instrumental learning task

Behavioural Brain Research 155 (2004) 275–282

Research report

Effects of the 5-HT7 receptor antagonists SB-269970 and DR 4004 in autoshaping Pavlovian/instrumental learning task Alfredo Meneses∗ Departamento Pharmacobiology, CINVESTAV-IPN, Tenorios # 235, Granjas Coapa, M´exico City 14330, M´exico Received 3 September 2003; received in revised form 30 April 2004; accepted 30 April 2004 Available online 19 June 2004

Abstract There is an important debate regarding the functional role of the 5-HT1A and 5-HT7 receptor in memory systems. Hence, the objective of this paper is to investigate the function of serotonin (5-hydroxytryptamine, 5-HT) in memory consolidation, utilising an autoshaping Pavlovian/instrumental learning test. Specific antagonists at 5-HT1A (WAY 100635) and 5-HT7 (SB-269970 or DR 4004) receptors administered i.p. or s.c.) after training, significantly decreased the improvement of performance produced by the 5-HT1A/7 agonist 8-OH-DPAT to levels lower than controls’. These same antagonists attenuated the decreased level of performance produced by mCPP, although they decrease the performance levels after p-chloroamphetamine (PCA) lesion of the 5-HT system, which has no effect on its own on the conditioned response. Moreover, SB-269970 or DR 4004 reversed amnesia induced by scopolamine and dizocilpine. These data confirm a role for 5-HT1A and 5-HT7 receptors in memory formation and support the hypothesis that serotonergic, cholinergic, and glutamatergic systems interact in cognitively impaired animals. These findings support a potential role for both 5-HT1A and 5-HT7 receptors in the pathophysiology and/or treatment of schizophrenia, cognitive deficits and the mechanism of action of atypical antipsychotic drugs. © 2004 Elsevier B.V. All rights reserved. Keywords: Autoshaping; 5-HT receptors; Learning rat

1. Introduction Compromised serotonergic function may play a role in the decline in cognitive function related to aging and/or Alzheimer’s disease [29]. Therefore, strategies targeting specific serotonin (5-hydroxytryptamine, 5-HT) receptors may be therapeutically useful in the treatment of cognitive dysfunction (see [2,6,8,19,21,26,37,40,41,43,46]). Investigation of the role of the 5-HT system in learning and memory has been greatly benefited from the identification, classification and cloning of multiple receptors for serotonin receptors (5HT1 to 5-HT7 ) and development of selective compounds [13,15,35]. Contradictory evidence regarding the role of the 5-HT system in normal and impaired memory in mammals has been reported ([37,39,40,46]; see [29] for references). Possible reasons for these discrepancies are differences in timing and site (systemic or central) of administration, the ∗ Tel.: +52 55 50612869; fax: +52 55 50612863. E-mail address: [email protected] (A. Meneses).

0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.04.026

type of behavioral test and drugs used (e.g., [22,28]; see [26,29,33]). For example, in the autoshaping learning task, post-training systemic injection of LY215840 (a 5-HT2/7 antagonist) or WAY 100635 (5-HT1A antagonist) had no effect on memory consolidation [30], whereas, in contrast, both drugs abolished the facilitatory effect of 8-OH-DPAT (a 5-HT1A/7 receptor agonist), with LY215840 being slightly more effective in this respect. Furthermore, selective 5-HT2A or 5-HT2B/2C receptor antagonists failed to alter the facilitatory 8-OH-DPAT effects, thus excluding participation of 5-HT2A and/or 5-HT2B/2C receptors. LY215840, at the doses that inhibited 8-OH-DPAT effect, reversed memory deficits induced by scopolamine or dizocilpine [30]. Notably, the 8OH-DPAT-facilitatory effect was induced by low doses, eliminated by 5-HT depletion or synthesis inhibition, suggesting a participation of presynaptic 5-HT1A receptors and, an additional mechanism probably involving 5-HT7 receptors [30]. On the basis of the above-mentioned studies, the present work was designed to further analyze the possible 5-HT1A and 5-HT7 receptor involvement in memory formation. The

