The effects of the 5-HT6 receptor agonist EMD and the 5-HT7 receptor agonist AS19 on memory formation

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Behavioural Brain Research 195 (2008) 112–119

Research report

The effects of the 5-HT6 receptor agonist EMD and the 5-HT7 receptor agonist AS19 on memory formation A. Meneses a,∗ , G. Perez-Garcia a , G. Liy-Salmeron a , D. Flores-Galvez b , C. Castillo c , E. Castillo c a

Dept Pharmacobiology, CINVESTAV, M´exico City 14330, Mexico b Universidad Metropolitana, M´ exico City 14330, Mexico c Escuela Superior de Medicina, CINVESTAV, M´ exico City 14330, Mexico Received 21 November 2007; accepted 22 November 2007 Available online 11 January 2008

Abstract Growing evidence indicates that 5-hydrohytryptamine (5-HT) receptors mediate learning and memory. Particularly interesting are 5-HT6 and 5-HT7 receptors, which are localized in brain areas involved in memory formation. Interestingly, recently selective 5-HT6 and 5-HT7 receptor agonists and antagonists have become available. Previous evidence indicates that 5-HT6 or 5-HT7 receptors antagonists had no effects, improved memory formation and/or reversed amnesia. Herein, the effects of EMD (a 5-HT6 receptor agonist) and AS19 (a 5-HT7 receptor agonist) in the associative learning task of autoshaping were studied. Post-training systemic administration of EMD (1–10 mg/kg) or AS19 (1–10 mg/kg) were tested in short-term memory (STM) and long-term memory (LTM). Results showed that only EMD 5.0 mg/kg impaired both STM and LTM. AS19 at 1–10 mg/kg significantly impaired STM but not LTM. In those groups used to test only LTM, EMD impaired it; while AS19 improved LTM. Moreover, in the interaction experiments, the STM EMD-impairment effect was partially reversed by the selective 5-HT6 receptor antagonist SB399885 (10 mg/kg). The STM AS19-impairment effect (5.0 mg/kg) was not altered by the selective 5-HT1A antagonist WAY 100635 (0.3 mg/kg) but reversed by the selective 5-HT7 receptor antagonist SB-269970 (10.0 mg/kg). The AS19-SB-269970 combination impaired LTM. Taken together these data suggest that the stimulation of 5-HT6 impaired both STM and LTM. 5-HT7 receptors stimulation impaired STM but improved LTM. And these results are discussed in the context of their possible neural bases. © 2007 Elsevier B.V. All rights reserved. Keywords: Autoshaping; 5-HT receptors; Memory; Rat

1. Introduction Using receptor binding profiles, common secondary messenger coupling and functional activity ligands, seven families of 5-hydroxytryptamine receptors (5-HT1A/1B/1D/1E/1F , 5HT2A/2B/2C , 5-HT3A/3B , 5-HT4A/4B/4C/4D , 5-HT5A/5B , 5-HT6 and 5-HT7A/7B/7C/7D ) had been identified [7,8]. The investigation about the role of 5-HT systems has been benefited from the identification, classification, cloning of multiple receptors for this monoamine and selective drugs. Actually, the administration of 5-HT drugs with diverse modes of action has been used to study the basic mechanisms of learning and memory under physiological and pathophysiological conditions,

∗

Corresponding author. Tel.: +52 55 50612869; fax: +52 55 50612863. E-mail address: [email protected] (A. Meneses).

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.11.023

aiming identified neural loci and therapeutic treatments for cognitive dysfunctions (see, e.g., [1,3,26,29,34] for a review). 5-HT pathways project to almost all brain areas [12] and apparently, diverse 5-HT mechanisms might be useful in the treatment of learning and memory dysfunctions. Indeed, 5-HT systems are involved in the physiological, pathophysiological and/or therapeutic mechanisms of cognitive processes, in disorders such as Alzheimer’s disease (AD) and schizophrenia [26,34,45]. In spite this encouraging information, the role of some 5-HT systems in learning and memory is not clear [26,35]. Importantly, several selective 5-HT receptor agonists and antagonists have become available for investigation (see, e.g., [2,5,7–9,16–18,32,42–47,51,53]) and particularly in recent times for 5-HT6 [2,5,16,25–28,34,37,45,47,53] and 5-HT7 receptors [14,18,44–46,51]. The topic of the study is highly interesting given the increasing availability of pharmacological tools to investigate the role of

