Modifications of 5-HT4 receptor expression in rat brain during memory consolidation

Brain Research 1042 (2005) 73 – 81 www.elsevier.com/locate/brainres

Research report

Modifications of 5-HT4 receptor expression in rat brain during memory consolidation L. Manuel-Apolinar a, L. Rochab, D. Pascoec, E. Castilloa, C. Castilloa, A. Menesesb,T a Escuela Superior de Medicina, IPN, Mexico City, Me´xico Depto. Farmacobiologı´a, CINVESTAV-IPN, Tenorios 235, Granjas Coapa, Me´xico City 14330, Me´xico c Centro Medico Nacional Siglo XXI, Mexico City, Me´xico

b

Accepted 7 February 2005 Available online 17 March 2005

Abstract Pharmacological evidence indicates a specific role of 5-HT4 receptors on memory function. These receptors are members of G-proteincoupled 7-transmembrane domain receptor superfamily, are positively coupled to adenylyl cyclase, and are heterogeneously located in some structures important for memory, such as the hippocampus and cortical regions. To further clarify 5-HT4 receptors’ role in memory, the expression of these receptors in passive (P3) untrained and autoshaping (A3) trained (3 sessions) adult (3 months) and old (P9 or A9; 9 months) male rats was determined by autoradiography. Adult trained (A3) rats showed a better memory respect to old trained (A9). Using [3H] GR113808 as ligand (0.2 nM specific activity 81 Ci/mmol) for 5-HT4 receptor expression, 29 brain areas were analyzed, 16 areas of A3 and 17 of A9 animals displayed significant changes. The medial mammillary nucleus of A3 group showed diminished 5-HT4 receptor expression, and in other 15 brain areas of A3 or 10 of A9 animals, 5-HT4 receptors were increased. Thus, for A3 rats, 5-HT4 receptors were augmented in olfactory lobule, caudate putamen, fundus striatum, CA2, retrosplenial, frontal, temporal, occipital, and cingulate cortex. Also, 5-HT4 receptors were increased in olfactory tubercule, hippocampal CA1, parietal, piriform, and cingulate cortex of A9. However, hippocampal CA2 and CA3 areas, and frontal, parietal, and temporal cortex of A9 rats, expressed less 5-HT4 receptors. These findings suggest that serotonergic activity, via 5-HT4 receptors in hippocampal, striatum, and cortical areas, mediates memory function and provides further evidence for a complex and regionally specific regulation over 5-HT receptor expression during memory formation. D 2005 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Learning and memory: systems and function—animals Keywords: Autoshaping; 5-HT; [3H] GR113808; Memory; Learning; Receptors; Rat

1. Introduction The 5-hydroxytryptamine4 (5-HT4) receptor has been identified in several tissues as a G-protein-coupled receptor positively linked to adenylyl cyclase activity [12,13]. Diverse evidence had led to suggest that 5-HT4 receptors may be targets to treat cognitive deficits, abdominal pain,

T Corresponding author. Fax: +52 55 50612863. E-mail address: [email protected] (A. Meneses). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.020

and feeding disorders [1]. The presence of this receptor has been described in the brain of several species, including rat, mouse, guinea-pig, pig, calf, monkey [8,14], and human [2,8,23,36,37]. Distribution of this receptor in the brain has been extensively studied using the labeled antagonists [3H] GR113808 and [125I] SB207710 [8,14,35], identifying 5HT4 receptor expression in limbic brain structures (hypothalamus, nucleus accumbens, and amygdala), Islands of Calleja, olfactory tubercule, fundus striatum, ventral pallidum, septum, hippocampus, basal ganglia (striatum, globus pallidus), etc. Apparently, the 5-HT4 receptor is

