Aniracetam enhances cortical dopamine and serotonin release via cholinergic and glutamatergic mechanisms in SHRSP

Brain Research 916 (2001) 211–221 www.elsevier.com / locate / bres

Research report

Aniracetam enhances cortical dopamine and serotonin release via cholinergic and glutamatergic mechanisms in SHRSP Masatoshi Shirane, Kazuo Nakamura* CNS Supporting Laboratory, Nippon Roche Research Center, 200 Kajiwara, Kamakura 247 -8530, Japan Accepted 11 June 2001

Abstract Aniracetam, a cognition enhancer, has been recently found to preferentially increase extracellular levels of dopamine (DA) and serotonin (5-HT) in the prefrontal cortex (PFC), basolateral amygdala and dorsal hippocampus of the mesocorticolimbic system in stroke-prone spontaneously hypertensive rats. In the present study, we aimed to identify actually active substances among aniracetam and its major metabolites and to clarify the mode of action in DA and 5-HT release in the PFC. Local perfusion of mecamylamine, a nicotinic acetylcholine (nACh) and N-methyl-D-aspartate (NMDA) receptor antagonist, into the ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) completely blocked DA and 5-HT release, respectively, in the PFC elicited by orally administered aniracetam. The effects of aniracetam were mimicked by local perfusion of N-anisoyl--aminobutyric acid (N-anisoyl-GABA), one of the major metabolites of aniracetam, into the VTA and DRN. The cortical DA release induced by N-anisoyl-GABA applied to the VTA was also completely abolished by co-perfusion of mecamylamine. Additionally, when p-anisic acid, another metabolite of aniracetam, and N-anisoyl-GABA were locally perfused into the PFC, they induced DA and 5-HT release in the same region, respectively. These results indicate that aniracetam enhances DA and 5-HT release by mainly mediating the action of N-anisoyl-GABA that targets not only somatodendritic nACh and NMDA receptors but also presynaptic nACh receptors.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Interactions between neurotransmitters Keywords: Aniracetam; N-anisoyl-g-aminobutyric acid; Stroke-prone spontaneously hypertensive rat; Microdialysis; Dopamine and serotonin release; Mecamylamine; Mesocortical pathway

1. Introduction Monoamine release in the brain is known to be regulated by multiple mechanisms. Regarding the cholinergic implication, previous in vivo microdialysis studies revealed that systemic administration of nicotine increased dopamine (DA) release in the nucleus accumbens and prefrontal cortex (PFC) [7,30] and serotonin (5-HT) release in the PFC [41] of freely moving rats. Nisell et al. [31] showed that the blockade of nicotinic acetylcholine (nACh) receptors prevented DA release in the nucleus accumbens induced by local infusion of nicotine into the ventral tegmental area (VTA). In addition, nicotine induced a dose-dependent increase of serotonergic neuronal firing *Corresponding author. Tel.: 181-467-47-2228; fax: 181-467-472219;. E-mail address: [email protected] (K. Nakamura).

rates and 5-HT release in the dorsal raphe nucleus (DRN) slice and the increased 5-HT release was competitively antagonized by mecamylamine, which was used as a nACh receptor antagonist [20]. The mesocorticolimbic dopaminergic pathway has been also suggested to be regulated by glutamatergic components, such as glutamate (Glu) release and N-methyl-D-aspartate (NMDA) receptor stimulation [7,12,42,44]. Recently, we have found that systemic administration of aniracetam, a cognition enhancer, increases extracellular levels of DA, 5-HT and their metabolites in the PFC, basolateral amygdala and dorsal hippocampus, but not in the striatum nor nucleus accumbens shell, of freely moving stroke-prone spontaneously hypertensive rats (SHRSP) that have a dopaminergic hypofunction throughout the brain [28]. The neurochemical evidence supports our previous behavioral results that aniracetam improved both functional attentional and vigilance loss in a two-lever