A. Meneses et al. / Behavioural Brain Research 195 (2008) 112–119

these receptors in memory behavior. The 5-HT6 receptor agonist 5-chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole hydrochloride (EMD 386088) [25] and the 5-HT7 receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol-4yl)-2-(dimethylamino)tetralin (AS19) [43–45] might be useful pharmacological tools in search of the functional role of these receptors. EMD [25] and AS19 [43–45] display high selectivity for 5-HT6 and 5-HT7 receptor, respectively. Apparently, aging, AD and amnesia are associated to decrements in 5-HT markers such as the raphe complex, the uptake/transporter complex, and in the number of 5-HT1A-1D, 5HT2A/2C , 5-HT4 , 5-HT6 and 5-HT7 receptors [4,15,20–22,34]. 5-HT6 receptors and 5-HT7 receptors are coupled to Gs proteins and stimulate AMPc production [8,23]. They are localized in brain areas involved in learning and memory processes [10,26–35,43], including hippocampus, amygdala, striatum, neocortex, where they alone might influence memory and neurotransmitters such as acetylcholine and glutamate [1,6,19,21,38]. In the case of 5-HT6 receptors, most of the drugs used to study them have been antagonists (see [26,34,53]), tested in learning tasks such as water maze, passive avoidance, autoshaping, novel object discrimination [53], showing facilitation of memory formation or by reversing amnesia. Notably, animals overexpressing 5-HT6 receptors in the striatum developed amnesia in a simple operant learning task (a purposely procedural memory), which was reversed by the selective 5-HT6 receptor antagonist SB-258585 [8,36,37,39]. More recently, scopolamine or dizocilpine produced amnesia in an autoshaping task and modified 5-HT6 receptors expression. These modifications were observed in brain areas thought to be in charge of procedural memory such basal ganglia [34] and declarative memory mediated by hippocampus (see below). In contrast, the selective 5-HT6 receptor antagonist SB-399885 improved memory and produced a moderate 5-HT6 receptors expression in basal ganglia. And in amygdala, septum and neocortex (brain areas in charge of memory storage) a similar expression occurred. However, amnesic- and SB-399885-treated groups relative to the control animals showed in the hippocampus, region where explicit memory is formed, a complex 5-HT6 receptors expression [34], including up- and down-regulation. These data clearly demonstrated that memory consolidation and amnesia are associated to 5-HT6 receptors regulation in key brain areas for memory. Notably, three putative selective 5-HT6 receptor agonists, EMDT, LY586713 and WAY-466 [2,5,17,25,37] have become available. Interestingly, recently several selective 5-HT6 receptor agonists appear to restore memory impairments in the novel object discrimination paradigm [5]. It is unclear why and how 5-HT6 receptor agonists and antagonists might improve memory and/or reverse amnesia (see also [35]). Moreover, 5-HT7 receptors occur in brain areas associated with cognitive processes, such as the raphe nuclei, hippocampus, amygdala and cortex [27,34]. In hamsters significant age-related decreases in mRNA expression of 5-HT7 receptors in cingulate cortex and paraventricular thalamic nucleus were reported [4]. In the dentate gyrus, hippocampal CA1 and CA2 areas, lateral septum, hypothalamus, amygdala and median raphe nuclei had substantial modifications. The few available behavioral reports

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using memory tasks [22,30,44] show that selective 5-HT7 receptor antagonists such as DR4004 or SB-269970 alone had no effect; nevertheless, both antagonists reversed scopolamineor dizocilpine-induced amnesia. Notably, expression of 5-HT7 receptor mRNA was modulated during memory consolidation and this expression was also altered by selective 5-HT agonists and antagonists or in amnesic animals, which almost did not express these receptors [9,45]. These latter results are, in part, in line with evidence that mice lacking 5-HT7 receptors showed selective impairment in contextual conditioning [49]. Therefore, in the present work the aim was to provide further evidence concerning 5-HT6 and 5-HT7 in short- and long-term memory, by testing the putative selective 5-HT6 receptor agonist 5-Chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1Hindole hydrochloride (EMD 386088; 25) and the putative selective 5-HT7 receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol4-yl)-2-(dimethylamino)tetralin (AS19; 44–46) given alone or in combination with selective 5-HT6 and 5-HT7 receptor antagonists [14,34,45,46]. Taking into account that no published report of a full pharmacological profile of AS19 is available and considering that this drug might display some physiological or pharmacological (i.e., affinity) interaction for 5-HT1A receptors [44,45] hence the selective 5-HT1A receptor antagonist WAY 100635 (see also [8,35]) was also included. For this work 1.5 (STM) and 24 h (LTM, memory consolidation) were selected considering that hippocampal gene expression is critical in two time windows around the time of training and 3–6 h after training (see [11,31]) and these time windows qualified to be periods of sensitivity to transcriptional and translational inhibitors as those were observed using the protein and mRNA synthesis inhibitors [11,31]. In fact, memory consolidation seems to have taken place within 0–5 h post-training in an autoshaping task [31]. 2. Methods 2.1. Animals Male Wistar rats (12-week old) weighing 250–300 g, were collectively housed in a temperature and light-controlled room under a 12:12 h light: dark cycle (light beginning at 7:00 A.M.) with water and food provided ad libitum for a week. After that period, body weights were reduced to 85% by gradually reducing the food intake during 7 days. The local institutional committee for the use of animal subjects approved the present experimental protocol (Project No. 047/02). 2.1.1. Autoshaping apparatus Autoshaping task [29], STM and LTM [30,31] protocols have been previously described. In short, the autoshaping learning task apparatus (Coulbourn Instruments, Lehigh Valley, PA) included a standard attenuation system, with the following inner dimensions: 25 cm × 29 cm × 25 cm (width × length × height) and consisted of a metal frame and transparent Perspex and aluminum walls and a bars floor. Solid-state programming equipment was used for control and recording. An acrylic retractable lever was mounted 4 cm above the floor and 10 cm from the right and left walls. The lever microswitch was adjusted to provide a 10 g force for operation. A food magazine for rat pellets (Bio-Serv, Frenchtown, NJ) was located 5 cm to the right of the lever and 3 cm above the floor. A photocell was mounted with the food magazine to measure head-pokes during the presence of conditioned stimulus (CS) and was defined as head-pokes/CS. A house light was located in the right top corner and maintained being turned on during session period.

