- Email: [email protected]
Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task
Behavioural Brain Research 163 (2005) 136–140
Short communication
Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task Georgina S. Perez-Garc´ıa, A. Meneses ∗ Department of Pharmacobiology, CINVESTAV-IPN, Tenorios #235, Granjas Coapa, M´exico City 14330, M´exico Received 25 February 2005; received in revised form 19 April 2005; accepted 20 April 2005 Available online 3 June 2005
Abstract This work aimed to evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Post-training administration of the potential 5-HT7 receptor agonist AS 19 or antagonist SB-269970 enhanced memory formation or had no effect, respectively. The AS 19 facilitatory effect was reversed by SB-269970, but not by the selective 5-HT1A antagonist WAY100635. Amnesia induced by scopolamine (cholinergic antagonist) or dizocilpine (NMDA antagonist) was also reversed by AS 19. Certainly, reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool. Having mentioned that, it should be noticed that together these data are providing further support to the notion of the 5-HT7 receptors role in memory formation. Importantly, this 5-HT7 receptor agonist AS 19 appears to represent a step forward respect to the notion that potent and selective 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer’s disease. © 2005 Elsevier B.V. All rights reserved. Keywords: Autoshaping; 5-HT receptors; Memory; Learning; Rat
1. Introduction A growing body of evidence from snails to humans shows that serotonin (5-hydroxytryptamine, 5-HT) input participates in memory formation [6,7]. It remains unclear if this 5-HT influence is tonic or phasic, involving different 5-HT receptors (for review, see [7]) and/or multiple cell signaling pathways [12]. Actually, compromised serotonergic function may have an important contribution to cognitive decline related to aging, Alzheimer’s disease (AD) [1,2] and schizophrenia. Serotonergic dysfunction represents a potential target for treatment of memory dysfunctions [6,7], and opens opportunities for the exploration of 5-HT agonists, antagonists, inverse agonists and mix activity ligands (agonists/antagonists). Recent evidence indicates that the 5-HT1A/7 receptor agonist 8-OH-DPAT facilitated memory formation, and such an effect was blocked by the ∗
Corresponding author. Tel.: +52 55 50612869; fax: +52 55 50612863. E-mail address: [email protected] (A. Meneses).
0166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.04.014
selective 5-HT1A receptor antagonist WAY 100635 and 5HT7 antagonist SB-269970 [8]. Until recently there were not selective 5-HT7 receptor agonists. Hence, in this work, we aim to re-evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Herein, post-training administration of the potentially selective 5-HT7 receptor agonist (2S)-(+)-8(1,3,5-trimethylpyrazolin-4-yl)-2-(dimethylamino) tetralin (AS 19) (binding IC50 = 0.83 nM; Tocris; see Sanin et al. [13] or the selective 5-HT1A (WAY 100635) or 5-HT7 receptor antagonist (SB-269970) [8] were tested. In addition, AS 19 was also evaluated in the pharmacological models of amnesia induced by scopolamine (cholinergic antagonist) and dizocilpine (NMDA antagonist).
2. Materials and methods Male Wistar rats (12-week-old) were collectively housed in a temperature- and light-controlled room under a 12:12 h light/dark
G.S. Perez-Garc´ıa, A. Meneses / Behavioural Brain Research 163 (2005) 136–140
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. The autoshaping test has been previously described (see [7]). Briefly, male Wistar rats (12-week-old) were collectively housed (12/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/group was 4–8 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. The local institutional committee for the use of animal subjects approved the present experimental protocol (project no. 047/02). 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. 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 [7], 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 [7]. All compounds and vehicle were injected (i.p. or s.c.) immediately after the first autoshaping session and the session test had place 24 h later. The results shown represent this latter autoshaping session. It is worth noting that extensive evidence exists [7; see 11 for references] which suggest 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. 2.1. Experimental protocol Except for dose–response of AS 19, all other doses were selected from previous works [7,8]. Immediately after the first autoshaping training session the animals receiving either vehicle propylene glycol (0.95%) in a volume of 1 ml/kg or AS 19 (0.5–10.0 mg/kg, s.c.)