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02939-0

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choice reaction task induced by apomorphine and 8-hydroxy-2-(di-n-propylamino) tetralin [23,25]. Moreover, orally given aniracetam and its locally perfused metabolites have been reported to increase ACh release in some brain regions [8,27]. Similarly, these results afford a neurochemical basis to the ameliorating effects of aniracetam on the scopolamine-induced poor performance of the choice reaction task [26]. The impairment of the task performance induced by those pharmacological manipulations could also be improved by the major metabolites of aniracetam, such as N-anisoyl-g-aminobutyric acid (Nanisoyl-GABA), p-anisic acid and 2-pyrrolidinone [23,25,26]. These results together indicate that the effects of aniracetam are mediated by its several metabolites but not aniracetam itself. As mentioned above, the enhancement of DA and 5-HT release by aniracetam was limited in specific brain regions in the mesocorticolimbic pathway, which play an essential role in the regulation of emotion and mood, motivation, sleep–wakefulness and cognition [9,13,14]. Therefore, aniracetam may ameliorate the monoaminergically induced behavioral hypofunction by site-specifically regulating monoamine release. However, there is no study that examined which substances among aniracetam and its metabolites are actually responsible for the monoamine release. Moreover, the mode of action underlying the selective activation of mesocorticolimbic dopaminergic and serotonergic pathways by aniracetam remained to be solved. Based on the previous reports that demonstrated the enhancing effects of N-anisoyl-GABA and p-anisic acid on ACh release in the brain regions of SHRSP [27], we hypothesize that ACh released by aniracetam or its metabolites may modulate dopaminergic and serotonergic neuronal activity via nACh receptors on somatodendritic membranes in the VTA and DRN, as suggested by others [20,31]. Thus, the present study aimed to identify the substances contributable to central dopaminergic and serotonergic activation by systemically given aniracetam and to clarify the action sites and the mechanism of action. For this purpose, DA and 5-HT release in the PFC of freely moving SHRSP was determined using by brain in vivo microdialysis.

2. Materials and methods

2.1. Animals Male SHRSP at 7 weeks of age were obtained from SLC Japan. They were housed in groups of three or four in a room with controlled temperature (22628C), relative humidity (55610%) and illumination from 07:00 to 19:00 h. Animals had free access to food (CRF-1, Charles River, Japan) and water. To accelerate the early development of severe hypertension and stroke, SHRSP received 1% NaCl

solution instead of water for 5 weeks [27]. Animals were used for the following experiments at 13 weeks of age.

2.2. Surgery Rats were anesthetized with sodium pentobarbitone (50 mg / kg i.p.) and then placed in a stereotaxic apparatus. A guide cannula was implanted just above the PFC (AP 3.3 mm, ML 3.3 mm, DV 1.5 mm, relative to the bregma and dura surface), VTA (AP 25.2 mm, ML 1.2 mm, DV 7.8 mm) and DRN (AP 27.8 mm, ML 3.2 mm, DV 6.6 mm at a 288 angle against the cross-section (diagonally)), according to the Brain Atlas of Paxinos and Watson [36]. A stainless steel obdurator was inserted into the guide cannula to prevent occlusion. The rats were allowed to recover for at least 3 days before the experiment. At the end of the dialysis experiment, the brain was removed, fixed in 30% formaline containing 20% sucrose, and then sectioned at a thickness of 50 mm. The slices were stained with cresyl violet and the probe location was confirmed. Data from probes that were located inappropriately were neglected (14 out of 118 total locations).

2.3. HPLC determination of extracellular DA and 5 -HT levels A concentric microdialysis probe (A-I-4-02 for the PFC, A-I-8-02 for the VTA or A-I-8-01 for the DRN: Eicom, Kyoto, Japan) was inserted into the guide cannula and perfused with normal Ringer solution (147 mM NaCl, 4 mM KCl, 1.2 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4) at a constant flow rate of 2 ml / min. After a 3-h equilibration period, the dialysates for every 20 min were collected into 200-ml test tubes containing 40 ml of 0.5 mM HCl to prevent degradation, and injected into the HPLC system (Eicom). DA and 5-HT in the injected dialysates were separated by Eicompack MA-5ODS column (4.63150 mm; Eicom) after passing Eicom Prepak (Eicom) and were determined by an electrochemical detector (ECD-100, Eicom) with a carbon electrode at 650 mV. The column was kept at 248C. The mobile phase, consisting of 0.1 M sodium acetate–0.1 M citrate buffer, pH 3.5, containing 0.016% sodium 1-octanosulfate and 0.001% EDTA-2Na was delivered at a flow rate of 1.0 ml / min. DA hydrochloride and 5-HT hydrochloride (Sigma; St. Louis, MO, USA) were used as external standards. The limit of sensitivity was typically 0.5 pg / sample for DA and 1.0 pg / sample for 5-HT and both amines were linear to 1000 pg / sample. The intra-assay imprecision was 2.1% (n510) and 6.3% (n5 10) as coefficients of variation for DA and 5-HT at a concentration of 50 pg / sample, respectively. The monoamine release was expressed as percent change over the average (basal level) of the first three samples before compound perfusion. AUC 180 data were calculated from the area under the curve ranging from 20 min to 3 h after the oral treatment or the beginning of compound perfusion.