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2.2. Behavioral protocols Behavioral protocols include food-magazine and autoshaping training and testing session for STM, LTM and memory consolidation (see below). 2.2.1. Food-magazine Individually each rat was placed in an experimental chamber for a habituation period (≈15 min), having access to 50 food pellets (45 mg each) previously placed inside the food-magazine. The criterion was that once the animal ate all 50 food-pellets and presented 150 nose-pokes (as measured by a photocell) into the food-magazine, immediately afterward the autoshaping training program was initiated. Testing sessions were realized later at 1.5 (for STM) and 24 h (for LTM) or only 24 h for memory consolidation 2.2.2. Autoshaping training The autoshaping program consisted of discrete trials. A trial consisted of the presentation of an illuminated retractable lever for 8 s (CS), followed by the delivery of a 45 mg food pellet (unconditioned stimulus, US) with a fixed time intertrials (ITT) of 60 s. Nonetheless, when the animal pressed the CS, the trial was then shortened, the lever was retracted, the light was turned off and a food pellet (US) was immediately delivered, thereby subsequently initiating ITT. The response during CS was regarded as a CR and its increase or decrease was considered as an enhancement or impairment measure of learning, respectively. Since, the possibility of memory manipulation is related to both the training amount and to the strength of post-training treatments and considering that 10 trials detected the drug-induced changes on autoshaped response [29], hence the training session consisted of 10 (lasting ≈12 min) trials and the following sessions (STM, LTM, memory consolidation) consistent in 20 trials (lasting ≈24 min). Increases or decreases in the head-pokes/CS were used as a motivation measure for foodpellets. The autoshaping training and testing sessions were conducted over two consecutive days. Following the first autoshaping training session, drugs were administered and the testing sessions took place 1.5 (STM), and 24 h (LTM) later for some groups and for others only 24 h later for memory consolidation; the results obtained represented the latter autoshaping sessions. It should be noticed that the earlier recognition of the memory time-dependent nature [29] and post-training drug administration allowed the exclusion of unspecific effects related to perception, motivation and motor activity, thus studying selectively the memory consolidation [29–31].

retention hours, days or years as (see e.g., [11,13,35,40,41,47]; however see [41]).

3. Materials 3.1. Drugs The drugs used in the present study were: 5-chloro-2-methyl-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydrochloride (EMD 386088) and (2S)-(+)-5-(1,3,5-Trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin (AS 19) both purchased from Tocris Cookson (Ellisville, MO, USA). N-[2[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanene carboxamide trihydrochloride (WAY 100635) (Sigma, USA), N-[3,5-dichloro2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide SB-388995 and (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl) pyrrolidine-1sulfonyl) phenol (SB-269970) [18,45]. Drugs were injected subcutaneously (SC) or intraperitoneally (IP) in a volume of 1 ml/kg. EMD, SB-388995 and AS19 were sc administered. All other drugs were dissolved in physiological saline or suspended in methyl cellulose (EMD) or propylene glycol (AS19).

3.2. Data acquisition and statistical analysis The responses in the presence of the CS (CR) were divided by the trials in the session, are expressed as a percentage. Multiple group comparisons were made using ANOVA followed by the Tukey test (e.g., vehicle vs. EMD or AS19). In all statistical comparisons, p < 0.05 was used as criterion for significance. The n per group was 8 animals. Twenty-two groups were used.

4. Results 4.1. 5-HT6 receptors: STM-LTM As depicted in Fig. 1, the post-training injection of EMD 1 or 10 mg/kg produced no significant effects, however at 5 mg/kg

2.3. Experimental protocol Immediately after the first autoshaping training session, the animals received either vehicle (either saline or propylene glycol), EMD (1–10 mg/kg) or AS19 (1–10 mg/kg). Also 5-HT receptor antagonists were tested alone and in combination with EMD or AS19 during STM and LTM using the same animals. They were then placed in their home cages until the autoshaping session test which was performed 1.5 (STM) and 24 h (LTM) later [30,31]. Independent animals received EMD or AS19 immediately following training and tested 24 h later for memory consolidation [43–45].

2.4. Interaction experiments: 5-HT6 or 5-HT7 receptors antagonists Other animals were given SB-388995 or SB-269970 immediately after the autoshaping training session and 1 min after that they received saline, EMD or AS19. After 1.5 and 24 h, the session tests were performed. Notably, since some anonymous referees have suggested that different animals should be used for STM and LTM experiments and considering that to our best knowledge no previous studies have reported this consideration we have included groups of animals receiving autoshaping training and 24 h later testing sessions. It should be noticed that this latter protocol of training and testing sessions corresponds to memory consolidation experiments (see [29,30]). Hence, these experiments used to test either LTM or memory consolidation protocol. Importantly, for mammalian and invertebrate species STM and LTM are defined as processes supporting memory for events across retention delays of several minutes to hours, while that those supporting memory for events across longer

Fig. 1. Effects on short-term memory (STM; top) 1.5 or long-term memory (LTM; bottom) 24 h following autoshaping training and post-training systemic administration of EMD (5-HT6 receptor agonist) on the autoshaping task in fasted rats. Data are plotted as conditioned responses (CR%). Values represent the means ± standard error (S.E.); n = 8/group, Tukey test, p < 0.05, *control vs. treated groups.