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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 and 5-HT7 receptors, AS 19 (5.0 mg/kg, s.c.) was administered immediately after the autoshaping training session and 5 min later they received WAY 100635 (0.3 mg/kg, s.c.), or SB-269970 (10.0 mg/kg, i.p.). In all cases, the session test was performed 24 h later. To further study AS 19 cognitive effects, scopolamine or dizocilpine-induced cognitive deficits were used, thus independent animals were given AS 19 (5.0 mg/kg) coadministered with either scopolamine (0.17 mg/kg) or dizocilpine (0.1 mg/kg) and the test session was performed 24 h later. The drugs used were: (2S)-(+)-8-(1,3,5-trimethylpyrazolin4-yl)-2-(dimethylamino) tetralin (AS 19) (Tocris, Ellisville, MO) [3,13], N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N(2-pyridinyl) cyclohexanene carboxamide trihydrochloride (WAY 100635), dizocilpine HCl, scopolamine HCl (from Sigma), (R)-3(2-(2-(4-methylpiperidin-1-yl) ethyl) pyrrolidine-1-sulfonyl) phenol (SB-269970) donated by Dr. D. Thomas, GlaxoSmithKline, Harlow Essex, UK). Responses in the presence of the CS (CR) were divided by the trials number during the last session, and were expressed as a percentage. Multiple group comparisons were made using ANOVA followed by Tukey’s test (e.g. vehicle versus AS 19 or SB-269970; or agonist plus antagonists and amnesics). In all statistical comparisons, P < 0.05 was used as criterion for significance (n = 8/group) and animals were used only once. 2.2. 5-HT7 receptors stimulation enhanced memory and reversed amnesia ANOVA revealed significant differences between vehicle and treated AS 19 groups [F(4, 24) = 24.18; P < 0.0001)], AS 19 at 5.0 mg/kg (upper Fig. 1) increased significantly CR%. In contrast, and as previously reported [7,8] neither WAY 100635 nor SB269970 administration had effect (bottom Fig. 1); nonetheless, SB269970 but not WAY 100635, significantly antagonized the AS 19 facilitatory effect [F(9,79) = 83.73 P < 0.0001]. In further interaction experiment series, 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 and these effects were significantly [F(9,79) = 83.73 P < 0.0001] reversed by AS 19 (bottom Fig. 1).
3. Discussion The major finding of the present work is that the 5HT7 receptor agonist AS 19 facilitated memory formation and significantly inhibited the amnesic-induced effects of scopolamine or dizocilpine, which is consistent with previous evidence (see below). These data support the notion that serotonergic, glutamatergic, and cholinergic systems interact in cognitively impaired animals [6,7]. Since a growing interest in 5-HT7 receptors investigation concerning a number of functions and pathologies, including AD and schizophrenia (see [2,15,16]), these findings prompt key questions: is AS 19 selectivity for 5-HT7 receptors? Does display AS 19 affinities for other 5-HT receptors? According with the information provided [13] by Tocris AS 19 is a selective
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G.S. Perez-Garc´ıa, A. Meneses / Behavioural Brain Research 163 (2005) 136–140
Fig. 1. Dose–response effects of post-training administration of AS 19 (upper) and AS 19 plus 5-HT1A (WAY 100635) and 5-HT7 (SB-269970) receptor antagonists and amnesic drugs (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’s test, * P < 0.05 control vehicle vs. antagonist; + AS 19 vs. AS + 5-HT antagonists or amnesic drugs.