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Table 1 Affinities (pKi ) of several 5-HT receptor compounds for various 5-HT receptor (sub)types

LY215840 mCPP MDL100907 8-OH-DPAT SB-200646 WAY 100635 DR 4004 SB-269970

5-HT1A

5-HT1B

5-HT1D

5-HT2A

5-HT2B

5-HT2C

5-HT3

5-HT7

7.4 6.5 6.0 8.7 <5.0 10.0 6.7 <5

6.0 6.6 5.2 4.2 – ? <6a <6

6.2 5.1 – 5.9 <5.0 ? <6a 5.8

7.7 6.7 9.4 5.0 5.2 ? 7.0 <5

8.7 7.6 – 5.4 7.5 ? ? 5.0

8.4 6.9 6.9 5.0 6.9 ? <6a <5

– 7.0 – <5.0 <5.0 ? <6a ?

7.8 6.6 – 7.5 – ? 8.7 8.9b

The data for 5-HT1A , 5-HT1B and 5-HT1D receptors are from [11,12,34,42,45]. Data for 5-HT2A , 5-HT2B and 5-HT2C receptors were taken from [3,7,10–13,17]. Data for 5-HT3 receptors were collected from [14]. Data for 5-HT7 receptors are from [15,16,18,36,38,43,45]. a T. Hiranuma, personal communication (2003). b D.R. Thomas, personal communication (2003).

selective 5-HT7 receptor antagonists (see Table 1) DR 4004 [18] and SB-269970 ([23,36]; for review see [43]), and the 5HT1A receptor-selective WAY 100635 administered alone or in combination with 8-OH-DPAT, were analyzed in a Pavlovian/instrumental autoshaping learning task, which represents a reliable model of learning and memory ([1,26]; however, see [22]). We also investigated whether 5-HT depletion (produced by PCA) plus DR 4004 or SB-269970 administration modify the 8-OH-DPAT-mediated facilitatory effects on memory. Finally, in order to gain further insights, we investigated available non-selective agonists for 5-HT7 receptors, including mCPP and mesulergine which display ranges of affinity and intrinsic activity for the receptor (see Table 1). 2. Materials and methods 2.1. Animals Male Wistar rats (12 weeks old) were collectively housed in a temperature- and light-controlled room under a 12:12 h light/dark cycle (light on 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 food intake (see further). 2.2. Autoshaping learning task The local institutional committee for the use of animal subjects approved the present experimental protocol (project no. 047/02). The autoshaping test has been previously described (see [24,26–31]; see [26] for review). Briefly, male Wistar rats (12 weeks old) were collectively housed (12 per cage) in a temperature controlled animal room (22 ± 11 ◦ C) and on an automatic 12:12 h light/dark cycle (light period: 7:00–19:00 h). Rats had ad libitum access to food and water for a week. Afterwards, body weights were reduced to 85% by gradually reducing the food intake during 7 days and animals were maintained in these weights during the behavioral training and testing period. The n per group was eight animals and used once. Autoshaping learning task was run on experimental chambers (Coulbourn Instruments, Lehigh Valley, PA),