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L. Manuel-Apolinar et al. / Brain Research 1042 (2005) 73–81

predominantly located in anatomical structures linked to cognition. Actually, the distribution pattern and neurochemical and behavioral studies indicate that 5-HT4 receptors may be involved in memory and anxiety processes [1,9]. Accumulated pharmacological evidence indicates that 5-HT4 receptor agonists may improve learning and memory (see Bockaert et al. [1]; Meneses [24]). Indeed, behavioral studies have confirmed a procognitive effect of 5-HT4 receptor ligands in several phases of memory. For instance, the 5-HT4 receptor agonist BIMU1 increased social olfactory memory in rats when supplied before training, and this effect was reversed by GR125487 (a 5-HT4 receptor antagonist) [19]. Also, the 5-HT4 receptor agonist RS67333 enhanced acquisition and consolidation of place recognition spatial memory [29]. In models of memory deficit, BIMU 1 and RS67333 were used to reverse working memory deficits induced by scopolamine [18]. Pre-training administration of the 5-HT4 receptor agonist BIMU 1 or BIMU 8 enhanced the acquisition of autoshaping response [27]. The above findings provide further support to the proposal that some 5-HT4 receptor agonists and antagonists may have beneficial effects in the treatment of memory disorders such as Alzheimer’s disease (AD). Moreover, growing evidence indicates that diverse serotonergic markers including receptors are altered in patients with AD. For instance, presynaptically, there are marked losses of dorsal and median raphe neurons [6], reduced concentrations of serotonin and metabolites [7], and altered binding to 5-HT re-uptake sites in the neocortex of AD patients [5,24,26]. Postsynaptically, 5-HT2A receptor densities seem to be consistently reduced, while disagreement remains as to whether 5-HT1A receptors are similarly affected [3], besides decrease densities of 5-HT4 receptor have been found in the hippocampus and neocortex [15,16,24,31,38]. Taken together, these studies suggest alterations in both pre- and postsynaptic serotonergic markers. Actually, in the course of AD, cholinergic deficits appear, and the serotonergic system is affected early [1,10]. However, the neurochemistry of the subtypes of serotonergic system and AD has not been thoroughly investigated [15,16,26]. Although there is evidence that 5-HT4 receptors may be associated to aging and/or AD [24,26], however, it is important to determine 5HT4 receptor expression changes in diverse brain areas during learning and memory tasks, as well as the modifications of these receptors with age. It should be noticed that, traditionally, the search for brain areas involved in learning and memory has been centered on examinations of amnesic and AD patients, cerebral lesions, and, more recently, neuroimaging. In this regard, a complementary alternative consists of using radioligands [20]. Hence, in the present study, we have measured 5-HT4 receptor expression using [3H] GR113808 as ligand to examine the distribution of this receptor in 29 brain areas by autoradiography during an autoshaped memory in adult (3-month-old) and old (9-month-old) rats compared with untrained adult (3-monthold) and old (9-month-old) rats.

2. Materials and methods 2.1. Animals Male Wistar rats of 3 and 9 months old were used. Animals were collectively (ten per cage) 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 previous to memory experiments. After that period, body weights were reduced to 85% by gradually reducing food intake during 7 days. 2.2. Autoshaping learning task The autoshaping task has been previously described (see Meneses [24–26]). Briefly, each rat was placed in an experimental chamber for a period of habituation (10–15 min) and having access to 50 food pellets (45 mg each) previously placed inside the food-magazine. Once the animals ate all food pellets and presented 150 nose-pokes (as measured by a photocell) into the food-magazine, immediately afterwards, the autoshaping program began, which consisted of discrete trials. A trial consisted in the presentation of an illuminated retractable lever for 8 s (conditioned stimulus, CS) followed by the delivery of a food pellet (unconditioned stimulus, US) and then there was an inter-trials time (ITT) of 60 s. However, whether the animal pressed the lever during the CS, the trial was then shortened, the lever was retracted, light was turned off, and a food pellet (US) was immediately delivered and ITT begun. The response during CS was regarded as a conditioned response (CR) and its increase or decrease was considered as an enhancement or impairment of learning, respectively. There were three autoshaping training sessions 24 h apart from each other. The first session consisted of 10 trials and the rest of 20 trials. The memory results correspond to this latter autoshaping session. 2.3. Experimental protocols The local institutional committee for the use of animal subjects approved the present experimental protocol (Project No. 047/02). Animals were divided into four groups as follows: an untrained or passive group (P3) and adult autoshaping trained (A3) group of 3 months old, and an old passive group (P9) and trained group (A9) of 9 months old. Both untrained and trained groups arrived from the colony room to the laboratory 2 weeks before initiation of autoshaping experiments and were handled daily for a week afterwards their body weighs were gradually reduced to 85% of their ad libitum body weight. While untrained groups were left into their home cage, autoshaping trained groups received autoshaping training during three consecutive days, between 8 and 12 A.M. everyday. Immediately following the last autoshaping session, animals were sacrificed by decapitation, their brains were removed and