5-HT7 receptor agonist with a potency of IC50 = 0.83 nM. Unfortunately Tocris was unable to provide us any further information about the affinity of AS 19 for 5-HT7 and other receptors. As Tocris gives as reference Sanin et al. [13], then we tried to email these authors; unfortunately, we were unable to contact them. Certainly, we lack precise information about AS 19 affinities for 5-HT7 receptors, pharmacokinetic profile in vivo, what brain levels of AS 19 would be achieved and degree of 5-HT7 receptor occupancy would be expected over the dose range tested. Although AS 19 shows some selectivity for 5-HT7 in terms of affinity, no data appear to be available regarding affinity for, e.g. the 5-HT1B/D , or 5HT2C subtypes, which are localized in brain regions implicated in learning and memory [5,6] and it is, therefore, not impossible that the effect of AS 19 (e.g. to reverse memory deficits) might be mediated at least in part via one or more of these subtypes. Doubtless, it is necessary to have precise information if AS 19 is or is not selective a 5-HT7 receptors agonist. AS 19 seems to have a reasonably good affinity for 5-HT7 receptors ([3], see also [13]), and herein we have tried a wide range of doses. Particularly relevant are the antiamnesics AS 19 effects, considering the centrality of the cholinergic deficit hypothesis in the study of Alzheimer’s disease (AD), along with, the glutamatergic tone which more recently has been shown to be integral to neuronal plasticity and memory [1,14]. Likewise, is important the growing evidence of 5-HT system role in physiological, pathophysiological and therapeutic in cognitive processes [6,7]. As noticed earlier, administration of AS 19 reversed the amnesic
effects induced by scopolamine (cholinergic antagonists) and dizocilpine (glutamate antagonists), which confirmed previous evidence produced by 8-OH-DPAT [8]. The facilitation of memory formation and the anti-amnesic effect produced by AS 19 is consistent with similar evidence reported with the 5-HT1A/7 receptor agonist 8-OH-DPAT, which was antagonized by selective 5-HT1A or 5-HT7 receptor antagonists [8]. In this context, it should be bear in mind that the above antiamnesic effects of AS 19 might appear inconsistent with a recent paper from our own laboratory [8], where we reported that the 5-HT7 antagonists SB-269970 and DR4004 reversed memory deficits. Notably, these 5-HT7 receptor antagonists had no effects when tested alone during memory formation [8]. A similar contradictory situation apparently occurs regarding 5-HT1A receptors. For instance, in normal learning and memory, 5-HT1A receptors blockade have no effect, while their stimulation (dependent on dosage) may impair or improve performance; in contrast, under amnesic conditions both 5-HT1A receptor antagonists and agonists may be effective antiamnesic (see [7,8]). We recently drawn attention to several key questions [8], being particularly relevant to the present context that the 5-HT7 (and of course, 5-HT1A ) receptors “plasticity” and/or “unmasked” activity is apparent under amnesic conditions. Notably, cortical and hippocampal increased on cAMP production was observed following administration of the 5-HT1A/7 agonist 8-OH-DPAT, while only the memory effect was, completely or partially, reversed by the selective antagonists WAY100635 (5-HT1A ) or DR4004 (5-HT7 ), respectively [5]. Ex vivo receptor au-
G.S. Perez-Garc´ıa, A. Meneses / Behavioural Brain Research 163 (2005) 136–140
toradiography using the ligand [3 H]8-OH-DPAT revealed [4] that the trained group relative to untrained animals showed increases of 5-HT1A receptor expression in 14 brain areas, decrements in 7, and no changes in 12. Thus, in contrast to untrained rats, 5-HT1A receptor expression of autoshaping trained rats was augmented in the tubercule olfactory, septal nucleus, nucleus accumbens, caudate putamen, globus pallidus, striate, and parietal [1,2], temporal cortex [1,3], granular retrosplenial cortex [1], amygdala, and median and dorsal raphe nuclei. In contrast, in the latter group, receptors were decreased in the CA1 area, hypothalamus dorsal, frontal cortex [1,3], occipital cortex, cingulate cortex [1,2], and cuneiform nucleus. These data suggest that upregulated, downregulated, and “silence” of 5-HT1A (and likely 5-HT7 ) receptors in brain areas form part of neural circuits engaged in memory formation by demonstrating a high degree of specificity and memory mapping. Importantly, 8-OH-DPATinduced phase-shift in the suprachiasmatic nucleus required of transcription and translation of proteins (see [16], for references). Actually, autoshaped long-term memory formation requires synthesis of proteins [9]. All together these data provide support to