being their inner dimensions 25 cm width, 29 cm in length, and 25 cm in height with standard sound-attenuation system. 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 require 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 house light was located in the right top corner and maintained turned on during session period. 2.2.1. Food-magazine training Individually each rat was placed in an experimental chamber for a habituation period (≈15 min) and having access to 50 food pellets (45 mg each) previously placed inside the food-magazine. The autoshaping program was initiated once animals ate all food pellets and presented 150 nose-pokes (as measured by a photocell) into the food-magazine. 2.2.2. Autoshaping training The autoshaping program consisted of discrete trials. A trial involved presenting an illuminated retractable lever for 8 s (conditioned stimulus, CS) followed by delivery of a 45 mg food pellet (unconditioned stimulus, US) with an intertrial time (ITT) of 60 s; however, when the animal pressed the lever during CS, the trial was then ended, the lever was retracted, light was turned off, and a food pellet (US) was immediately delivered, and then ITT began. The response during CS was regarded as a conditioned response (CR) and an increase or decrease of this was considered to be an enhancement or impairment of learning, respectively. Since, the possibility and degree of engram manipulation are related both to the training amount and post-training treatments [26], and considering that 10 rather than, 5 or 20 trials, better detected the drug-induced changes on autoshaped response, hence the first session consisted of 10 (lasting ≈12 min) trials and the second of 20 trials (lasting ≈24 min). Importantly, a testing session lasting between 10 and 20 min can be more sensitive to detect changes induced on memory [26]. All compounds and vehicle were injected (s.c. or i.p.) immediately after the first autoshaping session and the session test had

A. Meneses / Behavioural Brain Research 155 (2004) 275–282

place 24 h later. The results shown represent this latter autoshaping session. It should be noticed that is extensive evidence (see [26,29]) that post-training drug administration allows memory consolidation to be studied in isolation, thus excluding almost non-specific effects on perception, motivation and motor activity. 2.2.3. Experimental protocol Immediately after the first autoshaping training session the animals received either saline (0.9%) vehicle, 8-OH-DPAT (0.062 mg/kg, s.c.), DR 4004 (0.5–10 mg/kg, i.p.), or SB269970 (1.0–20.0 mg/kg, i.p.), and were then placed in their home cages and the autoshaping test was performed 24 h later. In the interaction experiments to investigate the role of 5-HT1A/7 receptors, 8-OH-DPAT (0.062 mg/kg, s.c.) was administered immediately after the autoshaping training session and 5 min later they received WAY 100635 (0.1 mg/kg, s.c.), DR 4004 (0.5 or 1.0 mg/kg, i.p.) or SB-269970 (3.0 or 10.0 mg/kg, i.p.). In all cases, the session test was performed 24 h later. To further study 5-HT7 receptors, independent animals were given DR 4004 immediately after the autoshaping training session and 5 min later they received WAY 100635 (0.1 mg/kg, s.c.) or mCPP (10.0 mg/kg, i.p). Other animals received 8-OH-DPAT (0.062 mg/kg) plus WAY 100635/SB-269970 or WAY 100635/DR 4004. PCA pretreatment (10.0 mg/kg, i.p., for two consecutive days) was 1 week before memory experiments, and PCA pretreated rats received post-training administration of 8-OH-DPAT and WAY 100635 or DR 4004 combinations. Finally, to investigate effects on scopolamine or dizocilpine-induced cognitive deficits, DR 4004 (1.0 mg/kg) or SB-267790 (3.0 or 10.0 mg/kg) were coadministered (i.p.) with either scopolamine (0.17 mg/kg) or dizocilpine (0.1 mg/kg) and the test session was performed 24 h later. 2.2.4. Drugs The drugs used were: (±)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), N-[2-[4-(2-methoxyphenyl)1-piperazinyl] ethyl]-N-(2-pyridinyl) cyclohexanene carboxamide trihydrochloride (WAY 100635), dizocilpine HCl, scopolamine HCl, p-chloroamphetamine (PCA), m-chlorophenylpiperazine (mCPP) HCl; (R)-3-(2-(2-(4methylpiperidin-1-yl) ethyl) pyrrolidine-1-sulfonyl) phenol (SB-269970 [23]; part purchased from Research Biochemical Inc., Wayland, MA, and part donated by Dr. D. Thomas, GlaxoSmithKline, Harlow Essex, UK); (2a-[4-4-phenyl-1,2,3,6-tetrahydropyridyl])-2a,3,4,5tetrahydrobenzo[ed]-indol[(iH)-1] (DR 4004; donated by Dr T. Hiranuma and co-workers [18]). All drugs were injected in a volume of 1 ml/kg and were dissolved in physiological saline or suspended in methylcellulose (at 25% of concentration/saline). 2.2.5. Statistical analysis Responses in the presence of the CS (CR) were divided by the trials number during the last session, and were expressed

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as a percentage. Multiple group comparisons were made using ANOVA followed by Tukey test (e.g., vehicle versus DR 4004 or SB-269970; or agonist plus antagonists and PCA). In all statistical comparisons, P < 0.05 was used as criterion for significance (n = 8 per group) and animals were used only once.