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Fig. 2. Effects on STM 1.5 (top) or LTM (bottom) 24 h following autoshaping training and post-training systemic administration of SB-399885 (5-HT6 receptor antagonist) plus EMD on the autoshaping task in fasted rats. Data are plotted as conditioned responses (CR%). Values represent the means ± standard error (S.E.); n = 8/group, Tukey test, p < 0.05, *control vs. treated groups or + EMD vs. SB-399 plus EMD.

significantly [F(3,31) = 5.9; P < 0.05] decreased CR% during both STM and LTM in comparison to control group. On the other hand, in the interaction experiment series (Fig. 2), this STMimpairment effect was significantly [F(3,31) = 4.5; P < 0.05] reversed by the selective 5-HT6 receptor antagonist SB-399885

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Fig. 4. Effects on STM 1.5 (top) or LTM (bottom) 24 h following autoshaping training and post-training systemic administration of WAY 100635 (5-HT1A receptor antagonist) or SB-269970 (5-HT7 receptor antagonist) plus AS19 on the autoshaping task in fasted rats. Data are plotted as conditioned responses (CR%). Values represent the means ± standard error (S.E.); n = 8/group, Tukey test, p < 0.05, *control vs. treated groups or + AS19 vs. SB-269970 plus AS19.

(10 mg/kg). SB-399885 alone did not affect CR% in STM and improved it in LTM (Fig. 2; see also [44]). 4.2. 5-HT7 receptors: STM-LTM Post-training administration of AS19 (1–10 mg/kg) produced significant effects [F(3,31) = 6.8; P > 0.05] in the percentage of CR in STM, but unaffecting LTM (Fig. 3). Moreover, in the interaction experiment series (Fig. 4) the decrements in the percentage of CR in STM produced by AS19 (5 mg/kg) were reversed [F(5,47) = 7.6; P < 0.05] by SB-269970 (10 mg/kg) but was not altered by WAY 100635 (0.3 mg/kg). While SB-269970 alone had no effects, WAY 100635 improved LTM (Fig. 4). Nonetheless, the SB-269970-AS19 combination significantly impaired LTM. 4.3. 5-HT6 and 5-HT7 receptors: memory consolidation Table 1 shows that animals treated with EMD (1–10 mg/kg) or AS19 (1–10 mg/kg) showed significant effects with the doses of 5.0 mg/kg. Thus, either EMD [F(3,31) = 6.1; P < 0.05] or AS19 [F(3,31) = 7.4; P < 0.05] showed significant effects impairing or improving memory consolidation, respectively. 5. Discussion

Fig. 3. Effects on STM 1.5 (top) or LTM (bottom) 24 h following autoshaping training and post-training systemic administration of AS19 (5-HT7 receptor agonist) on the autoshaping task in fasted rats. Data are plotted as conditioned responses (CR%). Values represent the means ± standard error (S.E.); n = 8/group, Tukey test, p < 0.05, *control vs. treated groups.

The present results indicate that EMD at 1–10 mg/kg doses tested, only 5.0 mg/kg impaired both STM and LTM or it impaired performance when animals tested only for LTM. In the following lines, in order to distinguish and for clarity STM/LTM from LTM protocols of training, to discuss the latter term the “memory consolidation” will be used. AS19 (1–10 mg/kg)

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Table 1 Effects of EMD and AS19 only during long-term memory Groups

CR (%)

Sal (mg/kg)

10 ± 3

(1.0) (5.0) (10.)

6±2 2 ± 1* 4±2

(1.0) (5.0) (10.0)

7±4 19 ± 5* 10 ± 4

EMD

AS19

Tukey test *p < 0.05 vehicle vs. 5-HT agonist; for other details see text.

impaired STM but did not affect LTM or (at 5.0 mg/kg) improved memory consolidation. In the interaction experiments, the STM and LTM EMD-impairment effect was partially reversed by the selective 5-HT6 receptor antagonist SB-399885 (10 mg/kg). The STM-induced amnesia effect by AS19 (at 5.0 mg/kg dose) was not altered by the selective 5-HT1A antagonist WAY 100635 (0.3 mg/kg) but reversed by the selective 5-HT7 receptor antagonist SB-269970 (10.0 mg/kg). To our best acknowledge these data showed for the first time that the stimulation of 5-HT6 or 5-HT7 receptors impaired STM and LTM or only STM, respectively. And these results are discussed in the following lines in the context of their neural bases. 5.1. 5-HT6 and 5-HT7 receptors With the identification and classification of 5-HT6 and 5-HT7 receptors into the subtypes 5-HT7A-7D , together with available new 5-HT receptor agonists and antagonists for these receptors (see Section 1), it has become possible to get insights of the functional role of 5-HT6 and 5-HT7 (see e.g., [9,47]) receptors. Although certainly there are not yet full pharmacological binding profiles of EMD or AS19, notwithstanding these limitations, the major findings of the present work clearly indicate that either EMD or AS19 impaired STM. Notably, when animals were trained for memory consolidation (i.e. only for LTM), again AS19 impaired it. All these effects involved 5-HT6 and 5-HT7 receptors, respectively (see below). The EMD and AS19 memory impairment effects are consistent with their distribution in hippocampus, amygdala, striatum, and neocortex. Expression of 5-HT6 and 5-HT7 receptors was modified by memory formation and amnesia [10,34,43]. Actually, hippocampus region is a temporary storing site for nonspatial memory engrams [38] and prefrontal cortex is in charge of executive function, STM or working memory and supports goaldirected behavior tasks [3,50,52]. Notably, autoradiography and mRNA studies (see e.g., [10,34,43]) have provided evidence that memory formation in a Pavlovian/instrumental autoshaping task involves protein and mRNA synthesis in the hippocampus [31] and 5-HT receptors expression in brain areas such as hippocampus, amygdala, prefrontal cortex, striatum, and raphe nuclei. Together this evidence confirmed the notion that Pavlovian/instrumental autoshaping involves brain areas mediating