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2.4. Drugs and treatment Aniracetam (Ro13-5057, lot No. 48259) and N-anisoylGABA (Ro13-6680, lot No. 966515) were synthesized in Hoffman-La Roche, Basel, Switzerland. The chemical purity determined by using HPLC was 100% for aniracetam and 96.2% for N-anisoyl-GABA. p-Anisic acid and 2-pyrrolidinone were purchased from Tokyo Kasei Kogyo (Tokyo, Japan) and Wako Pure Chemicals (Osaka, Japan), respectively. For oral administration, aniracetam was suspended in 0.25% carboxymethyl cellulose solution containing one to two drops of Tween 80. Test compounds for local perfusion were dissolved in Ringer solution and perfused into each region and nucleus at a final concentration of 0.1, 1 or 10 mM for 20 min through the microdialysis probe used for the collection of dialysates or inserted only for the perfusion. Mecamylamine hydrochloride obtained from Sigma was also dissolved in Ringer solution and perfused at a final concentration of 100 mM for 4 h from 1 h before the aniracetam treatment to the end of the experiment.

2.5. Statistical analysis All results were analyzed using either one-way analysis of variance (ANOVA) followed by Dunnett’s t-test or Student’s t-test. P values lower than 0.05 were considered statistically significant.

3. Results

3.1. Systemic administration of aniracetam The tip positions of dialysis probes for sample collection or compound perfusion inserted into the respective brain regions are illustrated in Fig. 1. In the PFC of freely moving SHRSP, the basal release of DA and 5-HT were 4.3460.61 pg / 20 min (n520) and 3.1760.38 pg / 20 min (n519), respectively. Systemic administration of aniracetam at 100 mg / kg elicited a slightly delayed but long-lasting enhancement of DA and 5-HT release in the PFC (Fig. 2), confirming our recent finding [28]. In the present study, we examined the involvement of nACh receptors in the effects of aniracetam. Continuous local perfusion of mecamylamine, a nACh receptor antagonist, at 100 mM into the VTA abolished DA release in the PFC after systemic treatment of aniracetam to SHRSP (F(2,17)55.14, P,0.01), whereas it showed only an early inhibition on 5-HT release. Similarly, local perfusion of mecamylamine into the DRN completely inhibited 5-HT release in the PFC elicited by aniracetam (F(2,14)56.97, P,0.05) and moderately decreased DA release (Fig. 3). The perfusion of mecamylamine alone into the VTA or DRN had no effect on the DA and 5-HT release in the PFC

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for 1 h, except for a transient but mild increase in 5-HT release following the VTA perfusion (Figs. 2 and 3).

3.2. Local perfusion into the VTA and DRN To identify actually active substances responsible for the effects of systemically given aniracetam, aniracetam and its metabolites were locally perfused into the VTA and DRN of SHRSP for 20 min. The drug solution used for the local perfusion was prepared at the each maximum concentration that is thought to be reachable to brain after the oral dose of 100 mg / kg [27,32]. Among them, local perfusion of N-anisoyl-GABA at 1 mM into the VTA significantly increased DA release in the PFC (F(4,19)56.92, P,0.01), whereas none of the aniracetam at 1 mM, p-anisic acid at 10 mM nor 2pyrrolidinone at 10 mM altered it (Fig. 4). In contrast, none of the compounds used affected 5-HT release in the PFC. The DA release in the PFC elicited by N-anisoylGABA locally perfused into the VTA was completely blocked by mecamylamine, although the pattern of 5-HT release was unchanged during the perfusion of N-anisoylGABA with or without mecamylamine (Fig. 5). When N-anisoyl-GABA at 1 mM was locally perfused into the DRN for 20 min, 5-HT release in the PFC was significantly enhanced with a successive increase in DA release (Fig. 6).

3.3. Local perfusion into the PFC Next, we examined the possibility that aniracetam and its metabolites may directly act on dopaminergic or serotonergic nerve terminals, as previously revealed on cholinergic nerve terminals [27,43]. Of test compounds locally perfused into the PFC, p-anisic acid at 1 and 10 mM significantly increased DA release (F(7,32)54.26, P,0.01) only at the higher concentration (Fig. 7). However, none of the aniracetam at 1 mM nor N-anisoylGABA at 0.1 or 1 mM affected it. 2-Pyrrolidinone at both doses (1 and 10 mM) tended to increase DA release. On the contrary, N-anisoyl-GABA dose-dependently enhanced 5-HT release (F(7,32)53.38, P,0.01) and p-anisic acid also tended to increase it at the lower concentration. Aniracetam and 2-pyrrolidinone failed to alter 5-HT release. In addition to the PFC, we also preliminarily examined the effects of the direct perfusion into the basolateral amygdala. Among the test compounds, only N-anisoyl-GABA at 1 mM showed a tendency to increase DA and 5-HT release in the basolateral amygdala (data not shown).

