Aging alters in a region-specific manner serotonin transporter sites and 5-HT1A receptor-G protein interactions in hamster brain

Neuropharmacology 43 (2002) 36–44 www.elsevier.com/locate/neuropharm

Aging alters in a region-specific manner serotonin transporter sites and 5-HT1A receptor-G protein interactions in hamster brain Marilyn J. Duncan a,∗, Julie G. Hensler b a

Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA b Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Received 24 January 2002; received in revised form 24 April 2002; accepted 29 April 2002

Abstract Key proteins regulating serotonergic activity, specifically the serotonin transporter and 5-HT1A receptor, were examined in the midbrain raphe nuclei of young (3–4 months) and old (17–19 months) hamsters (N=7–10/group). An age-related decrease in the maximal density of serotonin transporter sites labelled with [3H]paroxetine (fmol/mg protein, Old: 396±13; Young: 487±27) was observed in the dorsal raphe nucleus (DRN) but not the median raphe nucleus (MRN), without affecting the affinity of [3H]paroxetine. In the DRN and MRN, the stimulation of [35S]GTPγS binding by the 5-HT1A receptor agonist 8-OH-DPAT, or the number of 5-HT1A receptor sites labeled with [3H]MPPF, was not different in old versus young animals. Thus in the DRN, aging decreased serotonin transporter sites without changing 5-HT1A receptor activation of G proteins or 5-HT1A receptor density. In the CA1 region of hippocampus, 8-OH-DPAT-stimulated [35S]GTPγS binding was increased in the older animals (% above basal, Old: 141±21; Young: 81±17) without changing specific [3H]MPPF binding sites, suggesting that the capacity of 5-HT1A receptors to activate G proteins is enhanced. Aging also appears to enhance this capacity in the dentate gyrus, because this region exhibited a constant level of 8-OH-DPAT-stimulated [35S]GTPγS binding in spite of an age-related decrease in the number of [3H]MPPF binding sites (fmol/mg protein, Old: 203±21; Young: 429±51).  2002 Published by Elsevier Science Ltd. Keywords: Dorsal raphe nucleus; Median raphe nucleus; Hippocampus; Dentate gyrus; CA1; [35S]GTPγS binding; [3H]paroxetine binding; [3H]MPPF binding; Autoradiography

1. Introduction In brain, the neurotransmitter serotonin modulates many physiological and behavioral processes, including sleep, circadian rhythms, neuroendocrine function, and affective state. Serotonergic innervation of forebrain structures involved in these processes arises from serotonergic cell bodies localized to discrete groups or nuclei in the midbrain, predominantly in the median raphe nucleus and dorsal raphe nucleus (Moore et al., 1978; Molliver, 1987; Morin and Meyer-Bernstein, 1999). Serotonin itself controls the firing of the raphe serotonergic neurons through activation of somatodendritic 5-HT1A autoreceptors (Sprouse and Aghajanian, 1986; Sprouse and Aghajanian, 1987; Sotelo et al., 1990). In

Corresponding author. Tel.: +1-323-323-4718; fax: +1-323-3235946. E-mail address: [email protected] (M.J. Duncan). ∗

0028-3908/02/$ - see front matter  2002 Published by Elsevier Science Ltd. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 7 2 - 2

cell body areas, the serotonin transporter also influences serotonin neuronal firing by regulating the extracellular concentration of serotonin, and hence the activation of the 5-HT1A autoreceptors (Gallager and Aghajanian, 1975; Adell and Artigas, 1991; Invernizzi et al., 1992). In terminal field areas of serotonergic innervation, such as cortical and limbic areas, the 5-HT1A receptor is located postsynaptically (Verge´ et al., 1986; Hensler et al., 1991). Aging deleteriously affects many processes modulated by serotonin, including sleep, circadian rhythms, and affective state. For example, complaints of sleep problems are common among the elderly (Czeisler et al., 1992). Aging advances the timing of sleep, such that the major sleep interval occurs earlier, and also leads to sleep fragmentation, which impairs daytime alertness (Carskadon et al., 1982; Czeisler et al., 1992; Copinschi and Van Cauter, 1994). The advanced timing of sleep that occurs with aging is associated with altered timing of other circadian rhythms, such as the circadian body