3. Results 3.1. Learning during training and testing sessions Since the magnitude of learning elicited between training and testing sessions could be variable, some of these data are depicted in Table 2, showing that there were no significant differences between training session [F(1, 79) = 1.0; P > 0.05] among control and treated groups, displaying a CR% mean of 4.2 ± 1.0. However, during testing sessions groups differed significantly [F(1, 79) = 3.2; P < 0.05], thus the control group showed a mean of 12 ± 2 of CR% and the CR% mean of the treated groups ranged from 31 ± 4 (8-OH-DPAT) to 3 ± 1 (mCPP) [F(1, 39) = 4.9; P < 0.05]. These data indicate that there were modest but significant increases in the CR% between training and testing sessions across control groups, demonstrating that the rats were learning and mastering the task. In this context, it is important to mention that recently Tomie et al. [44] have reported that not all rats exposed to the Pavlovian autoshaping task acquire the autoshaping CR, and large between-subject differences in CR acquisition and asymptotic CR maintenance have been reported in a number of species. Indeed, as occurs with other learning tasks, Pavlovian autoshaping and Pavlovian/instrumental autoshaping trained rats display variability in CR acquisition [Meneses, unpublished observations], being larger in the former group. 3.2. 5-HT7 receptors blockade, 5-HT1A/7 stimulation and 5-HT depletion ANOVA revealed no significant differences between DR 4004 (0.5–10.0 mg/kg) and SB-269970 (1.0–20.0 mg/kg) Table 2 Effects of 5-HT1A/7 receptor agonists and antagonists and PCA on conditioned responses (%) during training and testing sessions Treatment (dose in mg/kg)

Control 8-OH-DPAT (0.062) WAY 100635 (0.1) mCPP (10) PCA (10 × 2 days)

Conditioned responses (%) Training session

Testing sessiona

3±2 5±2 4±1 3±2 5±1

12 ± 2 31 ± 4b 10 ± 3 3 ± 1b 12 ± 3

a Drugs were administered immediately after training session and 24 h later testing session took placed. b Values (±S.E.) are significantly different from control saline group (P < 0.05 by Tukey test).

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20

DR4004 Saline (mg/kg) DR4004 0.1) DR4004 (1.0) DR4004 (5.0) DR4004 (10.0)

Conditioned Responses (%)

10

0 20

SB-269970

Saline (mg/kg) SB-269970 (1.0) SB-269970 (3.0) SB-269970 (10.0) SB-269970 (20.0)

10

0 Fig. 1. Effects of post-training administration (i.p.) of DR 4004 (upper) and SB-269970 (bottom) in the autoshaping task. Data are plotted as mean (±S.E.) of conditioned responses percentage (CR%). All rats received injection immediately after the first training session. Values represent of eight different animals and were analyzed by ANOVA, followed by Tukey test, ∗ P < 0.05 control saline vs. antagonist.

Fig. 2. Effects of post-training administration (i.p.) of DR 4004 and SB-269970 on the autoshaping task in fasted animals and treated with scopolamine or dizocilpine. Data are plotted as conditioned responses (CR%). All rats received injection immediately after the first training session. Values represent the mean ± S.E.M. of eight different animals. Tukey test, ∗ P < 0.05 vs. control or + scopolamine + 5-HT antagonists.

