal cortex of conscious, freely moving, adult male rats. Nicotine (0.3 mg/kg, free base) was administered subcutaneously and the antagonists were infused locally via the microdialysis probe. Nicotine alone induced an increase of dopamine and its metabolites in all areas, an increase of norepinephrine in the cortex, and an increase of the norepinephrine metabolite 4–hydroxy-3-methoxy-phenylglycol in all areas. Serotonin was decreased in the hippocampus and increased in the cortex. Nicotine-induced dopamine increases were inhibited by nicotinic (mecamylamine 100 μM, methyllycaconitine 500 μM), muscarinic (atropine 100 μM), and dopaminergic D1 (SCH23390 100 μM) and D2 (eticlopride 100 μM) antagonists, in the hippocampal and cortical areas. In the hippocampal areas, these antagonists had less significant effect on norepinephrine and serotonin. However, in the cortical areas, all antagonists inhibited the nicotine-induced increase of serotonin to varying degrees; and some, primarily nicotinic and dopamine D1 antagonists, inhibited the induced increase of norepinephrine. In the hippocampal and cortical areas, the mechanisms of nicotine-induced dopamine increase seem to be similar, but the mechanisms seem to be different for noradrenergic and serotonergic systems, as shown by the fact that nicotine induces no change in norepinephrine and a decrease in serotonin in the hippocampus, while it induces an increase in both in the cortex. Nicotine-induced dopamine release seems to be mediated, in part locally, by nicotinic and muscarinic receptors on dopaminergic cells. In contrast, nicotine’s effect on norepinephrine and serotonin is at least partially mediated by initial changes at other than local sites, and through different receptors. Thus, the effects of nicotine and the mechanisms involved differ for different neurotransmitters and in different brain areas.     

Phencyclidine-Induced Dysregulation of Dopamine Response to Amphetamine in Prefrontal Cortex and Striatum

Phencyclidine (PCP) administration in rodents has been used to model aspects of schizophrenia. One aspect of such treatment has been the enhancement of amphetamine-induced increase of dopamine in the prefrontal cortex and striatum. To further characterize this mechanism rats were treated for 2 weeks with continuous PCP (15 mg/kg per day via Alzet minipump). Rats were implanted with a microdialysis probe into the prefrontal cortex (PFC) or striatum. Amphetamine was administered locally via the dialysis probe during one collection period and changes in extracellular dopamine were monitored. The effect of local administration of the dopamine uptake blocker nomifensine was also measured. Amphetamine (10 M) and nomifensine (10 M) increased the level of dopamine in both the PFC and striatum. PCP administration did not alter the response to amphetamine or nomifensine in the PFC, but reduced this response about 2-fold in striatum. To examine effects of continuous PCP administration on dopamine autoreceptor function, release of [³H]dopamine in response to electrical stimulation and in the presence of a dopamine agonist or antagonist was tested in striatal and prefrontal cortical tissue. Autoreceptor responses were similar in control and PCP-treated tissues. We conclude that the brain region-specific enhancement of dopamine release by peripheral amphetamine administration in rats after PCP is not likely mediated by alterations in the dopamine autoreceptors or changes in the dopamine transporter. The selective local responses of amphetamine indicates heterogeneous regional effects of continuous PCP on NMDA receptor function; effects that influence both regional excitatory responses and the overall dynamics of tonic excitatory/inhibitory inputs to the PFC and striatum.     

Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: Role of glutamic acid

We studied the effect of local administration of nicotine on the release of monoamines in striatum, substantia nigra, cerebellum, hippocampus, cortex (frontal, cingulate), and pontine nucleus and on the release of glutamic acid in striatum of rats in vivo, using microdialysis for nicotine administration and for measuring extracellular amine and glutamic acid levels. Following nicotine administration the extracellular concentration of dopamine, increased in all regions except cerebellum; serotonin increased in cingulate and frontal cortex; and norepinephrine increased in substantia nigra, cingulate cortex, and pontine nucleus. Cotinine, the major nicotine metabolite, had no effect at similar concentrations. The cholinergic antagonists mecamylamine and atropine, the dopaminergic antagonists haloperidol and sulpiride, and the excitatory amino acid antagonist kynurenic acid all inhibited the nicotine-induced increase of extracellular dopamine in the striatum. The fact that kynurenic acid almost completely prevented the effects of nicotine, and nicotine at this concentration produced a 6-fold increase of glutamic acid release, suggests that the effect of nicotine is mainly mediated via glutamic acid release.     

No evidence for association between a functional promoter variant of the Norepinephrine Transporter gene SLC6A2 and ADHD in a family-based sample

Noradrenergic neurotransmission influences executive functions, attentional performance, and general alertness, involving neuronal networks affected in attention deficit/hyperactivity disorder (ADHD). The norepinephrine transporter facilitates the reuptake of norepinephrine and dopamine in the prefrontal cortex and represents the main target of atomoxetine, an effective drug in the treatment of ADHD. Due to its influence on catecholaminergic signaling, variants of the coding gene (SLC6A2) have been widely investigated in ADHD. Several previous studies report an association between single nucleotide polymorphisms located in SLC6A2 and ADHD; however, the findings are inconsistent. The variant A-3081T (rs28386840) has been shown to have major influence on the expression levels of SLC6A2 due to sequence alteration at a repressor binding site, with the T-allele being associated with ADHD. We tested this potential association of A-3081T in a German family-based ADHD sample of 235 children from 162 families, which has a power >99% based on the previously reported odds ratios. There was no evidence for an overtransmission of the risk allele T (transmission rate: 48.5%, P = 0.55). We conclude that A-3081T is not a major risk variant in our ADHD sample, though SLC6A2 remains an interesting candidate gene in ADHD, especially for the inattentive subtype.     