the notion that 5-HT7 receptors play a role in normal and impaired memory and offer clues about the design of future experiments aimed to a better understanding of the functional role of these receptors. Certainly, the lack of a dose–response relation between cognitive facilitation and AS 19 dosage and considering that its active dose, both for procognitive and antiamnesic effects, was 5.0 mg/kg, then the range of dosage and potency of compounds like this should be significantly improved. Although the present study used only systemic treatments, some neuroanatomical considerations are worthy to notice. The 5-HT7 receptors have been localized in brain areas, such as anterior thalamus, the dentate gyrus, hypothalamus, anterior cingulate gyrus, hippocampus, amygdala and certain brainstem nuclei [15–17]. Notably, as previously mentioned autoshaping training involved basolateral amygdaloidal nucleus, hippocampal CA1 area and dentate gyrus, hypothalamic area and frontal, parietal, temporal, occipital, retrosplenial agranular and granular cortices, and medial raphe nucleus [4,5,10]. This is probably very important, regarding memory facilitation, since extensive studies have shown that the amygdala, and especially the basolateral amygdala is critically important in mediating post-training drug effects on memory formation itself ([1,14]; see [11] for references). Moreover, muscarinic cholinergic activation, in particular in amygdala and to lesser extent in the cortex, has been shown to be especially important in memory modulation during consolidation [11]. Indeed, muscarinic cholinergic activity has been shown to interact with other neurotransmitter systems in the basolateral amygdala. This evidence provides further support to notion that the 5HT7 receptor agonists may influence memory consolidation by interacting with muscarinic cholinergic receptor activity in the basolateral amygdala. Of course, other possible interactions involving the cortex, nucleus basalis, hippocampus/medial septum system could exist. Notably, in the con-
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text of instrumental acquisition, prefrontal cortex has been conceptualized to represent declaratively the contingencies between instrumental actions and their outcomes [7]. These conceptualizations have practical implications, including the fact that autoshaping Pavlovian/instrumental task combines, both Pavlovian and instrumental conditioning, which offers the opportunity to study hippocampus-mediated declarative memory and striatum-mediated S–R “habit formation” [7]. It detects little changes (increases or decreases) on diverse behavioral parameters not redundant (i.e., not measuring the same event), including the sign tracking (i.e., conditioned behavior directed toward the localized retractable and illuminated lever, CS), and the goal tracking (i.e., the place where the US is delivered) [7]. These characteristics are quite important, since they allow the study of bi-directional expression of an enhanced or impaired memory formation, and maybe more importantly, is to study separately motivated behavior (i.e., goal tracking) and learned responses (i.e., autoshaped responses) [7]. In conclusion, administration of AS 19 improved memory consolidation in na¨ıve animals. Modeling the potential therapeutic benefits of 5-HT7 receptor stimulation, acute administration of AS 19 reversed memory deficits produced by scopolamine or dizocilpine. Hence, potent 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer’s disease. Certainly reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo, are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool.
Acknowledgements Authors thank GlaxoSmithKline for its generous gifts (see drugs section). This work was partially supported by the CONACYT Scholarship 185583 (G.S.P.G.) and Grant 39534M (A.M.). We thank Sofia Meneses-Goytia for revised language, and Roberto Gonzalez for his expertise and assistance. Finally, authors want to deeply thank the reviewer’s comments, which were very useful and their incorporation significantly improved the original version of our manuscript.
References [1] Berger-Sweeney J, Caliguri EJ, Frick KM. Neurochemical systems involved in learning and memory. In: Charney DS, Nestler EJ, editors. Neurobiology of mental illness. 2nd ed. New York: Oxford University Press; 2004. p. 813–20. [2] Hedlund PB, Sutcliffe JG. Functional, molecular and pharmacological advances in 5-HT7 receptor research. Trends Pharmacol Sci 2004;25:481–6. [3] Holmberg P, Sohn D, Leideborg R, Caldirola P, Zlatoidsky P, Hanson S, et al. Novel 2-aminotetralin and 3-aminochroman derivatives as selective serotonin 5-HT7 receptor agonists and antagonists. J Med Chem 2004;47:3927–30.