A. Meneses / Behavioural Brain Research 155 (2004) 275–282 Table 3 Effects of 8-OH-DPAT, 5-HT7 receptor antagonists and PCA pretreatment in conditioned responses (%) in the consolidation of memory Treatment (dose in mg/kg)

Conditioned responses (%)

Control 8-OH-DPAT (0.062) WAY 100635 (0.1) mCPP (10) PCA (10 × 2 days) 8-OH-DPAT (0.062) + WAY 100635 (0.1) 8-OH-DPAT (0.062) + SB-269970 (3.0) 8-OH-DPAT (0.062) + SB-269970 (10.0) 8-OH-DPAT (0.062) + DR 4004 (0.5) 8-OH-DPAT (0.062) + DR 4004 (1.0) DR 4004 (0.5) + WAY 100635 (0.1) DR 4004 (1) + mCPP (10) SB-269970 (10.0) + mCPP (10.0)

10 ± 1 32 ± 5a 10 ± 3 2 ± 1a 10 ± 3 14 ± 3b 0 ± 0a b 2 ± 2a b 3 ± 2a b 18 ± 5a 13 ± 4 10 ± 3b 7 ± 2b

PCA (10 × 2 days) + 8-OH-DPAT (0.062) PCA (10 × 2 days) + WAY 100635 (0.1) PCA (10 × 2 days) + DR 4004 (1.0) PCA (10 × 2 days) + SB-269970 (10.0)

16 ± 2b 12 ± 3 4 ± 3a 3 ± 2a

8-OH-DPAT (0.062) + DR 4004 (1.0) + WAY 100635 (0.1) 8-OH-DPAT (0.062) + SB-269970 (3.0) + WAY 100635 (0.1) PCA (10 × 2 days) + WAY 100635 (0.1) + 8-OH-DPAT (0.062) PCA (10 × 2 days) + DR 4004 (1) + 8OH-DPAT (0.062)

15 ± 4b

a b

18 ± 2b 18 ± 5b 12 ± 8b

Values are significantly different from control saline. P < 0.05 by Tukey test, vs. 5-HT drug alone.

[F(1, 79) = 1.9; P > 0.05] (Fig. 1). In contrast, and as previously reported [30,31], 8-OH-DPAT administration significantly [F(2, 23) = 3.8; P < 0.05)] increased the CR%, while mCPP decreased performance. In the interaction experiments, the CR% 8-OH-DPAT-induced increment was significantly [F(6, 55) = 7.4; P < 0.05)] reversed by DR 4004 (both doses), SB-269970 (at both doses tested), and WAY 100635 (Table 3). DR 4004 and WAY 100635 did not modify performance when administered together. In contrast, DR 4004 and SB-267790 both eliminated the mCPPinduced decrement in CR% [F(3, 31) = 3.2; P < 0.05)]. Although, PCA pretreatment had no effect alone or in combination with WAY 100635 (Table 3), PCA reversed the 8-OH-DPAT-induced increment and in the presence of DR 4004 or SB-269970, diminished it to a small but significant degree [F(9, 79) = 12.6; P < 0.001)]. Finally, compared with 8-OH-DPAT alone or in combination with WAY 100635 or DR 4004, PCA in combination with 8-OH-DPAT and WAY 100635 or 8-OH-DPAT and DR 4004 produced significant [F(14, 119) = 64.011; P < 0.001] changes in the CR% (Table 3), the latter combinations tending to reverse the WAY 100635-blockade or to facilitate the DR 4004-blockade, respectively.

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3.3. Effect of SB-269970-A and DR-4004 on scopolamine and dizocilpine-induced memory impairment Post-training (i.p.) administration of scopolamine (a centrally acting anticholinergic agent) (0.17 mg/kg) or dizocilpine (a non-competitive NMDA receptor antagonist) (0.1 mg/kg) significantly decreased the CR percentage (Fig. 2) [F(2, 23) = 4.1; P < 0.05]. When either scopolamine or dizocilpine were injected immediately after autoshaping training session and were followed by the administration of SB-269970 (3 and 10 mg/kg, i.p.) or DR 4004 (1 mg/kg, i.p.), their amnesic effects were reversed [F(8, 55) = 7.3; P < 0.01] (Fig. 2).