explicit/implicit memory [30]. Hence, the present EMD and AS19 findings suggest that they affected explicit/implicit STM and/or LTM, which would function in lineal manner (i.e., STM is a step towards LTM; [11]), regarding stimulation of 5-HT6 receptors and parallel manner (STM and LTM are separately formed) respect to 5-HT7 receptors stimulation. Thus, while 5HT6 receptors stimulation affected both STM and LTM, 5-HT7 receptors stimulation affected only STM. It should be noted here that while prefrontal cortex supports working memory and STM, memory consolidation occurs in the hippocampal formation and memory is stored in neocortex (see e.g., [30,35,50,54–55]). In all these brain areas 5-HT6 and 5-HT7 receptors are present (see Section 1 and below). Certainly, both 5-HT6 and 5-HT7 receptors are positively coupled stimulatory Gs -proteins [8]. Nevertheless, considering the result that stimulation of 5-HT6 or 5-HT7 receptors differentially affected STM and LTM. Hence it seems likely that their temporal and molecular mechanistic requirements are fundamentally different. These and other questions will require further investigation. 5.2. 5-HT6 receptors The EMD memory-impairment effects are consistent with 5HT6 receptors distribution in hippocampus, amygdala, striatum, and neocortex. Notably, expression of 5-HT6 receptors was modified by memory formation and amnesia [10,34,43]. Actually, autoradiography and mRNA studies (see, e.g., [10,34,43]) have provided evidence that memory formation involves protein and mRNA synthesis 5-HT6 receptors expression in hippocampus, amygdala, prefrontal cortex, striatum, and raphe nuclei. Hence, the present EMD findings suggest that stimulation of 5-HT6 receptors affect STM and LTM in lineal manner regarding. Supporting the notion that EMD these cognitive processes via 5-HT6 receptors is the finding that the selective 5-HT6 receptor antagonist SB-399885 [45] reversed the EMD-induced STM and LTM impairment effect (Fig. 2). It should be noticed that at dose tested SB-399885 alone facilitated (see also [45]) and this effect was eliminated when SB-399885 and EMD were co-administered. Moreover, the finding that EMD at 5.0 mg/kg impaired STM (Fig. 1) or memory consolidation (Table 1) might appear an unusual result, nonetheless it should be noticed that in experiments of learning and memory regular dose-dependent effect curves are infrequently reported. Indeed, frequently one or two doses are reported. Another reason could be due to the narrow involvement of such as neurotransmitter or therapeutic window. For instance, up- or down-regulation of 5-HT6 receptors is associated to amnesia (see [34,36] and below). 5.3. 5-HT7 receptors On the other hand, AS19 on all doses tested impaired only STM and even improved LTM, which might mean that AS19 affected either STM or expression of memory at 1.5 h after injection. This AS19-induced STM amnesic effect was reversed by the selective 5-HT7 receptor antagonist SB-269970 but not by the selective 5-HT1A receptor antagonist WAY 100635. These data clearly showed that 5-HT7 receptors mediated the STM-

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impairment effect induced by AS19. Nevertheless, it should be noticed that previous evidence indicates that AS19 facilitated memory consolidation (see, e.g., [43,45]) and this effect was antagonized by both WAY 100635 and SB-269970 suggesting a participation of both 5-HT1A and 5-HT7 receptors. These contrasting results suggest that STM and LTM and memory consolidation protocols of training involve different mechanisms related to its sensibility to memory formation and/or drugs effects. Indeed, the 5-HT1A and 5-HT7 receptor agonists 8-OHDPAT, at low doses facilitated memory consolidation but only at high doses impaired STM and LTM [31]. It is also possible that these contrasting results are likely due to different underlying timing, molecular, neural and pharmacological mechanisms mediating memory tasks. As is illustrated by the well-known fact that spaced training produces a better memory and overtraining might masked drug effects (see below and [28,34]). The AS19 memory impairment effects are consistent with their distribution in hippocampus, amygdala, striatum, and neocortex. Indeed, expression of 5-HT7 receptors was modified by memory formation and amnesia [10,34,43]. As noted above, autoradiography and mRNA studies (see, e.g., [10,34,43]) have showed that memory formation involved protein and mRNA synthesis of 5HT7 receptors expression in hippocampus, amygdala, prefrontal cortex and striatum. Hence, the AS19 findings suggest that 5HT7 receptors stimulation affected in parallel manner memory formation, affecting only STM. 5.4. 5-HT6 receptors neuromodulation of memory For a number of years 5-HT involvement in memory formation and/or amnesia has been linked directly and indirectly, via GABAergic interneurons, in inhibiting cholinergic or glutamatergic neurotransmission (see, e.g., [1,53]) or by enhancing hippocampal/cortical cholinergic and glutamatergic neurotransmissions [19]. Indeed, selective cholinergic lesion with the immunotoxin 192-IgG-Saporin into the nucleus basalis magnocellularis, in untrained animals failed to alter the density of 5-HT6 receptors mRNA or protein expression in the deafferentated frontal cortex, suggesting that these receptors are not located on cholinergic neurons [21]. The 5-HT6 receptor antagonist SB-357134 induced a concentration-dependant K+ evoked [3 H]acetylcholine (ACh) release in vitro in rat cortical and striatal slices. Also SB-357134 stimulated glutamate release in cortical and striatal slices. In cortex, riluzole blocked the SB-357134-induced K+ -stimulated [3 H]ACh release, and simultaneous administration of dizocilpine and SB-357134 elicited an increase in K+ -evoked ACh release [21]. Notably, in a time-related retention deficit in an object recognition task scopolamine pretreatment) or tryptophan (TRP) depletion impaired memory and Ro4368554 a selective 5-HT6 receptor antagonist reversed both effects, suggesting that both mechanisms may be mediating the 5-HT6 receptor antagonism [16]. The later finding is relevant to the present results, inasmuch as they provide further support to the notion that 5-HT manipulations affect memory formation [26,28,33,34]. As already mentioned expression of 5-HT6 receptors is linked to memory consolidation [10,44,45], improved memory and reversed amnesia induced