4. Discussion The present study demonstrated that local perfusion of mecamylamine into the VTA and DRN completely blocked

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Fig. 1. Schematic drawing of the sites where the microdialysis probes were inserted into the rat brain.

DA and 5-HT release, respectively, in the PFC elicited following systemic administration of aniracetam. Local perfusion of N-anisoyl-GABA into the VTA elicited a mecamylamine-sensitive DA release in the PFC, whereas the perfusion into the DRN enhanced both DA and 5-HT release. Furthermore, local perfusion of N-anisoyl-GABA and p-anisic acid into the PFC produced 5-HT and DA release in the same region, respectively. These findings suggest that both metabolites are responsible components for orally given aniracetam-induced DA and 5-HT release in the PFC and the effects of N-anisoyl-GABA appears to be mediated by nACh and NMDA receptors in the VTA and DRN.

4.1. Mechanisms underlying DA release Central cholinergic stimulation, as well as glutamatergic stimulation, is known to induce monoaminergic transmission in different brain regions. Somatodendritic nACh receptor activation elicits depolarization and neuronal firing, whereas presynaptic nACh receptor stimulation enhances the terminal release of neurotransmitters [40,48]. Indeed, systemic administration of nicotine to rats enhanced DA release in the nucleus accumbens and PFC and increased burst activity of DA neurons in the VTA [7,30,31], and its intrategmental infusion increased DA release in the nucleus accumbens [4,31]. Recently, it has

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Fig. 2. Effects of mecamylamine perfusion into the VTA on DA and 5-HT release in the PFC enhanced by aniracetam in freely moving SHRSP. Aniracetam (100 mg / kg) or vehicle was orally administered (arrow) and mecamylamine at 100 mM or vehicle was continuously perfused into the VTA from 1 h before systemic treatment of aniracetam. AUC 180 data were calculated from the area under the curve ranging from 20 min to 3 h after the aniracetam treatment. Data represent means6S.E.M. obtained from the number indicated in the parentheses. *P,0.05 and **P,0.01 versus vehicle control and [ P,0.05 and [[ P,0.01 versus aniracetam alone.

been demonstrated that orally given aniracetam, a cognition enhancer, accelerated ACh release in the rat brain [8] and the microinjection of N-anisoyl-GABA into the peduculopontine tegmental nucleus (PPTg), one of the ACh cell body groups, increased ACh release in the nucleus reticularis thalami, which is cholinergically innervated primarily by the PPTg [19,27]. We also preliminarily observed that the microinjection of N-anisoyl-GABA into the laterodorsal tegmental nucleus (LDTg), another ACh cell body group, significantly increased ACh release in the VTA. Systemic administration of aniracetam dose-dependently enhances DA release in specific brain regions of freely moving SHRSP [28]. Cholinergic projections into the VTA, a DA cell body, originate from the PPTg and / or LDTg [19]. Nilson et al. [31] have reported that nACh

receptor blockade in the VTA antagonizes DA release in the nucleus accumbens observed after systemic nicotine administration. Similarly, in the present study, intrategmental perfusion of mecamylamine completely prevented DA release in the PFC elicited by systemic administration of aniracetam, suggesting that aniracetam increases ACh release in the VTA and consequently activates nACh receptors on the somatodendritic membranes and presynaptic terminals. Systemic effects of aniracetam on DA release in the PFC appeared to be mediated by its metabolites, largely Nanisoyl-GABA and partly p-anisic acid. In fact, microinjection of N-anisoyl-GABA into the cholinergic nuclei increased ACh release in the respective projection area and nucleus [27] (unpublished data). Aniracetam itself is an

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Fig. 3. Effects of mecamylamine perfusion into the DRN on DA and 5-HT release in the PFC enhanced by aniracetam in freely moving SHRSP. Aniracetam (100 mg / kg) or vehicle was orally administered (arrow) and mecamylamine at 100 mM or vehicle was continuously perfused into the DRN from 1 h before systemic treatment of aniracetam. AUC 180 data were calculated from the area under the curve ranging from 20 min to 3 h after the aniracetam treatment. Data represent means6S.E.M. *P,0.05 and **P,0.01 versus vehicle control and [ P,0.05 and [[ P,0.01 versus aniracetam alone.