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temperature rhythm (Czeisler et al., 1992). Aging also induces other changes in circadian rhythms, including decreased amplitude and reduced responsiveness to phase resetting signals (Van Reeth et al., 1992; Zee et al., 1992; Scarbrough et al., 1997). Affective disorders, which are associated with alterations in circadian rhythms, are also more common among the elderly than the young adult population (Barefoot et al., 2001). Because aging adversely affects processes mediated by serotonin, the main objective of the current project was to examine age-related changes in key proteins involved in the regulation of serotonin neuronal activity, specifically the serotonin transporter and 5-HT1A receptor in cell body areas, the dorsal and median raphe nuclei. We hypothesized that aging leads to a decrease in serotonin reuptake sites in the dorsal and median raphe nuclei that is accompanied by a reduction in the sensitivity or function of 5-HT1A receptors in these areas. In these experiments we examined young (3–4 months old) and old (17–19 month old) hamsters, in view of previous demonstrations that these groups exhibit differences in serotonergic regulation of circadian rhythms (Penev et al., 1995) and in another serotonin receptor subtype (i.e. 5-HT7 receptor) in the dorsal raphe nucleus (Duncan et al., 1999). In order to test our hypothesis, we used quantitative autoradiography to determine the effect of aging on the serotonin transporter and 5-HT1A receptors in the hamster dorsal and median raphe nuclei. The binding of the selective radioligand [3H]paroxetine to serotonin reuptake sites was measured in saturation binding experiments, allowing us to determine the affinity of this radioligand for the serotonin transporter and the density of binding sites in young and old hamsters. Stimulation of [35S]GTPγS binding by the 5-HT1A receptor agonist 8OH-DPAT was quantified in the raphe nuclei of young and old hamsters, allowing us to determine the effect of aging on 5-HT1A receptor function at the level of receptor-G protein interaction. We also examined the effect of aging on 5-HT1A receptor-G protein interactions in the hippocampus, in view of the importance of this structure in affective state and memory, and the deleterious effects of aging on these functions. 5-HT1A receptor density was assessed in autoradiographic studies using a single saturating concentration of the selective antagonist radioligand [3H]MPPF.

2. Materials and methods 2.1. Animals and tissue preparation Young (3–4 months old) and old (17–19 months old), male Syrian hamsters (Harlan Sprague Dawley, SYRHSD), were used for these studies because they are a well-established animal model for the effects of aging

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on circadian rhythms (Van Reeth et al., 1992; Penev et al., 1995) and they exhibit age-related changes in several parameters of serotonergic function (Duncan et al., 1999, 2000). Because hamsters live for approximately 2 years, the 17–19 month old hamsters would not be expected to be senescent (Kirkman and Yau, 1972). The hamsters were decapitated at 1300–1400 h, after exposure to a 14L:10D photoperiod (lights on at 0600 h) for at least 1 week. The brains were dissected, frozen on powdered dry ice, and stored for ⬍ 3 weeks at ⫺80 °C. Coronal sections (20 µm thick) were cut with a cryostat microtome and mounted on positively charged slides (Superfrost Plus, Curtin Matheson Scientific, Inc.). The use of these animals was approved by the University of Kentucky Institutional Care and Use Committee and was in accordance with the guidelines provided by the National Institutes of Health. For [3H]paroxetine autoradiography, tissue sections from young hamsters (N=10) and old hamsters (N=8) were randomly assigned to sets for each age group (young: 7 sets, old: 6 sets); each set was incubated with a different concentration of [3H]paroxetine, as described below. For [35S]GTPγS autoradiography and [3H]MPPF autoradiography, brain sections from the same young hamsters (N=10) and old hamsters (N=10) were used. Brain sections corresponding to Figures 28 to 30 (hippocampus) and 40 to 42 (median and dorsal raphe nuclei) of a stereotaxic atlas of the hamster brain (Morin and Wood, 2001) were selected. For some animals, due to problems with sectioning, there were not enough sections representing these areas to conduct both the [35S]GTPγS autoradiography and [3H]MPPF autoradiography. Therefore, the final samples sizes, which are indicated on the figures shown in Section 3, are somewhat lower. Brain regions were identified on the autoradiograms by comparison with Nissl-stained tissue sections and reference to a stereotaxic atlas of the hamster brain (Morin and Wood, 2001). 2.2. [3H]Paroxetine autoradiography Serotonin reuptake sites were quantified using the selective radioligand [3H]paroxetine, as described previously (Hrdina et al., 1990; Duncan et al., 2000). Saturation binding experiments were performed using eight concentrations of [3H]paroxetine, ranging from 0.07 to 5.07 nM. Slide-mounted brain sections were pre-incubated in Tris–HCl buffer (50 mM, pH=7.4) at 22 °C for 15 min. The slides were then incubated with [3H]paroxetine dissolved in assay buffer (50 mM Tris–HCl with 120 mM NaCl and 5 mM KCl) at 22 °C for 60 min. Nonspecific binding was determined in adjacent sections by co-incubation with clomipramine (10 µM). At the end of the incubation period, the slides were washed in assay buffer at 37 °C (2×20 min each), dipped in ice-cold deionized water and dried under cool air. The slides and