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by scopolamine and/or dizocilpine [34]. Hence, it is probable that the SB-399885 improved memory formation and/or reversing amnesia effects involved, in part, hippocampal/cortical cholinergic and glutamatergic neurotransmissions. Notably, in the hippocampus where explicit memory is formed, systemic administration of the selective 5-HT6 receptor antagonist SB399885 reduced 5-HT6 receptors expression. Scopolamine- or dizocilpine augmented 5-HT6 receptors expression [34]. This augmented 5-HT6 expression might be consistent with the present finding with EMD STM-LTM impairment effects. In addition, since 5-HT6 receptor overexpression was associated with amnesia [34,36] or its downregulation appears associated with AD [6], hence, an optimal level of these is required for memory formation. Moreover, the three putative selective 5HT6 receptor agonists, EMDT, LY586713 and WAY-466 (see Section 1) should allow to explore the interesting finding that several selective 5-HT6 receptor agonists apparently was able to restore memory impairments in the novel object discrimination paradigm [5]. It is unclear why and how 5-HT6 receptor agonists and antagonists might improve memory or reverse amnesia (see [35] for a similar situation for 5-HT1A and 5-HT7 receptor agonists and antagonists). Actually, the 5-HT6 receptor agonist WAY-181187 significantly impaired social recognition [17] or facilitated executive function in an extra-dimensional shifting model and had no effects on reversal learning or intra-dimensional shifting [2]; these effects were abolished by selective 5-HT6 receptor antagonists [2,17]. Also, WAY-181187 into the frontal cortex (FCX) significantly impaired social recognition, while conversely, SB-271046 into the FCX, but neither into the striatum nor the NBM, significantly reversed spontaneous deficit [17]. 5.5. 5-HT7 receptors neuromodulation of memory In the case of 5-HT7 receptors, it should be noticed that we recently reported that reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that there was a higher level of expression of the investigated 5-HT receptor mRNAs in autoshaping-trained relative to untrained groups [43]. Actually, pharmacologically naive autoshaping-trained rats showed, in decreasing order, 5-HT1A < 5-HT6 < 5-HT4 ≤ 5-HT7 receptors mRNA in prefrontal cortex and hippocampus. In order to determine more precisely mRNA expression and memory consolidation, we combined selective 5-HT7 receptors stimulation or blockade in the same animals, and brain areas were individually analyzed. 5-HT7 receptors were strongly expressed in hippocampus, prefrontal cortex and raphe nuclei of vehicletrained rats relative to untrained group. The selective 5-HT7 receptor agonist AS19 enhanced memory consolidation and attenuated mRNA receptors expression. The AS19 facilitatory effect was reversed by SB-269970. AS19 also reversed scopolamine- or dizocilpine-induced amnesia and receptor down-regulation [43,44]. Hippocampal 5-HT1A and/or 5-HT7 receptors have been recently associated to cAMP production [22,23], constitutive activity [24] and gene expression such as CREB [20], which is important for memory formation [13]. The AS19 facilitatory effect in memory consolidation is a puzzle

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result with regard to the present findings. It should be noticed nonetheless that, in the memory consolidation protocol, both WAY 100635 and SB-269970 reversed the AS19 facilitatory effect [44,45]. Herein WAY 100635 was unable to modify AS19 STM-impairment effect, which excludes 5-HT1A receptors participation. It should be noted nevertheless that, WAY 100635 is able to reverse amnesia in some memory tasks (see [35]), likely by decreasing 5-HT1A receptors activity and/or modulate cholinergic and glutamatergic neurotransmission. Moreover, it is important to highlight here that memory consolidation and STMLTM training protocols of training, though producing similar results with some drugs (e.g., SB-399885 facilitated both memory consolidation and LTM; [30,45]) while with others elicited contrasting effects. For instance, the 5-HT1A/7 receptor agonist 8-OH-DPAT facilitated memory consolidation [29,43,43] but had no effect at low doses or, at high doses, impaired both STM and LTM [31]. The most heuristic explanation is that memory consolidation and STM-LTM training protocols of training involve different neural mechanisms (see also [48] about the interaction between amount and pattern of training in invertebrate species). Notably, some unselective 5-HT receptor agonists impaired memory, thus, the 5-HT1A/1B/2A-2C/3 agonist m-chlorophenylpiperazine (mCPP) significantly greater worsening in recent memory and knowledge memory in patients with AD than in controls [15] and this drug impaired memory consolidation in autoshaping task ([31] see also [43]; see also [35]) as well as STM and LTM [29]. In conclusion, the present data clearly showed 5-HT6 or 5HT7 receptors stimulation impaired STM, LTM and memory consolidation or only STM, respectively. STM and LTM effects were reversed by selective antagonists. Doubtless, further studies with selective 5-HT6 , and 5-HT7 receptors agonists and antagonists and other testing times are needed to confirm the present results. Acknowledgments Special thanks to those private laboratories for having provided drugs used in this work. We thank Sofia Meneses-Goytia for revised language and Roberto Gonzalez for his expertise assistance. The author thanks the GlaxoSmithKline for their generous gifts of 5-HT receptor antagonists (see Section 3.1). References [1] Buhot MC, Wolff M, Segu L. In: Riedel G, Platt B, editors. Serotonin. In memories are made of these: from messengers to molecules. Eurekah.com and Kluwer Academic/Plenum Publishers; 2003. p. 1–19. [2] Burnham KE, Baxter MG, Dawson LA, Southam E, Sharp T, Bannerman DM. Effect of the 5-HT6 receptor agonist WAY181187 on prefrontal cortical function in the rat. Neurosci Abstr 2007, 741.28.AAA1. [3] Chudasama Y, Robbins TW. Functions of frontostriatal systems in cognition: comparative neuropsychopharmacological studies in rats, monkeys and humans. Biol Psychol 2006;73:19–38. [4] Duncan MJ, Franklin KM. Expression of 5-HT7 receptor mRNA in the hamster brain: effect of aging and association with calbindin-D28K expression. Brain Res 2007;1143:70–7.