Pharmacological Profile of the “Triple” Monoamine Neurotransmitter Uptake Inhibitor, DOV 102,677

Summary 1. The molecular and behavioral pharmacology of DOV 102,677 is characterized.2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, “triple” uptake inhibitor that suppresses [³H]dopamine (DA), [³H]norepinephrine (NE) and [³H]serotonin (5-HT) uptake by recombinant human transporters with IC₅₀ values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k _i values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of β-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days).4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity.5. In summary, DOV 102,677 is an orally active, “balanced” inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.     

Effect of co-administration of varenicline and antidepressants on extracellular monoamine concentrations in rat prefrontal cortex

Since a substantial proportion of smokers have comorbid mood disorders, the smoking cessation aid varenicline might occasionally be prescribed to patients who are simultaneously treated with antidepressants. Given that varenicline is a selective nicotinic acetylcholine receptor partial agonist and not a substrate or inhibitor of drug metabolizing enzymes, pharmacokinetic interactions with various classes of antidepressants are highly unlikely. It is, however, conceivable that varenicline may have a pharmacodynamic effect on antidepressant-evoked increases in central monoamine release. Interactions resulting in excessive transmitter release could cause adverse events such as serotonin syndrome, while attenuation of monoamine release could impact the clinical efficacy of antidepressants. To investigate this we examined whether varenicline administration modulates the effects of the selective serotonin reuptake inhibitor sertraline and the monoamine oxidase inhibitor clorgyline, given alone and combined, on extracellular concentrations of the monoamines serotonin, dopamine, and norepinephrine in rat brain by microdialysis. Given the important role attributed to cortical monoamine release in serotonin syndrome as well as antidepressant activity, the effects on extracellular monoamine concentrations were measured in the medial prefrontal cortex. Responses to maximally effective doses of sertraline or clorgyline and of sertraline plus clorgyline were the same in the absence as in the presence of a relatively high dose of varenicline, which by itself had no significant effect on cortical monoamine release. This is consistent with the binding profile of varenicline that has insufficient affinity for receptors, enzymes, or transporters to inhibit or potentiate the pharmacologic effects of antidepressants. Since varenicline neither diminished nor potentiated sertraline- or clorgyline-induced increases in neurotransmitter levels, combining varenicline with serotonergic antidepressants is unlikely to cause excessive serotonin release or to attenuate antidepressant efficacy via effects on cortical serotonin, dopamine or norepinephrine release.     

Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation

The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5mg/kg) robustly increased dopamine and norepinephrine release (to ～175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ～135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.     

Repeated exposure to methamphetamine, cocaine or morphine induces augmentation of dopamine release in rat mesocorticolimbic slice co-cultures

Repeated intermittent exposure to psychostimulants and morphine leads to progressive augmentation of its locomotor activating effects in rodents. Accumulating evidence suggests the critical involvement of the mesocorticolimbic dopaminergic neurons, which project from the ventral tegmental area to the nucleus accumbens and the medial prefrontal cortex, in the behavioral sensitization. Here, we examined the acute and chronic effects of psychostimulants and morphine on dopamine release in a reconstructed mesocorticolimbic system comprised of a rat triple organotypic slice co-culture of the ventral tegmental area, nucleus accumbens and medial prefrontal cortex regions. Tyrosine hydroxylase-positive cell bodies were localized in the ventral tegmental area, and their neurites projected to the nucleus accumbens and medial prefrontal cortex regions. Acute treatment with methamphetamine (0.1-1000 μM), cocaine (0.1-300 μM) or morphine (0.1-100 μM) for 30 min increased extracellular dopamine levels in a concentration-dependent manner, while 3,4-methylenedioxyamphetamine (0.1-1000 μM) had little effect. Following repeated exposure to methamphetamine (10 μM) for 30 min every day for 6 days, the dopamine release gradually increased during the 30-min treatment. The augmentation of dopamine release was maintained even after the withdrawal of methamphetamine for 7 days. Similar augmentation was observed by repeated exposure to cocaine (1-300 μM) or morphine (10 and 100 μM). Furthermore, methamphetamine-induced augmentation of dopamine release was prevented by an NMDA receptor antagonist, MK-801 (10 μM), and was not observed in double slice co-cultures that excluded the medial prefrontal cortex slice. These results suggest that repeated psychostimulant- or morphine-induced augmentation of dopamine release, i.e. dopaminergic sensitization, was reproduced in a rat triple organotypic slice co-cultures. In addition, the slice co-culture system revealed that the NMDA receptors and the medial prefrontal cortex play an essential role in the dopaminergic sensitization. This in vitro sensitization model provides a unique approach for studying mechanisms underlying behavioral sensitization to drugs of abuse.