a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor desensitization inhibitor [16,24] and AMPA receptor desensitization process is present in the medial PFC [3]. Intrategmental infusion of AMPA increased DA release in the nucleus accumbens of rats [42]. Nevertheless, aniracetam directly applied to the VTA and PFC failed to increase DA release in the PFC, indicating no contribution of aniracetam mediated by AMPA receptors. In contrast, local perfusion of p-anisic acid into the PFC enhanced cortical DA release, probably by the activation of ACh release from cholinergic terminals [27] and leading to the stimulation of presynaptic nACh receptors on dopaminergic terminals [40,48]. There may be another assumable mechanism in the effects of aniracetam on DA release. There is growing evidence that nACh receptors are presynaptically localized

on glutamatergic afferents in the VTA and regulate the mesocorticolimbic dopaminergic activity via Glu release and NMDA receptor stimulation [7,12,42,44]. Moreover, several recent reports have indicated that mecamylamine may interact with NMDA receptors in vitro and in vivo [17,35]. Mecamylamine at the concentration of 100 mM that was used in the present study only transiently inhibits NMDA receptors [35]. However, it seems more likely that the continuous perfusion for 4 h involves the significant blockade of NMDA receptors in the effects of mecamylamine. In addition, it has been recently found that aniracetam at nanomolar concentrations abolished the kynurenic acid antagonism of NMDA-evoked noradrenaline release in the rat hippocampal slice, suggesting a positive modulation of NMDA receptor function [37]. Further interestingly, systemic treatment of aniracetam

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Fig. 4. Effects of local perfusion of aniracetam and its metabolites into the VTA on DA and 5-HT release in the PFC of freely moving SHRSP. Test compounds were perfused for 20 min at the indicated concentrations. AUC 180 data were calculated from the area under the curve ranging from 20 min to 3 h after the compound perfusion. Data represent means6S.E.M. *P,0.01 versus vehicle control.

elicited Glu release in the PFC of freely moving SHRSP (personal communication with Dr. Togashi) and altered NMDA receptor expression in the rat hippocampus [11]. Taken together, it may be speculated that ACh released by orally administered aniracetam stimulates presynaptic nACh receptors on glutamatergic afferents in the VTA leading to Glu release and then activates somatodendritic NMDA receptors on DA cells and subsequent increase in DA release at the mesocorticolimbic terminals. However, the possibility that aniracetam itself modulates NMDA receptors through AMPA receptors was ruled out because of the ineffectiveness of aniracetam perfused into the VTA on cortical DA release. N-Anisoyl-GABA perfused into the PFC has been proven to enhance ACh release in the PFC via group II metabotropic Glu (mGlu) receptors [43]. However, since mRNA expression of group II mGlu receptors (subtypes 2 and 3) in the VTA was very few [33,34], it seems unlikely that N-anisoyl-GABA acts with the same mechanism in the VTA. Indeed, our preliminary study showed that N-

anisoyl-GABA perfused for 20 min into the VTA failed to increase ACh release in the VTA, despite that its intrategmental perfusion increased a mecamylamine-sensitive DA release in the PFC. Moreover, N-anisoyl-GABA did not directly interact with nACh receptors [16]. Thus, it would be reasonable to speculate the presence of non-cholinergic, glutamatergic activation mechanism of dopaminergic neurons in the effects of N-anisoyl-GABA in the VTA.

4.2. Mechanisms underlying 5 -HT release We recently found that orally given aniracetam elicited regionally specific 5-HT release in the PFC, basolateral amygdala and dorsal hippocampus, but not in the striatum nor nucleus accumbens shell, of freely moving SHRSP [28]. In the present study, the aniracetam-elicited 5-HT release in the PFC was mimicked by local perfusion of N-anisoyl-GABA into the DRN and PFC and was almost completely blocked by the intra-raphe perfusion of mecamylamine. Systemic or local administration of nico-

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Fig. 5. Effects of mecamylamine perfusion into the VTA on DA and release in the PFC enhanced by N-anisoyl-GABA in freely moving SHRSP. N-Anisoyl-GABA at 1 mM was locally perfused into the VTA for 20 min (bar) and mecamylamine at 100 mM was continuously perfused into the same area from 1 h before an application of N-anisoyl-GABA (open column). Data represent means6S.E.M. *P,0.05 and **P,0.01 versus vehicle control.