4. Discussion The major finding of this work is that selective 5-HT1A and 5-HT7 receptor antagonists had no effect when tested alone on memory formation but significantly inhibited the facilitatory 8-OH-DPAT effect, consistent with the involvement of 5-HT1A/7 receptors in this 8-OH-DPAT effect. Furthermore, these data support the notion that serotonergic, glutamatergic, and cholinergic systems interact in cognitively impaired animals [26,27]. These findings prompt a number of key questions: firstly, why do the 5-HT7 receptor antagonists decrease the 8-OH-DPAT enhancing effects below the baseline response seen for control animals? Why do they compensate for the mCPP decreasing effects? And why do they decrease the performance in control animals controls following PCA treatment? These apparently contradictory findings should help in the design of future experiments aimed at better understanding the functional role of the 5-HT7 receptors, particularly its “plasticity” and/or its “unmasked” activity. 4.1. Why 5-HT7 antagonists decrease the 8-OH-DPAT enhancing memory effects far below controls? In the present study, WAY 100635, DR 4004 and SB269970-A had no effect alone on memory formation but, consistent with previous findings [30,31], reversed the facilitatory effects of 8-OH-DPAT. Since these antagonists display (see Table 1) high affinity for 5-HT1A (WAY 100635) or 5-HT7 (DR 4004, SB-269970) receptors, then it likely that, both 5-HT1A and 5-HT7 receptors, mediated the memory enhancing effects of 8-OH-DPAT. Furthermore, the 5HT7 receptor antagonists decreased the 8-OH-DPAT memory enhancing effects below the baseline level for control animals. The mechanism underlying this inhibition of baseline response is unclear at present. Evaluation of further 5-HT receptor-selective ligands may help to better understand the mechanisms involved. Interestingly, in a previous study [30] DR 4004, tested at 1.0 mg/kg, partially blocked the facilitatory 8-OH-DPAT effect, which would lead to the postulate of a minor involvement of the 5-HT7 receptor in the 8-OH-

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DPAT-mediated effects. In contrast, in the present study, both SB-269970 and DR 4004 reduced performance below the level for control animals suggesting a greater degree of efficacy of these 5-HT7 receptor antagonists, relative to 5-HT1A receptors, during memory formation, likely due to differences like couple transduction systems [13,20,38], 5-HT1A receptor hyperpolarization [13] versus 5-HT7 receptors depolarization [4,5]. Notably, silent 5-HT1A receptor antagonists reversed the facilitatory effects of 8-OH-DPAT, down to the control level [30,31]. Hence, the present data would suggest a dominant role for the 5-HT7 receptor during Pavlovian/instrumental memory formation, which would be “unmasked” under an altered memory state (i.e., either facilitated or impaired memory). In this regard, cortical and hippocampal cyclic adenosine monophosphate (cAMP) production has been reported to be associated with memory consolidation via 5-HT1A and 5-HT7 receptors [24]. Furthermore, age-related memory deficits are associated with changes in 5-HT receptors expression, as labelled with [3 H]-5-HT, in cortical and hippocampal areas ([33]; see [26]). Moreover, 5-HT1A or 5HT7 receptor antagonists ([30,31]; this work) did not alter learning and memory in normal animals notwithstanding, reverse deficits in memory. Hence, under these conditions the role of either 5-HT1A or 5-HT7 receptors changes from silence to active ones. Notably, 5-HT1A −/− receptor knockout mice showed hippocampal and memory dysfunction [39] and 5-HT7 −/− receptor mice show a selective impairment in contextual fear conditioning [9], suggesting a task-dependent impairment. This latter finding appears consistent with the data from the present study showing that 5-HT7 receptor antagonists have no effect in normal rats during memory consolidation. Therefore, what mechanisms underlie the effect of 5-HT7 receptor antagonists under amnesic conditions? 4.2. Why do 5-HT7 receptor antagonists compensate for mCPP decreasing effects? 5-HT7 receptor antagonists reversed the cognitive deficits induced by scopolamine (a muscarinic antagonist), dizocilpine (a glutamatergic non-competitive antagonist) and mCPP (a 5-HT agonist/antagonist). Since mCPP displays affinity for several 5-HT receptors (see Table 1), including 5-HT1A , 5-HT2 and 5-HT7 receptors, this may explain the antagonism by WAY 100635, SB-2669970 and DR 4004 of the mCPP-induced amnesic effects. Further characterization of the role of 5-HT7 receptors in memory formation, using a number of drugs displaying varying affinity for and intrinsic activity at the 5-HT7 receptor (Table 1), ranging from agonists to antagonists, revealed that whereas SB-269970 or DR 4004 selectively reversed the memory deficit induced by mCPP, mesulergine and ketanserin, 5-HT2A/2C receptor antagonists with lower affinity for the 5-HT7 receptor, were inactive (data not shown). These findings suggest that during memory formation, the effect of mCPP (but not mesulergine or ketanserin) is partly via 5-HT7 receptors. However, it should be noted that, mCPP displays affinity for multiple