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radioactive standards ([3H]microscales, Amersham Corp.) were exposed to 3H-sensitive Hyperfilm, (Amersham Corporation) for 6–8 weeks. The autoradiograms of [3H]paroxetine binding were quantitated by computer-assisted microdensitometry (M4, Imaging Corp., Ontario, Canada), as described previously (Duncan et al., 1993). Specific binding was calculated by subtracting nonspecific binding from total binding on adjacent sections. 2.3. [35S]GTPgS autoradiography Autoradiography of 5-HT1A receptor-stimulated [35S]GTPγS binding in brain sections was performed as described previously (Hensler and Durgam, 2001). Slide-mounted sections were thawed in a dessicator at 4 °C under vacuum and then equilibrated in HEPES buffer (50 mM, pH 7.4), supplemented with 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, and 0.2 mM dithiothreitol, at room temperature for 10 min at 30 °C. Sections were then pre-incubated in HEPES buffer containing GDP (2 mM) for 15 min at 30 °C. Sections were subsequently incubated in pre-warmed HEPES assay buffer containing GDP (2 mM) and 80 pM [35S]GTPγS either in the absence or in the presence of the 5-HT1A receptor agonist (±)8-OH-DPAT for 45 min at 30 °C. Basal [35S]GTPγS binding was defined in the absence of agonist. Nonspecific [35S]GTPγS binding was defined in the absence of agonist and in the presence of 10 µM GTPγS. The incubation was stopped by two washes for 2 min each in ice-cold 50 mM Tris–HCl buffer (pH 7.4), followed by a brief immersion in ice-cold deionized water. Sections were dried on a slide-warmer. Slides and [14C] standards (ARC-146, American Radiochemicals, St. Louis, MO) were exposed to Kodak Biomax MR film (Amersham) for 24 h. Autoradiograms of 5-HT1A receptor-stimulated [35S]GTPγS binding were quantified using the image analysis program NIH Image, version 1.47 (NIH, Bethesda, MD), as described (Hensler and Durgam, 2001). Nonspecific binding of [35S]GTPγS was subtracted from basal binding and from binding in the presence of agonist. Specific, agonist-stimulated binding was expressed as percentage above basal.

the presence of 10 µM WAY-100635. Incubation was terminated by two washes for 5 min each in ice-cold 170 mM Tris–HCl buffer (pH 7.6), followed by a dip in icecold deionized water. Sections were dried on a slide warmer. Slides and [3H] standards (ART-123, American Radiochemicals, St. Louis, MO), which had been calibrated using brain-mash sections according to the method of Geary and Wooten (Geary and Wooten, 1983; Geary et al., 1985), were exposed to Kodak BioMax MR film for a period of 5 weeks. Autoradiograms of [3H]MPPF binding were quantified as described (Hensler and Durgam, 2001), using NIH image, version 1.47 (NIH, Bethesda, MD). Specific binding was calculated by subtracting nonspecific binding from total binding on adjacent sections. 2.5. Data analysis For saturation binding experiments, binding data were analyzed by nonlinear regression analysis, using the computer program Prism, in order to determine the affinity (Kd) and maximal density (Bmax) of the specific [3H]paroxetine binding sites. For each brain region, a ttest was performed to assess the statistical significance (P⬍0.05) of differences in specific binding between age groups. The level of significance was P⬍0.05 for all experiments. 2.6. Materials [3H]Paroxetine (15 Ci/mmol), [35S]GTPγS (1250 4-(2⬘-Methoxy)-phenyl-1Ci/mmol) and [3H] [2⬘-(N-2⬙-pyridinyl)-p-fluorobenzamido]ethyl-piperzin ([3H]MPPF) (66.2 Ci/mmol) were purchased from Dupont/NEN (Boston, MA). Chlomipramine and WAY 100635 maleate were purchased from Sigma RBI (Natick, MA). GDP (disodium salt) was purchased from ICN (Costa Mesa, CA). 8-hydroxy-dipropylaminotetralin ((±)8-OH-DPAT) hydrobromide was purchased from Tocris (Ballwin, MO). GTPγS (tetralithium salt) was purchased from Roche/Boehringer-Manheim (Indianapolis, IN).