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Table 1 Incubation conditions used for the binding of 5-HT4 receptor [37] Ligand

[3H] GR113808

Preincubation protocol

30 min at room temperature, incubation buffer

Incubation protocol Ligand concentration (nM)

Buffer

Temperature

Time (min)

0.2

50 mM Tris–HCl, 10 AM pargyline, 0.01% ascorbic acid pH 7.4

Room temperature

30

placed on ice, and prepared for autoradiography essay (Table 1). 2.4. Preparation of brains Animals were decapitated and their brains were quickly frozen in pulverized dry ice and stored at 70 8C until used. For 5-HT receptor autoradiography, coronal sections through the rat brain were cut at 20 Am in a cryostat (Leica Model CM 1510), thaw-mounted on gelatin-coated slides, and stored at 70 8C until the day of incubation and only during ligand incubation the tissue was rehydrated at room temperature. The interval of time between sacrifice and the performance of the binding assay was 6 months. Although we do not have an estimate of tissue decay, we used the standard conditions according to Waeber et al. [37], such as concentration of tritium ligand (equivalent to its Kd), concentration of ligand for non-specific binding, temperature and incubation time, and washing time.

Washing

Exposure time (months)

2  5 min (4 8C)

6

tical densities into receptor densities values expressed in fmol/mg protein [37]. The presented results herein were obtained from six animals per group. Brain areas and nuclei were identified using Paxinos and Watson (see Ref. [30]) atlas. Twenty-nine brain areas were analyzed (see Table 2 for a detailed list). 2.6. Materials The following substances and reagents were used: [3H] GR113808 (1-[(2-methylsulphonyl) amino] ethyl-4-piperidinyl] methyl 1-methyl-1H-indole-3-carboxylate; Grossman et al. [11]) (specific activity 81 Ci/mmol) (Amersham Biosciences). The following substances were obtained from Sigma: BIMU 8 (endo-N-(8-azabicyclo (3.2.1)-oct-yl)-2, 3dihydro-(1-methyl) ethyl-2oxo-1H-benzimidazol-1 carboxamide hydrochloride), chromium potassium sulfate, gelatin, trizma base (tris [hydroxymethyl] aminomethane), Tris– HCl, ascorbic acid, and pargyline. Hyperfilm-3H, Kodak D11 developer, and fixer (K.R. Amtmann).

2.5. Receptor autoradiography

2.7. Statistical analysis

Following Waeber et al. [37] (see Table 1) for an autoradiographic study, a concentration of [3H] GR113808 0.2 nM was used to label the 5-HT4 receptor binding sites in brain slices. Tissue sections were preincubated in Coplin jars for 30 min at room temperature in 40 ml of a buffer solution consisting of 50 mM Tris–HCl, 10 AM pargyline, and 0.01% acid ascorbic, pH 7.4. Sections were then incubated in the same buffer containing the radioligand, [3H] GR113808, at a final 0.2 nM concentration (specific activity 81 Ci/mmol). Nonspecific binding was generated by the addition of BIMU 8 10 AM. After incubation, the sections were washed in ice-cold (4 8C) buffer solution twice for 5 min and then quickly dipped into cold distilled water to remove any salts. The sections were dried under a gentle stream of cool air. The slides were arrayed in X-ray cassettes together with tritium standards (Amersham) and exposed to tritium-sensitive film (Kodak hyperfilm) at room temperature for 6 months. The films were developed using Kodak D11 developer and fixer at room temperature. Optical densities of selected areas appearing on autoradiograms were determined using a video-computer enhancement program (Jandel Video Analysis Software JAVA), which transforms the values of op-

2.7.1. Memory scores As previously reported (see Ref. [26]), the CR number of the third autoshaping session for each group was transformed into percentage. These data are expressed as means F SEM and were analyzed comparing adult autoshaping trained vs. old autoshaping trained rats by Student’s t test. Six animals were used per group and used only once. 2.7.2. Autoradiographic studies Planed comparison used an experimental design relating to between-subjects or groups is a 2  2 factorial with 2 levels of age (adult vs. old) and 2 levels of untrained vs. trained. Thus, 5-HT receptor density was determined in fmol/mg protein and these values were analyzed by analysis of variance (ANOVA) comparing autoradiographic results of adult and old passive (untrained) vs. autoshaping trained and untrained rats. ANOVA was followed by post hoc analysis using Tukey test where trained animals were compared with untrained animals, and the values of the former were considered as a fraction of the density of 5-HT receptors of passive (either adult or old animals). In all comparisons, P b 0.05 was considered as significant. In