[5] Fone KCF. Selective 5-HT6 compounds as a novel approach to the treatment of Alzheimer’s disease. J Pharmacol Sci 2006;101:53. [6] Garcia-Alloza M, Gil-Bea FJ, Diez-Ariza M, Chen CP, Francis PT, Lasheras B, et al. Cholinergic-serotonergic imbalance contributes to cognitive and behavioral symptoms in Alzheimer’s disease. Neuropsychologia 2005;43:442–9. [7] Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, et al. VII. International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharma Rev 1994;46:157– 203. [8] Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 2002;71(4):533–54. [9] Huang YY, Kandel ER. 5-Hydroxytryptamine induces a protein kinase A/mitogen-activated protein kinase-mediated and macromolecular synthesis-dependent late phase of long-term potentiation in the amygdala. J Neurosci 2007;27:3111–9. [10] Huerta-Rivas A, Gonz´alez-Espinosa C, P´erez-Garc´ıa G, Meneses A. 5-HT6 receptor role during memory formation. Soc Neurosci Abstr 2007. [11] Izquierdo I, Medina JH, Vianna MR, Izquierdo LA, Barros DM. Separate mechanisms for short- and long-term memory. Behav Brain Res 1999;103:1–11. [12] Jacobs BL, Azmitia EC. Structure and function of the brain serotonin system. Physiol Rev 1992;72:165–229. [13] Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 2001;294(5544):1030–8. [14] Kikuchi C, Nagaso H, Hiranuma T, Koyama M. Tetrahydrobenzindoles: selective antagonists of the 5-HT7 receptor. J Med Chem 1999;42:533– 5. [15] Lawlor BA, Sunderland T, Mellow AM, Hill L, Molchan SE, Murphy DL. Hyperresponsivity to the serotonin agonist m-chlorophenylpiperazine in Alzheimer’s disease. Arch Gral Psychiatry 1989;46:542–9. [16] Lieben CK, Blokland A, Sik A, Sung E, van Nieuwenhuizen P, Schreiber R. The selective 5-HT6 receptor antagonist Ro4368554 restores memory performance in cholinergic and serotonergic models of memory deficiency in the rat. Neuropsychopharmacol 2005;30:2169–79. [17] Loiseau F, Dekeyne A, Millan MJ. Pro-cognitive effects of 5-HT6 receptor antagonists in the social recognition procedure in rats: implication of the frontal cortex. Psychopharmacology (Berl) 2007 Oct 6 [Epub ahead of print] PMID: 17922111. [18] Lovell PJ, Bromidge SM, Dabbs S, Duckworth DM, Forbes IT, Jennings AJ, et al. A novel, potent, and selective 5-HT7 antagonist: (R)-3(2-(2-(4methylpiperidin-1-1yl)ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970). J Med Chem 2000;43:342–5. [19] Madjid N, Tottie EE, Luttgen M, Meister B, Sandin J, Kuzmin A, et al. 5-Hydroxytryptamine1A receptor blockade facilitates aversive learning in mice: interactions with cholinergic and glutamatergic mechanisms. J Pharmacol Exp Ther 2006;316:581–91. [20] Mahgoub MA, Sara Y, Kavalali ET, Monteggia LM. Reciprocal interaction of serotonin and neuronal activity in regulation of cAMPresponsive element-dependent gene expression. J Pharmacol Exp Ther 2006;317:88–96. [21] Marcos B, Gil-Bea FJ, Hirst WD, Garcia-Alloza M, Ramirez MJ. Lack of localization of 5-HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5-HT6 receptor-mediated acetylcholine release. Eur J Neurosci 2006;24:1299–306. [22] Manuel-Apolinar L, Meneses A. 8-OH-DPAT facilitated memory consolidation and increased hippocampal and cortical cAMP production. Behav Brain Res 2004;148:179–84. [23] Markstein R, Hoyer D, Engel G. 5-HT1A -receptors mediate stimulation of adenylate cyclase in rat hippocampus. Naunyn Schmiedebergs Arch Pharmacol 1986;333:335–41. [24] Martel JC, Ormiere AM, Leduc N, Assie MB, Cussac D, Newman-Tancredi A. Native rat hippocampal 5-HT1A receptors show constitutive activity. Mol Pharmacol 2007;71:638–43. [25] Mattsson C, Sonesson C, Sandahl A, Greiner HE, Gassen M, Plaschke J, et al. 2-Alkyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles as novel 5-HT6 receptor agonists. Bioorg Med Chem Lett 2005;15:4230–04.