2.4. [3H]MPPF autoradiography

3. Results

Autoradiography of the binding of [3H]MPPF to 5HT1A receptors in brain sections was performed as described (Hensler and Durgam, 2001). Briefly, slidemounted sections were thawed in a dessicator at 4 °C for 30 min. Sections were preincubated for 30 min at room temperature in assay buffer (170 mM Tris–HCl, pH 7.6 at room temperature). Sections were subsequently incubated in assay buffer containing 10 nM [3H]MPPF for 90 min at room temperature. Nonspecific binding was defined by incubating adjacent sections in

The effect of aging on the binding of [3H]paroxetine to the serotonin transporter was examined in the dorsal and median raphe nuclei using saturation binding analysis. Saturation isotherms for the specific binding of [3H]paroxetine to serotonin reuptake sites in the dorsal and median raphe nuclei were analyzed and the data are summarized in Table 1. Aging induced a significant (P⬍0.02) decrease of approximately 20% in the maximal density (Bmax) of specific [3H]paroxetine binding sites in the dorsal raphe nucleus. A trend toward an age-

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Table 1 Effect of aging on the binding of [3H]paroxetine to serotonin transporter sites in the dorsal and median raphe nuclei Brain region Dorsal raphe nucleus Young animals Old animals Median raphe nucleus Young animals Old animals

Kd (pM)

Bmax (fmol/mg protein)

342±44 311±19

487±27 396±13∗

294±33 247±50

294±12 247±20

Kd and Bmax values were derived from the analysis of saturation binding data. Eight concentrations of [3H]paroxetine (0.07–5.07 nM) were used as described in Section 2. Shown are the mean±S.E.M. of six to seven individual experiments carried out in duplicate. ∗P⬍0.02 versus young animals.

related decrease in Bmax was observed in the median raphe nucleus, but this trend did not reach statistical significance (P= 0.07). There was no significant difference between the age groups in the apparent affinity (Kd) of specific [3H]paroxetine binding in either the dorsal or median raphe nuclei (Table 1). Representative autoradiographic images are shown in Fig. 1. The effect of aging on 5-HT1A receptor-stimulated [35S]GTPγS binding was examined in the dorsal raphe and median raphe nuclei. A single, maximal concentration of the 5-HT1A receptor agonist (±)8-OH-DPAT (1 µM) (Hensler and Durgam, 2001) was used to stimulate [35S]GTPγS binding in tissue sections. Aging did not alter (±)8-OH-DPAT-stimulated [35S]GTPγS binding in either the dorsal or median raphe nucleus (Fig. 2). These data indicate that in these serotonergic cell body areas, the capacity of the 5-HT1A receptor to activate G proteins is not altered in old animals versus young animals. Furthermore, aging did not alter the specific binding of a single saturating concentration of the 5-HT1A receptor antagonist radioligand [3H]MPPF (10 nM) to 5-HT1A

Fig. 2. Effect of aging on 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal and median raphe nuclei. Coronal sections were incubated with [35S]GTPγS (80 pM). Nonspecific binding was defined in the presence of 10 µM GTPγS. [35S]GTPγS binding was stimulated by (±)8-OH-DPAT (1 µM). Specific binding of [35S]GTPγS is expressed as % above basal. Shown are the mean±S.E.M. MRN, median raphe nucleus; DRN, dorsal raphe nucleus.

receptor sites in the dorsal and median raphe nuclei (Fig. 3). The effect of aging on 5-HT1A receptor-stimulated [35S]GTPγS binding was also examined in the hippocampus. Autoradiograms of the binding of [35S]GTPγS to sections of hamster brain taken at the level of the hippocampus are shown in Fig. 4. Interestingly, [35S]GTPγS binding stimulated by (±)8-OH-DPAT (1 µM) was significantly increased in the CA1 region of old hamsters by approximately 75% as compared to young hamsters (Fig. 5). There was no significant effect of aging on (±)8OH-DPAT-stimulated [35S]GTPγS binding in the dentate gyrus (Fig. 5). These data suggest that aging selectively increases postsynaptic 5-HT1A receptor-G protein interaction in the CA1 region of the hippocampus. The effect of aging on 5-HT1A receptor number in hippocampus was examined by measuring the binding the 5-HT1A receptor antagonist radioligand [3H]MPPF (10