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Table 2 [3H] GR113808 binding (fmol/mg protein) in specific brain regions of rat of 3 and 9 months Area

Abbreviations

3 months Mean F SEM

TU LO ICJ VP AcbC Cpu FStr AMYG LSV

334 20 136 225 126 52 38 196 30

LSI

102 F 7

150 F 3*

118 F 2

DG CA1 CA2 CA3 DA

101 F 11 183 F 6 110 F 9 239 F 26 216 F 14

44 154 440 285 172

148 149 276 246 153

VMH

223 F 15

373 F 36**

133 F 14yyy

LH

168 F 8

284 F 18**

MPA MP

115 F 19 148 F 26

FR PAR RSA

P3 Olfactory system

Basal ganglia

Amygdala Septum

Hippocampus

Hypothalamus

Cortical areas

Mid brain

Olfactory tubercule Olfactory lobule Islands of Calleja Ventral pallidum Accumbens nucleus Caudate putamen Fundus striatum Lat. septal nucleus, ventral part Lat. septal nucleus, intermediate part Dentate gyrus Ammon’s horn, area 1 Ammon’s horn, area 2 Ammon’s horn, area 3 Dorsal hypothalamic area Ventromedial hypothalamic nucleus Lateral hypothalamic area Medial preoptic area Medial mammillary nucleus Frontal cortex Parietal cortex Retrosplenial agranular cortex Retrosplenial granular cortex Temporal cortex Entorhinal cortex Occipital cortex Piriform cortex Cingulate cortex Substantia nigra, reticular part