5-HT receptors (Table 1) and multiple 5-HT receptors appear to mediate its effects in memory consolidation, including 5-HT1B , 5-HT2A/2C , 5-HT3 and 5-HT7 receptors [26]. Notably, SB-200646 (a selective 5-HT2B/2C receptor antagonist) and LY215840 (a non-selective 5-HT2/7 receptor antagonist) had no effect on autoshaped memory consolidation [30]; however, both drugs significantly antagonized the memory-improvement (DOI and ketanserin) or impairment (mCPP, mesulergine, N-(3-trifluoromethylphenyl) piperazine [TFMPP]) [27,30,31]. However, SB-200646 failed to modify the facilitatory procognitive effect produced by DOI or ketanserin, which were sensitive to the selective 5-HT2A receptor antagonist MDL100907 and the 5-HT2/7 receptor antagonist LY215840 (high dose) [27]. More importantly in this context, MDL100907 or SB-200646A failed to modify the 8-OH-DPAT-facilitatory effect [27,30,31]. Moreover, since these 5-HT7 receptor antagonists have no effect in normal animals, it should be noted that, when modeling dysfunctional memory formation, as observed with cholinergic or glutamatergic blockade and aging, serotonergic function appears to be modified [26]. Indeed, under altered memory conditions in humans and animal models, a number of markers, including 5-HT receptors expression are also altered ([26,32]; see [29] for review). For example, trained adult rats showed better retention than old animals and the hippocampal CA1 area and dentate gyrus of adult trained rats showed lower 5-HT receptor relative to adult untrained rats, as defined using [3 H]-5-HT binding [32]. Since, 5-HT receptors display different affinities for 5-HT (see [26]), it would helpful to study [3 H]-5-HT (or other selective marked ligands) binding data under pharmacologically ’blocked’ conditions to define specifically 5-HT7 and/or 5HT1A and clarify the difference between changes in receptor expression level and receptor function. The above data suggest that, memory formation modulates 5-HT receptors expression in brain areas relevant to memory systems. In contrast to adult trained rats, in aged animals the dentate gyrus and basomedial amygdaloid nucleus showed a higher expression of 5-HT receptors relative to aged untrained and trained animals, which would suggest that hippocampal 5-HT receptor expression is related to memory formation and dysfunctional memory in aging [32]. If, receptor function also changes under altered memory conditions then it is possible that that otherwise ‘silent’ 5-HT1A and 5-HT7 receptors (as showed herein), could become responsive to agonists and antagonists for these receptors, so normalizing memory consolidation. That is, under procognitive or amnesic conditions 5-HT7 receptors activity would be “unmasked”, leading to a modulatory role in memory formation. Interestingly, 5-HT7 −/− mice showed a selective impairment in contextual fear conditioning [9]. Doubtless, it will be important to determine in naturalistic or pharmacological models of amnesia the performance of 5-HT1A −/− and 5-HT7 −/− mice or animals overexpressing them. The data in the present study suggest that, animal models of the cognitive deficits seen in AD appear to reveal the interac-