A. Meneses et al. / Behavioural Brain Research 195 (2008) 112–119 [26] Meneses A. Role of 5-HT6 receptors in memory formation. Drug News Perspect 2001;4:396–400. [27] Meneses A. 5-HT system and cognition. Neurosci Biobehav Rev 1999;23:1111–25. [28] Meneses A. Effects of the 5-HT6 receptor antagonist Ro 04-6790 on learning consolidation. Behav Brain Res 2001;118:107–10. [29] Meneses A. A pharmacological analysis of an associative learning task: 5-HT1 to 5-HT7 receptor subtypes function on a Pavlovian/instrumental autoshaped memory. Learn Mem 2003;10:363–72. [30] Meneses A. Do serotonin1-7 receptors modulate short and long-term memory? Neurobiol Learn Mem 2007;87:561–72. [31] Meneses A. Stimulation of 5-HT1A , 5-HT1B , 5-HT2A/2C , 5-HT5 and 5-HT4 receptors or 5-HT uptake inhibition: short- and long-term memory. Behav Brain Res 2007;184:81–90. [32] Meneses A, Hong E. Role of 5-HT1B , 5-HT2A and 5-HT2C receptors in learning. Behav Brain Res 1997;87:105–10. [33] Meneses A, Manuel-Apolinar L, Rocha L, Castillo E, Castillo C. Expression of the 5-HT receptors in rat brain during memory consolidation. Behav Brain Res 2004;152:425–36. [34] Meneses A, Manuel-Apolinar L, Castillo C, Castillo E. Memory consolidation and amnesia modify 5-HT6 receptors expression in rat brain: an autoradiographic study. Behav Brain Res 2007;178:53–61. [35] Meneses A, Perez-Garcia G. 5-HT1A receptors and memory. Neurosci Biobehav Rev 2007;3:705–27. [36] Mitchell ES, Sexton T, Neumaier JF. Increased expression of 5-HT receptors in the rat dorsomedial striatum impairs instrumental learning. Neuropsychopharmacol 2007;32:1520–30. [37] Mitchell ES, Neumaier JF. 5-HT6 receptors: a novel target for cognitive enhancement. Pharmacol Ther 2005;108:320–33. [38] Myhrer T. Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioral tasks. Brain Res Rev 2003;41:268–87. [39] Neumaier JF, Mitchell ES. Increased expression of 5-HT6 receptors in rat striatum impairs instrumental learning. J Pharmacol Sci 2006;101: 52. [40] Perrin G, Ferreira G, Meurisse M, Verdin S, Mouly AM, Levy F. Social recognition memory requires protein synthesis after reactivation. Behav Neurosci 2007;121:148–55. [41] Ranganath C, Blumenfeld RS. Doubts about double dissociations between short- and long-term memory. Trends Cogn Sci 2005;9:374–80.

119

[42] Riekkinen M, Sirvi¨o J, Riekkinen P. Treatment with 5-HT2A/2C receptor agonist disrupts initial development of spatial navigation strategies in rats. Soc Neurosci Abstr 1994;20:1546. [43] Perez-Garcia G, Gonzalez-Espinosa C, Meneses A. An mRNA expression analysis of stimulation and blockade of 5-HT7 receptors during memory consolidation. Behav Brain Res 2006;169:83–92. [44] Perez-Garcia GS, Meneses A. Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task. Behav Brain Res 2005;163:136–40. [45] Perez-Garcia G, Meneses A. Oral administration of the 5-HT6 receptor antagonists SB-357134 and SB-399885 improves memory formation in an autoshaping learning task. Pharmacol Biochem Behav 2005;81(3): 673–82. [46] Sanin A, Brisander M, Rosqvist S, Mohell N, Malberg A, Johansson A. 5-Aryl substituted (S)-2-(dimethylamino)-tetralins novel serotonin 5HT7 receptor ligands. 14th Camerino-Noord Symposium, ongoing progress in the receptor chemistry. 2004; p. 27. [47] Svenningsson P, Tzavara ET, Qi H, Carruthers R, Witkin JM, Nomikos GG, et al. Biochemical and behavioral evidence for antidepressant-like effects of 5-HT6 receptor stimulation. J Neurosci 2007;27:4201–9. [48] Sutton MA, Ide J, Masters SE, Carew TJ. Interaction between amount and pattern of training in the induction of intermediate- and long-term memory for sensitization in Aplysia. Learn Mem 2002;9:29–40. [49] Roberts AJ, Krucker T, Levy CL, Slanina KA, Sutcliffe JG, Hedlund PB. Mice lacking 5-HT7 receptors show specific impairments in contextual learning. Eur J Neurosci 2004;19:1913–22. [50] Robbins TW. Chemistry of the mind: neurochemical modulation of prefrontal cortical function. J Comp Neurol 2005;493:140–6. [51] Thomas DR, Hagan JJ. 5-HT7 receptors. Curr Drug Targets-CNS Neurol Dis 2004;3:81–90. [52] Vertes RP. Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse 2004;51:32–58. [53] Woolley ML, Marsden CA, Fone KCF. 5-HT6 receptors. Curr Drug TargetsCNS Neurol Dis 2004;3:81–90. [54] Meneses A. Stimulation of 5-HT1A , 5-HT1B , 5-HT2A/2C , 5-HT3 and 5-HT4 receptors or 5-HT uptake inhibition: short- and long-term memory. Behav Brain Res 2007;184:81–90. [55] Liy-Salmeron G, Meneses A. Role of 5-HT1-7 receptors in short- and longterm memory for an autoshaping task: intrahippocampal manipulations. Brain Res 2007;1147:140–7.