A3 72 5 13 14 17 13 7 33 11

283 314 301 413 143 149 153 401 47

F 29 F 85** F 10 F 14*** F7 F 19* F 15** F 34** F5

9 months Mean F SEM

ANOVA

P9

F

202 190 170 175 128 122 201 247 27

A9 25 15+ 14 10yyy 23 5 20+++ 7y 8

502 F 57**,y 234 F 25y 438 F 80**,++ 209 F 28yyyy 115 F 68 80 F 33 434 F 32***,+++,yyy 219 F 30yy 94 F 16

P value

6.50 8.72 11.13 35.53 0.09 4.57 65.27 10.42 0.08

0.0073 0.0024 0.0009 0.0001 0.9609 0.0200 0.0001 0.0012 0.9501

4.30

0.0281

9.68 13.31 381.3 8.77 53.06

0.0016 0.0004 0.0001 0.0024 0.0001

207 F 30yy

15.42

0.0002

103 F 7yyy

251 F 27***,+

24.56

0.0001

295 F 25** 33 F 9*

229 F 23yy 210 F 30yyy

129 F 21***,+++,yyy 42 F 10***,+

46.20 16.38

0.0001 0.0002

25 F 4 123 F 5 115 F 16

102 F 23* 167 F 24 249 F 12

205 F 19+++,yyy 205 F 32 49 F 23y

119 F 20*,+ 44 F 9***,yy 265 F 85*

16.74 11.39 5.35

0.0001 0.0008 0.0143

RSG

27 F 5

219 F 19**

397 F 60***,+++,yy

30.42

0.0001

TE Ent OC PIR CG SNR

130 F 5 118 F 7 136 F 8 47 F 11 22 F 5 276 F 21

10.40 2.69 18.26 37.07 23.27 22.88

0.0012 0.0929 0.0001 0.0001 0.0001 0.0001

F F F F F F F F F

254 228 323 28 163 268

F F F F F

F F F F F F

12 5 13*** 46 29

44* 33 31*** 9 6*** 25

F F F F F F F F F

F F F F F

4yyy 7 3+++,yyy 24 14

44 F 5yy 189 175 85 159 21 156

F F F F F F

31 15 23yyy 19y 5yyy 11++,yy

107 F 20 107 368 51 73 473

46 138 133 440 175 76

F F F F F

F F F F F F

22y 56***,++,yyy 8***,++,yyy 26*,+,yy 20***,+++,yyy

6*,yyy 45 29yyy 58***,+++,yyy 34***,+++ 19+++,yyy

Data are plotted representing the mean F SE of six animals. *P b 0.05, **P b 0.01, ***P b 0.001 indicate significant difference between control passive (P) vs. autoshaping (A) trained; or +P b 0.05, ++P b 0.01 significantly different control passive (3 months; P3) vs. control passive (P9) or autoshaping (A9) trained groups 9-month-old; or yP b 0.05, yyP b 0.01, yyyP b 0.001 significantly different autoshaping (A3) vs. P9 or A9; Tukey test.

order to facilitate the results description of 5-HT receptor expression in trained groups, henceforth these will be express as percentage of untrained control groups values.

3. Results 3.1. Effects on memory formation Since control passive groups had no autoshaping training, they lack memory scores. Moreover, comparing percentage of CR% scores between adult vs. old rat groups during the third autoshaping session, significant differences were observed, showing 9 F 2 of CR% compared to 2 F 1 of CR%, respectively (P b 0.01) (Fig. 1).

3.2. Autoradiography of 5-HT4 receptors When adult and old passive (P3 and P9) or autoshaping (A3 and A9) trained groups were compared, there were significant differences on 5-HT4 receptor expression. ANOVA showed significant changes in 5-HT4 receptor expression among adults vs. old groups [ F(3,28) = 21.2; P b 0.01], trained vs. passive [ F(3,28) = 70.7; P b 0.01], and the interaction among them [F(28,84) = 13.3; P b 0.01]. Twenty-seven out of 29 brain areas showed significant changes displaying two or more changes (Fig. 2). In the following sections, results of specific brain areas are described, firstly comparing trained vs. their respective passive control, and secondly, comparing adult and old passive and trained groups.

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Fig. 1. Effects on memory consolidation in an autoshaping learning task. Data are plotted as percentage of conditioned responses representing the mean (CR% F SEM) from 6 animals per group. **P b 0.01, indicates significantly different from group by Student’s t test.

3.2.1. Olfactory system 5-HT4 receptors in the olfactory tubercule (TU) of A3 group were decreased with regard to P3 (+200%, P b 0.01; Table 2), but they showed a significant increment (+150%) in A9 relative to P9 (P b 0.01; Table 2). Both trained groups presented increase 5-HT4 receptor expression in olfactory lobule (LO), Islands of Calleja (ICJ), and ventral pallidum (VP) regarding their respective passive groups. Comparing TU area of P3 group vs. P9 or A3 vs. A9, a decrease or increase, respectively, in the 5-HT4 receptor expression was observed (P b 0.05). An increase or decrease of 5-HT4 receptor in LO area of P9 group relative to P3 or A9 vs. A3 groups was revealed. While ICJ area of P9 vs. P3 or A9 vs. A3 showed an increased expression of 5-HT4 receptors, the opposite effect was observed in VP areas (P b 0.05; to see Table 2).

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3.2.2. Basal ganglia The distribution of 5-HT4 receptors in the basal ganglia was heterogeneous, for example, in the Accumbens Nucleus (AcbC), there were no differences among trained vs. untrained and among ages. The caudate putamen (Cpu) of A3 compared to P3 showed an increase (+183%, P b 0.05) of 5-HT4 receptors. In fundus striatum (FStr) of A3 group, there was a high density of 5-HT4 sites (+300%, P b 0.01; Table 2) with regard to P3, and A9 increased 5-HT4 receptors compared with P9 group (+116%, P b 0.01; Table 2). There was a tendency of increments in P9 and A9 groups with regard to P3 (P b 0.01) (Table 2). A9 group relative to A3 displayed an increment of 5-HT4 receptors (P b 0.01). 3.2.3. Amygdala In amygdala (AMYG), the binding of 5-HT4 receptors in A3 group was increased significantly (+107%, P b 0.01) compared to P3 group (Table 2). In addition, P9 group compared with A3 ( 45%, P b 0.05) and A9 ( 38%, P b 0.01; Table 2) showed decreases. 3.2.4. Septum In the ventral part of the lateral septal nucleus (LSV), there were no differences in the density of 5-HT4 receptors among trained vs. untrained and ages. With regard to the intermediate part of the lateral septal (LSI) of A3, there was a significant increase of 5-HT4 receptors (+25%, P b 0.05) relative to P3 group (Table 2). 3.2.5. Hippocampus 5-HT4 receptors in the CA2 hippocampal area of A3 group showed a significant increase (+300%, P b 0.01) regarding P3 group (Table 2). Similarly, in the CA1 area of