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tion between 5-HT and other neurotransmitter systems implicated in memory (such as glutamatergic and cholinergic) as well as possible key mechanisms underlying age-related to memory decline. Notably, following PCA pretreatment, 5-HT7 receptor antagonists decreased animals performance below the controls level, demonstrating that at least under an impaired-memory condition 5-HT7 receptors function may be unmasked, as observed under AD amnesia and 5-HT depletion. Indeed, Lawlor et al. [20] found that mCPP produced a significantly greater deficit in recent memory and knowledge memory in AD patients than in controls and the authors argued that these findings could not be explained by pharmacokinetic differences across populations; indeed, mCPP may be associated with damage to brain serotonergic neurons or other neuronal systems that interact with them [20]. 4.3. Why do 5-HT7 receptor antagonists decrease the performance under controls following PCA treatment? Previous evidence [26] suggests that neither 5-HT depletion nor synthesis inhibition altered an autoshaping Pavlovian/instrumental learning task, however, these drugs have allowed changes induced by 5-HT receptor agonists and antagonists to be detected. Thus, comparing with the 5-HT1A receptor, the present data suggest a dominant role for 5-HT7 during memory formation which was “unmasked” under either procognitive or amnesic conditions. Under amnesic conditions, 5-HT1A and/or 5-HT7 or both receptors, may have a functional role. As reported in the present study, 5-HT7 antagonists decrease the 8-OH-DPAT enhancing effects far below the control level and decreased the performance under controls after PCA. As already mentioned, there is evidence that serotonergic receptors could be altered under amnesic conditions such as AD [26]. Interestingly, 5-HT receptors may need to be differentially modulated to optimally enhanced cognition and, these in turn, depend on the underlying pathology (see [26,37]), which suggests 5-HT1A receptor ligands could show therapeutic utility in the treatment of memory dysfunctions (see, e.g., [31,40], respectively). Finally, PCA together with 8-OH-DPAT, WAY 100635 or DR 4004 normalized learning consolidation, had no effects or slightly decreased performance, respectively; thus suggesting the involvement of presynaptic 5-HT1A and (or 5-HT7 ?) receptor mechanism during memory formation.

5. Conclusions The present data have confirmed and extended previous findings regarding: (1) 8-OH-DPAT-facilitatory effects on learning consolidation mediate by 5-HT1A and 5-HT7 receptors; (2) the lack of effect of 5-HT7 receptors antagonists on memory formation in normal animals; (3) a possible presynaptic 5-HT1A (and/or 5-HT7 ) receptors involvement in amnesic conditions; and (4) the normalization by 5-HT7 receptor antagonists of memory deficits associated with sero-

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tonergic, cholinergic and glutamatergic systems. Importantly, the presence of 5-HT7 receptors in the hippocampus and the entorhinal cortex, the major input to the hippocampus, supports a potential involvement of 5-HT7 receptors in learning and memory [25].

Acknowledgements The authors thank the pharmaceutical companies for their generous gifts (see Section 2.2.4). This work was partially supported by CONACYT Grant 39534-M. I thank Sofia Meneses-Goytia for revising language, and Roberto Gonzalez for his expertise assistance. I want to thank David R. Thomas for revised the language in final version and for his excellent comments. Finally, I would like to thank anonymous reviewers for their helpful comments, and Prof. J.P. Huston for his support to our work.

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