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[4] Luna-Munguia H, Manuel-Apolinar L, Rocha L, Meneses A. 5-HT1A receptor expression during memory formation. Psychopharmacology (Berl) 19 March 2005 [Epub ahead of print]. [5] 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. [6] Meneses A. 5-HT system and cognition. Neurosci Biobehav Rev 1999;8:1111–25. [7] Meneses A. A pharmacological analysis of an associative learning task: function of 5-HT1 to 5-HT7 receptor subtypes on a Pavlovian/instrumental autoshaped memory. Learn Mem 2003;10:363–72. [8] Meneses A. The effects of the 5-HT7 receptor antagonists SB-269970 and DR 4004 in autoshaping Pavlovian/instrumental learning task. Behav Brain Res 2004;155:275–82. [9] Meneses A, Huerta AG, Valdez D. 5-HT system function on shortand long-term memory. Int J Nueropsychopharmacol 2000;7(Suppl. 1):14. [10] 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. [11] Perez-Garc´ıa G, Meneses A. Oral administration of the 5-Ht6 receptor antagonists Sb-357134 and SB-399885 improves memory forma-
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tion in an autoshaping learning task. Pharmacol Biochem Behav, in press. Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, et al. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther 2001;92:179–212. Sanin A, Brisander M, Rosqvist S, Mohell N, Malberg A, Johansson A. 5-Aryl substituted (S)-2-(dimethylamino)-tetralins novel serotonin 5HT7 receptor ligands. In: Proceedings of the 14th Camerino-Noord Symposium. Ongoing Progress in the Receptor Chemistry; 2004. p. 27. Santucci AC, Haroutunian V. Pharmacological and transgenic animal models of Alzheimer’s disease. In: Charney DS, Nestler EJ, editors. Neurobiology of mental illness. 2nd ed. New York: Oxford Unversity Press; 2004. p. 791–806. Slassi A, Isaac M, Xin T. Recent progress in 5-HT7 receptors: potential treatment of central and peripheral nervous system diseases. Expert Opin Ther Patents 2004;14:1009–27. Thomas DR, Hagan JJ. 5-HT7 receptors. Curr Drug Targets, CNS Neurol Dis 2004;3:81–90. Varn¨as K, Thomas DR, Tupala E, Tiihonen J, Hall H. Distribution of 5-HT7 receptors in the human brain: a preliminary autoradiographic study using [3 H]SB-269970. Neurosci Lett 2004;367:313–6.
Short communication
Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task Georgina S. Perez-Garc´ıa, A. Meneses ∗ Department of Pharmacobiology, CINVESTAV-IPN, Tenorios #235, Granjas Coapa, M´exico City 14330, M´exico Received 25 February 2005; received in revised form 19 April 2005; accepted 20 April 2005 Available online 3 June 2005
Abstract This work aimed to evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Post-training administration of the potential 5-HT7 receptor agonist AS 19 or antagonist SB-269970 enhanced memory formation or had no effect, respectively. The AS 19 facilitatory effect was reversed by SB-269970, but not by the selective 5-HT1A antagonist WAY100635. Amnesia induced by scopolamine (cholinergic antagonist) or dizocilpine (NMDA antagonist) was also reversed by AS 19. Certainly, reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool. Having mentioned that, it should be noticed that together these data are providing further support to the notion of the 5-HT7 receptors role in memory formation. Importantly, this 5-HT7 receptor agonist AS 19 appears to represent a step forward respect to the notion that potent and selective 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer’s disease. © 2005 Elsevier B.V. All rights reserved. Keywords: Autoshaping; 5-HT receptors; Memory; Learning; Rat
1. Introduction A growing body of evidence from snails to humans shows that serotonin (5-hydroxytryptamine, 5-HT) input participates in memory formation [6,7]. It remains unclear if this 5-HT influence is tonic or phasic, involving different 5-HT receptors (for review, see [7]) and/or multiple cell signaling pathways [12]. Actually, compromised serotonergic function may have an important contribution to cognitive decline related to aging, Alzheimer’s disease (AD) [1,2] and schizophrenia. Serotonergic dysfunction represents a potential target for treatment of memory dysfunctions [6,7], and opens opportunities for the exploration of 5-HT agonists, antagonists, inverse agonists and mix activity ligands (agonists/antagonists). Recent evidence indicates that the 5-HT1A/7 receptor agonist 8-OH-DPAT facilitated memory formation, and such an effect was blocked by the ∗
Corresponding author. Tel.: +52 55 50612869; fax: +52 55 50612863. E-mail address: [email protected] (A. Meneses).