Fig. 2. Autoradiographic distribution of [3H] GR113808 receptors in the hippocampus, temporal cortex, occipital cortex, and hypothalamus of passive and autoshaping trained rats. (A) Passive 3-month-old, (B) autoshaping trained of 3-month-old, (C) passive 9-month-old, and (D) autoshaping trained 9-month-old.

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A9, there was an increase (+146%, P b 0.01) with respect to P9; however, in CA2 ( 82, P b 0.01) and CA3 ( 70, P b 0.05) areas, significantly diminished 5-HT4 receptors of A9 vs. P9 group (Table 2). In the dentate gyrus (DG) of P9 (P b 0.01) and A9 (P b 0.05) groups, 5-HT4 receptors were increased in comparison with the A3 group. Similar increases in the CA1 area of A9 increased (P b 0.01) with regard to P3 and A3 (P b 0.01) were observed. In contrast, in the CA2 (P b 0.01) and CA3 (P b 0.01) areas of the A9 group, there were significantly diminished 5-HT4 receptors regarding A3 (see Table 2). 3.2.6. Hypothalamus In the A3 group regarding P3, there were increases of 5HT4 receptors in the region hypothalamic and in the ventromedial hypothalamic nucleus (VMH) (+67%, P b 0.01), the lateral hypothalamic area (LH) (+69%, P b 0.01), and preoptic medial area (MPA) (+156%, P b 0.01), but diminished in the mammillary medial nucleus (MP) ( 78%, P b 0.05) (see Table 2). In the A9 group relative to the P9 group, 5-HT4 receptors were increased in the dorsal hypothalamic area (DA) (+208%, P b 0.01) and LH (+143%, P b 0.01), but decrease them in MPA ( 44%, P b 0.01) and MP ( 80%, P b 0.01) (Table 2). With respect to A3 animals, in MP area of P9 and DA of A9 groups, 5-HT4 receptors were increased, but diminished in VHM, LH, MPA of P9 group and VHM and MPA of A9 group (Table 2). 3.2.7. Cortical areas The 5-HT4 receptor expression in the cortical area of A3 group relative to P3 was increased in the frontal cortex (FR) (+300, P b 0.05), retrosplenial granular (RSG) (+100%, P b 0.01), temporal (TE) (+95%, P b 0.05), occipital (OC) (+137%, P b 0.01), and cingulate (CG) (+300%, P b 0.01) (see Table 2). A9 group expressed an increment in the density of receptors in the retrosplenial agranular (RSA) (+400%, P b 0.05), RSG (+400%, P b 0.01), piriform cortex (PIR) (+176%, P b 0.01), and CG (+400%, P b 0.01) compared to P9 group. In contrast, relative to P9, the A9 group showed a decrease of 5-HT4 receptors in FR ( 42, P b 0.05), PAR ( 78, P b 0.01), and TE ( 75, P b 0.05) (Table 2). On the other hand, comparing groups of 9 months (P9 and A9) with the P3 group, significant increments of 5-HT4 receptors in FR of P9 group (P b 0.01) and of A9 group in RSG (P b 0.01), PIR (P b 0.01), and CG (P b 0.01) were observed. Comparing A3 group with P9 and A9 groups, there were increments in 5HT4 receptors in FR (P b 0.01) and PIR (P b 0.05) of P9 and in RSG (P b 0.01) and PIR (P b 0.01) of A9 group. In contrast, RSG (P b 0.01), RSA (P b 0.05), TE (P b 0.01), and OC (P b 0.01) of A9 group showed a diminution of 5-HT4 receptor expression (Table 2). 3.2.8. Mid brain In the substantia nigra (SNR) of P9 and A9 groups, a diminished expression of 5-HT4 receptors relative to the P3

group was observed (P b 0.01). Similarly, A3 group compared with P9 and A9 groups presented significant decrements in the expression of 5-HT4 receptors ( P b 0.01) (Table 2).