tine increased 5-HT release in the cortical regions of freely moving rats [41,47]. Local perfusion of NMDA into the DRN increased extracellular 5-HT levels in the DRN and nucleus accumbens, probably by activating serotonergic neurons with a depolarization-dependent manner in the DRN [45]. Thus, the effects of aniracetam on 5-HT release may be basically explained with similar mechanisms to those mentioned in the above section. Systemic aniracetam administration may increase ACh release in the DRN by activating the cholinergic nuclei (PPTg and LDTg). The released ACh stimulates not only somatodendritic but also presynaptic nACh receptors and the latter produces Glu release from glutamatergic terminals and stimulates somatodendritic NMDA receptors. However, it is unclear whether or not the effects of N-anisoyl-GABA in the DRN are mecamylamine-sensitive. N-Anisoyl-GABA directly perfused into the PFC may

elicit nACh receptor-mediated 5-HT release from serotonergic terminals by accelerating ACh release via group II mGlu receptors [43]. The suggestion is further supported by other findings, such as 5-HT release evoked by a mGlu receptor agonist [15] and abundant existence of group II mGlu receptors in the PFC [33,34].

4.3. Neural circuits between the VTA and DRN The mechanisms involved in monoamine release elicited by orally given aniracetam are multiple, since aniracetam has at least three different target sites, such as cholinergic cells, monoaminergic cells and their projection terminals. In addition to simple cholinergic–monoaminergic interaction mechanism, there appears to be a cholinergic– glutamatergic–monoaminergic interaction. Moreover, serotonergic–dopaminergic or its reciprocal interaction

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Fig. 6. Effects of local perfusion of N-anisoyl-GABA into the DRN on DA and 5-HT release in the PFC of freely moving SHRSP. N-Anisoyl-GABA at 1 mM was perfused for 20 min (bar). Data represent means6S.E.M. *P,0.05 and **P,0.01 versus vehicle control.

may be involved in the neuronal regulation between the VTA and DRN. Electrophysiological, biochemical and neuroanatomical evidence indicates the importance of serotonergic afferent regulation on dopaminergic neuronal activity in the VTA [2,10,18,38,46]. 5-HT2A, 5-HT3A and 5-HT3 receptors may be implicated in the regulatory process [1,5,21,38]. The findings of the DA release increased by N-anisoylGABA perfused into the DRN and of the moderate inhibition of aniracetam-elicited DA release by mecamylamine applied to the DRN clearly indicate a strong serotonergic–dopaminergic interaction and the necessity of 5-HT to maintain dopaminergic tone in the VTA. Thus, aniracetam (mainly by N-anisoyl-GABA) may alternatively trigger dopaminergic activity in the VTA via the serotonergic–dopaminergic circuit. Aniracetam indeed represents anxiolytic effects in mice [22] and antidepressant-like effects in rats [29] by the sequential facilitation of cholinergic (nACh receptors), serotonergic (5-HT2A receptors) and dopaminergic (DA D 2 receptors) systems. On

the contrary, the dopaminergic afferent modulation of serotonergic cells in the DRN was only slightly seen as the early inhibition of aniracetam-elicited 5-HT release by mecamylamine applied to the VTA, as reported previously [6]. Together with the results that the perfusion of Nanisoyl-GABA into the VTA increased only DA release and had no effect on 5-HT release, it seems likely that the dopaminergic–serotonergic interaction between the VTA and DRN is weak. Perhaps, the inhibition of early 5-HT release by mecamylamine may result from the blockade of the glutamatergic pathway originated from the VTA, which projects to the DRN and evokes a fast excitatory synaptic response [12,39]. In conclusion, DA and 5-HT release in the PFC elicited after systemic treatment of aniracetam to SHRSP was mimicked by local perfusion of N-anisoyl-GABA into the VTA and DRN and of N-anisoyl-GABA and p-anisic acid into the PFC. These effects of aniracetam and N-anisoylGABA appeared to be mediated by the functional activation of nACh and NMDA receptors located in the VTA and

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Fig. 7. Effects of local perfusion of aniracetam and its metabolites into the PFC on DA and 5-HT release in the PFC of freely moving SHRSP. Data represent means6S.E.M. *P,0.05 and **P,0.01 versus vehicle control.

DRN. Thus, it is suggested that aniracetam increases monoamine release through multiple mechanisms, such as cholinergic–monoaminergic, cholinergic–glutamatergic– monoaminergic or serotonergic–dopaminergic interaction.

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