0166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.04.014
selective 5-HT1A receptor antagonist WAY 100635 and 5HT7 antagonist SB-269970 [8]. Until recently there were not selective 5-HT7 receptor agonists. Hence, in this work, we aim to re-evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Herein, post-training administration of the potentially selective 5-HT7 receptor agonist (2S)-(+)-8(1,3,5-trimethylpyrazolin-4-yl)-2-(dimethylamino) tetralin (AS 19) (binding IC50 = 0.83 nM; Tocris; see Sanin et al. [13] or the selective 5-HT1A (WAY 100635) or 5-HT7 receptor antagonist (SB-269970) [8] were tested. In addition, AS 19 was also evaluated in the pharmacological models of amnesia induced by scopolamine (cholinergic antagonist) and dizocilpine (NMDA antagonist).
2. Materials and methods Male Wistar rats (12-week-old) were collectively housed in a temperature- and light-controlled room under a 12:12 h light/dark
G.S. Perez-Garc´ıa, A. Meneses / Behavioural Brain Research 163 (2005) 136–140
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. The autoshaping test has been previously described (see [7]). Briefly, male Wistar rats (12-week-old) were collectively housed (12/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/group was 4–8 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. The local institutional committee for the use of animal subjects approved the present experimental protocol (project no. 047/02). 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. 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 [7], 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 [7]. All compounds and vehicle were injected (i.p. or s.c.) immediately after the first autoshaping session and the session test had place 24 h later. The results shown represent this latter autoshaping session. It is worth noting that extensive evidence exists [7; see 11 for references] which suggest 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. 2.1. Experimental protocol Except for dose–response of AS 19, all other doses were selected from previous works [7,8]. Immediately after the first autoshaping training session the animals receiving either vehicle propylene glycol (0.95%) in a volume of 1 ml/kg or AS 19 (0.5–10.0 mg/kg, s.c.)
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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 and 5-HT7 receptors, AS 19 (5.0 mg/kg, s.c.) was administered immediately after the autoshaping training session and 5 min later they received WAY 100635 (0.3 mg/kg, s.c.), or SB-269970 (10.0 mg/kg, i.p.). In all cases, the session test was performed 24 h later. To further study AS 19 cognitive effects, scopolamine or dizocilpine-induced cognitive deficits were used, thus independent animals were given AS 19 (5.0 mg/kg) coadministered with either scopolamine (0.17 mg/kg) or dizocilpine (0.1 mg/kg) and the test session was performed 24 h later. The drugs used were: (2S)-(+)-8-(1,3,5-trimethylpyrazolin4-yl)-2-(dimethylamino) tetralin (AS 19) (Tocris, Ellisville, MO) [3,13], N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N(2-pyridinyl) cyclohexanene carboxamide trihydrochloride (WAY 100635), dizocilpine HCl, scopolamine HCl (from Sigma), (R)-3(2-(2-(4-methylpiperidin-1-yl) ethyl) pyrrolidine-1-sulfonyl) phenol (SB-269970) donated by Dr. D. Thomas, GlaxoSmithKline, Harlow Essex, UK). Responses in the presence of the CS (CR) were divided by the trials number during the last session, and were expressed as a percentage. Multiple group comparisons were made using ANOVA followed by Tukey’s test (e.g. vehicle versus AS 19 or SB-269970; or agonist plus antagonists and amnesics). In all statistical comparisons, P < 0.05 was used as criterion for significance (n = 8/group) and animals were used only once. 2.2. 5-HT7 receptors stimulation enhanced memory and reversed amnesia ANOVA revealed significant differences between vehicle and treated AS 19 groups [F(4, 24) = 24.18; P < 0.0001)], AS 19 at 5.0 mg/kg (upper Fig. 1) increased significantly CR%. In contrast, and as previously reported [7,8] neither WAY 100635 nor SB269970 administration had effect (bottom Fig. 1); nonetheless, SB269970 but not WAY 100635, significantly antagonized the AS 19 facilitatory effect [F(9,79) = 83.73 P < 0.0001]. In further interaction experiment series, 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 and these effects were significantly [F(9,79) = 83.73 P < 0.0001] reversed by AS 19 (bottom Fig. 1).