4. Discussion The present results clearly showed that old rats showed poor memory formation relative to adult animals. Autoshaping training and age produced specific changes in particular regions of brain areas, relevant to memory formation, and provided further support to the notion that serotonin, via 5-HT4 receptors, may contribute to the modulation of learning and memory. Out of twenty-nine brain areas analyzed for 5-HT4 receptor expression in adult autoshaping trained rats relative to untrained group, there were changes on sixteen, fifteen showing augmented 5-HT4 receptors and one diminished them (Fig. 3). In old autoshaping trained rats, seventeen areas showed significant differences, ten displayed increased 5-HT4 receptors and seven decreased them. Overall, age augmented 5-HT4 receptor expression. Fig. 3 summarizes the more significant changes of 5-HT4 receptor expression. Since we did not measure Kd values, the possibility that some of the above changes could be due to modifications in affinity cannot rule out. Nonetheless, it should be borne in mind that 5-HT4 receptor stimulation in a two-trial recognition task designed to test place or object recognition memory produced an enhancement place and object recognition in young adult rats. In old rats, RS 67333 (a higher dose) improved place recognition when injected before the acquisition phase and object recognition when injected before the acquisition or in the consolidation phase of information processing [17]. Thus, these data are consistent with the notion of 5-HT4 receptor changes might be related to aging. Regardless of age or training condition, the present patterns of 5-HT4 receptor localization are consistent with previous studies (see Ref. [1], for recent a review). For instance, as herein (Fig. 2), 5-HT4 receptors are expressed in several species including rat, mouse, monkey, etc., in hypothalamus, nucleus accumbens, amygdala, Islands of Calleja, olfactory tubercule, fundus striati, ventral pallidum, septum, hippocampus, basal ganglia, striatum, globus pallidus, and substantia nigra [1]. Regarding dorsal and medial raphe nuclei, we were unable to detect any change. a similar pattern of 5-HT4 receptors was observed in normal humans [31], and more importantly in the present context, decreases of 5-HT4 receptor expression were observed in AD patients, in hippocampus and frontal cortex. Notably, in this study, 9-month-old rats (Table 2) showing poor memory retention relative to adult rats displayed diminished expression of these receptors in CA2 and CA3 hippocampal areas and temporal cortex. Although this latter brain area showed a non-significant diminished expression of 5-HT4 receptors in AD brains

L. Manuel-Apolinar et al. / Brain Research 1042 (2005) 73–81

relative to its control age group in the Reynolds et al. study, notwithstanding there is an interesting parallel result in frontal cortex (area 11) of AD patients and old rats (Table 2), in both groups expression of 5-HT4 receptors was decreased. Notably, Pavlovian autoshaping seems to be associated to an increase in 5-HT content in the prefrontal cortex in rats, likely due to enhance enzymatic activity of tryptophan hydroxylase increase, which would increase synthesis of 5-HT [33,34]. Notably, it is wellknown that serotonergic activity depends on tryptophan availability, the rate-limiting enzyme tryptophan hydroxylase, monoamine oxidase, re-uptake sites, and 5-HT receptors, which also might be influenced by learning, memory, and aging [28]. Together, these data are in line with the notion of contribution of 5-HT4 receptors in the physiology, pathophysiology, and therapeutic of learning and memory [1]. Further support for this conclusion comes from a recent work in which we aimed to determine, by autoradiography and using [3H] 5-HT as ligand, 5-HT receptor expression in passive (untrained) and autoshaping trained (3 sessions) adult (3 months) and old (9 months) male rats. Trained adult rats presented better retention than old animals. Raphe nuclei of adult and old trained rats

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expressed less receptors on medial and dorsal, respectively, and hippocampal CA1 area and dentate gyrus of adult trained rats expressed less 5-HT receptors, while dentate gyrus of old animals increased them. Basomedial amygdaloid nucleus in old trained rats expressed more 5-HT receptors; while in the basolateral amygdaloid nucleus, they were augmented in both groups. Autoshaping training decreased or did not change 5-HT receptors in caudate putamen of adult or old animals. This profile of 5-HT receptor localization is consistent with previous reports and suggests that memory formation and age may modulate 5-HT receptor expression in brain areas relevant to memory systems. Moreover, it should be noticed that recently Campan et al. [4] reported that 5-HT4 receptor-null mice are less reactive in novel environments. The 5-HT4 receptor-null mice were placed in an open field and their locomotion was monitored for 30 min and the test was repeated for three consecutive days to evaluate habituation to a novel environment, which could also represent a simple learning task. The mutant mice displayed an overall decrease in the traveled path length only during the first day of exposure, as compared with wild-type animals in both the periphery and center of the open field [4].