Fig. 1. Age-related differences in specific binding of [3H]paroxetine (1.2 nM) to serotonin reuptake sites in hamster raphe. The photograph represents the digitized images of specific [3H]paroxetine binding created by subtraction non-specific [3H]paroxetine binding (in the presence of 10 µM chlomipramine) from total [3H]paroxetine binding. (A) 3 months old; (B) 17 months old.

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Fig. 3. Effect of aging on the specific binding of [3H]MPPF to 5HT1A receptor sites in the dorsal and median raphe nuclei. Coronal sections of hamster brain were incubated with [3H]MPPF (10 nM). Nonspecific binding was defined in the presence of 10 µM WAY100635. Specific binding is expressed as fmol/mg protein. Shown are the mean±S.E.M. MRN, median raphe nucleus; DRN, dorsal raphe nucleus.

Fig. 5. Effect of aging on 5-HT1A receptor-stimulated [35S]GTPγS binding in hippocampus. Coronal sections were incubated with [35S]GTPγS (80 pM). Nonspecific binding was defined in the presence of 10 µM GTPγS. [35S]GTPγS binding was stimulated by (±)8-OHDPAT (1 µM). Specific binding of [35S]GTPγS is expressed as % above basal. CA1, CA1 region; DG, dentate gyrus. ∗P⬍0.05 versus young animals.

Fig. 4. Autoradiograms of [35S]GTPγS binding to sections of hamster brain taken at the level of the hippocampus. (A) 3 months old; (B) 17 months old. Coronal sections were incubated with [35S]GTPγS (80 pM). Nonspecific binding was defined in the presence of 10 µM GTPγS. The binding of [35S]GTPγS was stimulated by (±)8-OH-DPAT (1 µM).

nM). Representative autoradiographic images are shown in Fig. 6. As shown in Fig. 7, the binding of [3H]MPPF to 5-HT1A receptor sites in the CA1 region of the hippocampus was not altered in old versus young animals. These data indicate that the age-related increase in 8OH-DPAT-stimulated [35S]GTPγS binding observed in the CA1 region of the hippocampus was not due to an increase in 5-HT1A receptor number. Interestingly, the

binding of [3H]MPPF to 5-HT1A receptor sites in the dentate gyrus of the hippocampus was reduced by approximately 53% in old versus young animals (Fig. 7). In view of our finding that 5-HT1A receptor-stimulated [35S]GTPγS binding is not altered in dentate gyrus, these data suggest that the capacity of the remaining 5-HT1A receptors to activate G proteins is increased in this hippocampal region as well.

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Fig. 6. Age-related differences in specific binding of [3H]MPPF to 5-HT1A receptor sites in hamster hippocampus. The photograph represents the digitized images of specific [3H]MPPF binding created by subtraction non-specific [3H]MPPF binding (in the presence of 10 µM WAY 100635) from total [3H]MPPF binding. (A) 3 months old; (B) 17 months old.

Fig. 7. Effect of aging on the specific binding of [3H]MPPF to 5HT1A receptor sites in the hippocampus. Coronal sections of hamster brain were incubated with [3H]MPPF (10 nM). Nonspecific binding was defined in the presence of 10 µM WAY100635. Specific binding is expressed as fmol/mg protein. Shown are the mean±S.E.M. CA1, CA1 region; DG, dentate gyrus. ∗P⬍0.05 versus young animals.