3. Discussion The major finding of the present work is that the 5HT7 receptor agonist AS 19 facilitated memory formation and significantly inhibited the amnesic-induced effects of scopolamine or dizocilpine, which is consistent with previous evidence (see below). These data support the notion that serotonergic, glutamatergic, and cholinergic systems interact in cognitively impaired animals [6,7]. Since a growing interest in 5-HT7 receptors investigation concerning a number of functions and pathologies, including AD and schizophrenia (see [2,15,16]), these findings prompt key questions: is AS 19 selectivity for 5-HT7 receptors? Does display AS 19 affinities for other 5-HT receptors? According with the information provided [13] by Tocris AS 19 is a selective
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Fig. 1. Dose–response effects of post-training administration of AS 19 (upper) and AS 19 plus 5-HT1A (WAY 100635) and 5-HT7 (SB-269970) receptor antagonists and amnesic drugs (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’s test, * P < 0.05 control vehicle vs. antagonist; + AS 19 vs. AS + 5-HT antagonists or amnesic drugs.
5-HT7 receptor agonist with a potency of IC50 = 0.83 nM. Unfortunately Tocris was unable to provide us any further information about the affinity of AS 19 for 5-HT7 and other receptors. As Tocris gives as reference Sanin et al. [13], then we tried to email these authors; unfortunately, we were unable to contact them. Certainly, we lack precise information about AS 19 affinities for 5-HT7 receptors, pharmacokinetic profile in vivo, what brain levels of AS 19 would be achieved and degree of 5-HT7 receptor occupancy would be expected over the dose range tested. Although AS 19 shows some selectivity for 5-HT7 in terms of affinity, no data appear to be available regarding affinity for, e.g. the 5-HT1B/D , or 5HT2C subtypes, which are localized in brain regions implicated in learning and memory [5,6] and it is, therefore, not impossible that the effect of AS 19 (e.g. to reverse memory deficits) might be mediated at least in part via one or more of these subtypes. Doubtless, it is necessary to have precise information if AS 19 is or is not selective a 5-HT7 receptors agonist. AS 19 seems to have a reasonably good affinity for 5-HT7 receptors ([3], see also [13]), and herein we have tried a wide range of doses. Particularly relevant are the antiamnesics AS 19 effects, considering the centrality of the cholinergic deficit hypothesis in the study of Alzheimer’s disease (AD), along with, the glutamatergic tone which more recently has been shown to be integral to neuronal plasticity and memory [1,14]. Likewise, is important the growing evidence of 5-HT system role in physiological, pathophysiological and therapeutic in cognitive processes [6,7]. As noticed earlier, administration of AS 19 reversed the amnesic
effects induced by scopolamine (cholinergic antagonists) and dizocilpine (glutamate antagonists), which confirmed previous evidence produced by 8-OH-DPAT [8]. The facilitation of memory formation and the anti-amnesic effect produced by AS 19 is consistent with similar evidence reported with the 5-HT1A/7 receptor agonist 8-OH-DPAT, which was antagonized by selective 5-HT1A or 5-HT7 receptor antagonists [8]. In this context, it should be bear in mind that the above antiamnesic effects of AS 19 might appear inconsistent with a recent paper from our own laboratory [8], where we reported that the 5-HT7 antagonists SB-269970 and DR4004 reversed memory deficits. Notably, these 5-HT7 receptor antagonists had no effects when tested alone during memory formation [8]. A similar contradictory situation apparently occurs regarding 5-HT1A receptors. For instance, in normal learning and memory, 5-HT1A receptors blockade have no effect, while their stimulation (dependent on dosage) may impair or improve performance; in contrast, under amnesic conditions both 5-HT1A receptor antagonists and agonists may be effective antiamnesic (see [7,8]). We recently drawn attention to several key questions [8], being particularly relevant to the present context that the 5-HT7 (and of course, 5-HT1A ) receptors “plasticity” and/or “unmasked” activity is apparent under amnesic conditions. Notably, cortical and hippocampal increased on cAMP production was observed following administration of the 5-HT1A/7 agonist 8-OH-DPAT, while only the memory effect was, completely or partially, reversed by the selective antagonists WAY100635 (5-HT1A ) or DR4004 (5-HT7 ), respectively [5]. Ex vivo receptor au-