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According to the same authors, in contrast, there was no difference in locomotion between the mutant and wild-type animals in their home cages and less reactivity in 5-HT4 receptor-null mice has also been found in the elevated plus maze and alley tests. In addition, the mutant mice did not exhibit impairment in the rotarod test, which demonstrates that there is no deficit in motor coordination in 5-HT4 receptor-null mice. In these mice, pentylenetetrazolinduced convulsive responses were enhanced, suggesting an increase in neuronal network excitability. Campan et al.’s [4] conclusion was that these results suggest that the 5-HT4 receptor-null mice display decreased reactivity to novelty rather than locomotor impairment. But could these same data be also suggesting an improve habituation? Apparently, the absence of 5-HT4 receptors reduced the time of habituation during the first 15 min of observation. Of course, though more information on other learning and memory tasks is necessary, wide pharmacological evidence indicates that while 5-HT4 receptor agonists facilitate learning and memory, antagonists for these receptors have no effect or may impair performance [1,22,24,26]. For instance, in an olfactory associative discrimination task [17], injection of the selective 5-HT4 receptor antagonist RS 67532 (1 (4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl) 5-(1-piperidinyl) 1-pentanone) before the third training session induced a consistent deficit in associative memory during the following training sessions, and this deficit was absent when the antagonist was injected together with either the selective 5-HT4 receptor agonists RS 17017 (1-(4-amino-5-chloro-2-methoxyphenyl) 5-(piperidin-1-yl) 1-pentanone hydrochloride) and RS 67333 (1(4-amino-5-chloro-2-methoxyphenyl)-3(1-n-butyl-4-piperidinyl)-1-propanone) [17]. Moreover, in models of memory deficit, BIMU 1 and RS67333 were used to reverse working memory deficits induced by scopolamine, which further confirm the therapeutic potential of such ligands in the treatment of cognitive alterations associate to short-term working memory disorders and cholinergic hypofunction [18]. Relevant to the present data, it is the fact that pre-training administration of the 5HT4 receptor agonist BIMU 1 or BIMU 8 enhanced the acquisition of autoshaping response, while post-training administration of either of these agents impaired memory consolidation [27]. In the same study, further experiments revealed that the post-training injections of the 5-HT4 receptor antagonist SDZ 205–557 or GR 125487D by themselves did not alter the performance; nevertheless, when BIMU 1 or BIMU 8 was administered to pretreated rats with SDZ 205–557 or GR 125487D, the decrement induced by both agonists was reversed. Interestingly, Lamirault and Simon found an increased consolidation of object recognition in old rats with RS 67333 (see Ref. [17]). Although the source for discrepancies between these works is unclear, age of rats, drugs, and nature of behavioral tasks used are noteworthy. In fact, trying recently RS 67333 on autoshaping learning task, its pre-

training administration improved learning of acquisition of autoshaping response, while post-training had no effect (Meneses, unpublished results). In conclusion, the present results may constitute, to our knowledge, the first report of the regional distribution of 5-HT4 receptor expression during memory formation in autoshaping task, which will help to clarify the sites of synthesis of 5-HT4 receptors and provide further clues for future works. Of course, the present results must be taken with caution, since memory formation studies have identified memory phases or learning tasks, which differ on relative contribution basis of brain areas, neurotransmitters, receptors, signal transduction pathways, protein synthesis, and genes induction. Actually, multiple 5-HT receptors have been identified on brain areas relevant to cognitive processes, and, it is possible that during memory formation and amnesia the expression of some of these receptors may have task- and regional-dependent variations. Notably, receptors [32] and transduction signals [21] are dynamic, time-dependent patterns of learning-induced regulatory processes.

Acknowledgments This work was partially supported by CONACYT Grant 39534-M. L.M.A. was supported by CONACYT scholarship No. 165460. We thank Sofia Meneses-Goytia for language review and Roberto Gonzalez for his expertise assistance.

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