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toradiography using the ligand [3 H]8-OH-DPAT revealed [4] that the trained group relative to untrained animals showed increases of 5-HT1A receptor expression in 14 brain areas, decrements in 7, and no changes in 12. Thus, in contrast to untrained rats, 5-HT1A receptor expression of autoshaping trained rats was augmented in the tubercule olfactory, septal nucleus, nucleus accumbens, caudate putamen, globus pallidus, striate, and parietal [1,2], temporal cortex [1,3], granular retrosplenial cortex [1], amygdala, and median and dorsal raphe nuclei. In contrast, in the latter group, receptors were decreased in the CA1 area, hypothalamus dorsal, frontal cortex [1,3], occipital cortex, cingulate cortex [1,2], and cuneiform nucleus. These data suggest that upregulated, downregulated, and “silence” of 5-HT1A (and likely 5-HT7 ) receptors in brain areas form part of neural circuits engaged in memory formation by demonstrating a high degree of specificity and memory mapping. Importantly, 8-OH-DPATinduced phase-shift in the suprachiasmatic nucleus required of transcription and translation of proteins (see [16], for references). Actually, autoshaped long-term memory formation requires synthesis of proteins [9]. All together these data provide support to the notion that 5-HT7 receptors play a role in normal and impaired memory and offer clues about the design of future experiments aimed to a better understanding of the functional role of these receptors. Certainly, the lack of a dose–response relation between cognitive facilitation and AS 19 dosage and considering that its active dose, both for procognitive and antiamnesic effects, was 5.0 mg/kg, then the range of dosage and potency of compounds like this should be significantly improved. Although the present study used only systemic treatments, some neuroanatomical considerations are worthy to notice. The 5-HT7 receptors have been localized in brain areas, such as anterior thalamus, the dentate gyrus, hypothalamus, anterior cingulate gyrus, hippocampus, amygdala and certain brainstem nuclei [15–17]. Notably, as previously mentioned autoshaping training involved basolateral amygdaloidal nucleus, hippocampal CA1 area and dentate gyrus, hypothalamic area and frontal, parietal, temporal, occipital, retrosplenial agranular and granular cortices, and medial raphe nucleus [4,5,10]. This is probably very important, regarding memory facilitation, since extensive studies have shown that the amygdala, and especially the basolateral amygdala is critically important in mediating post-training drug effects on memory formation itself ([1,14]; see [11] for references). Moreover, muscarinic cholinergic activation, in particular in amygdala and to lesser extent in the cortex, has been shown to be especially important in memory modulation during consolidation [11]. Indeed, muscarinic cholinergic activity has been shown to interact with other neurotransmitter systems in the basolateral amygdala. This evidence provides further support to notion that the 5HT7 receptor agonists may influence memory consolidation by interacting with muscarinic cholinergic receptor activity in the basolateral amygdala. Of course, other possible interactions involving the cortex, nucleus basalis, hippocampus/medial septum system could exist. Notably, in the con-
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text of instrumental acquisition, prefrontal cortex has been conceptualized to represent declaratively the contingencies between instrumental actions and their outcomes [7]. These conceptualizations have practical implications, including the fact that autoshaping Pavlovian/instrumental task combines, both Pavlovian and instrumental conditioning, which offers the opportunity to study hippocampus-mediated declarative memory and striatum-mediated S–R “habit formation” [7]. It detects little changes (increases or decreases) on diverse behavioral parameters not redundant (i.e., not measuring the same event), including the sign tracking (i.e., conditioned behavior directed toward the localized retractable and illuminated lever, CS), and the goal tracking (i.e., the place where the US is delivered) [7]. These characteristics are quite important, since they allow the study of bi-directional expression of an enhanced or impaired memory formation, and maybe more importantly, is to study separately motivated behavior (i.e., goal tracking) and learned responses (i.e., autoshaped responses) [7]. In conclusion, administration of AS 19 improved memory consolidation in na¨ıve animals. Modeling the potential therapeutic benefits of 5-HT7 receptor stimulation, acute administration of AS 19 reversed memory deficits produced by scopolamine or dizocilpine. Hence, potent 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer’s disease. Certainly reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo, are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool.
Acknowledgements Authors thank GlaxoSmithKline for its generous gifts (see drugs section). This work was partially supported by the CONACYT Scholarship 185583 (G.S.P.G.) and Grant 39534M (A.M.). We thank Sofia Meneses-Goytia for revised language, and Roberto Gonzalez for his expertise and assistance. Finally, authors want to deeply thank the reviewer’s comments, which were very useful and their incorporation significantly improved the original version of our manuscript.
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