4. Discussion Aging induces alterations and deficits in many processes that are modulated by serotonin, including circadian rhythms, sleep, affective state, and hormone secretion (Carskadon et al., 1982; Van Reeth et al., 1992; Czeisler et al., 1992; Zee et al., 1992; Copinschi and Van Cauter, 1994; Barefoot et al., 2001). The midbrain raphe nuclei are important neuroanataomical substrates for the regulation of these processes, because they are the primary sources of serotonergic innervation of the forebrain (Moore et al., 1978; Molliver, 1987; Morin and Meyer-Bernstein, 1999). The main focus of the current study was the effect of aging on the serotonin transporter and 5-HT1A receptors in the raphe, since these proteins regulate the activity of the serotonergic neurons (Gallager and Aghajanian, 1975; Sprouse and Aghajanian, 1986, 1987; Sotelo et al., 1990; Adell and Artigas, 1991; Invernizzi et al., 1992). We have found that aging decreased the density of serotonin transporter sites in the dorsal raphe nucleus. A trend towards an age-related decrease in serotonin transporter sites in the median

raphe nucleus was also observed, although this effect was not statistically significant. There was no effect of aging on 5-HT1A receptor density or 5-HT1A receptorstimulated [35S]GTPγS binding in the dorsal or median raphe nuclei. These results confirm and extend our previous finding that aging did not alter specific binding of the agonist radioligand [3H]8-OH-DPAT to 5-HT1A receptors in the raphe nuclei (Duncan et al., 1999) by showing that aging does not alter the total number of 5HT1A receptors or 5-HT1A receptor function at the level of receptor-G protein interaction in serotonergic cell body areas. The raphe nuclei are emerging as an important locus of regulation of circadian rhythms. Electrical stimulation of the either the median or dorsal raphe nuclei during the midsubjective daytime stimulates serotonin release in the suprachiasmatic nucleus, the site of the mammalian circadian pacemaker, and induces circadian phase advances (Dudley et al., 1999; Glass et al., 2000; Ehlen et al., 2001). Furthermore, administration of 8-OHDPAT into the median and dorsal raphe nuclei induces phase advances during the midsubjective day, similar to systemic administration of 8-OH-DPAT (Mintz et al., 1997). In previous studies, we have observed age-related changes in the serotonin transporter and serotonin receptors in the hamster dorsal raphe and other brain regions (Duncan et al., 1999, 2000). Some changes in the serotonergic system appear in middle age, while others are not evident until old age. For example, we have previously found an increase in 5-HT1B receptors and serotonin transporter sites in the SCN of middle-age hamsters (12 months old) (Duncan et al., 2000). A decrease in 5-HT7 receptors in the dorsal raphe nucleus is not evident in middle age (12 or 15 months old), but appears in old age (17–19 months old) (Duncan et al., 1999, 2000). Similarly, we did not detect any changes in the binding of [3H]paroxetine to serotonin transporter sites in raphe nuclei of middle-age hamsters (12 months old) (Duncan et al., 2000), but do observe a decrease in specific [3H]paroxetine binding in the raphe nuclei of old

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hamsters (17–19 months old) (current study). A decrease in the serotonin transporter sites would be expected to lead to a decrease in serotonin reuptake and thus an increase in the extracellular levels of serotonin. 5-HT7 receptors are down-regulated following administration of selective serotonin reuptake inhibitors which increase extracellular serotonin levels (Mullins et al., 1999; Thomas et al., 2001). Thus, the age-related decline in the serotonin transporter sites in the dorsal raphe may be related to the age-related decrease in 5-HT7 receptors we previously observed in this region (Duncan et al., 1999), and in this way, contribute to the reduced sensitivity of old hamsters to circadian phase shifts induced by serotonergic drugs (Penev et al., 1995). Experimentally-induced reductions in the serotonin transporter in the raphe nuclei have been shown to cause changes in 5-HT1A receptor function. For example, injection of an antisense coding sequence for the 5-HT transporter gene into the dorsal raphe nucleus of the rat results in a 28% reduction in serotonin transporter sites and a decrease in 5-HT1A receptor-stimulated [35S]GTPγS binding in this brain region (Fabre et al., 2000b). In knock-out mice lacking the 5-HT transporter, 5-HT1A receptor-stimulated [35S]GTPγS binding is reduced in the dorsal raphe nucleus; however 5-HT1A receptor density is also significantly reduced in this brain region (Fabre et al., 2000a). Following chronic administration of selective serotonin re-uptake inhibitors, serotonin transporter binding is down-regulated by approx. 50% in the dorsal raphe nucleus (S. Benmansour and A. Frazer, personal communication), and somatodendritic 5-HT1A receptor function is decreased (see Blier and De Montigny, 1994; Kreiss and Lucki, 1995; Le Poul et al., 2000). This desensitization of somatodendritic 5-HT1A receptors following chronic selective serotonin re-uptake inhibitor treatment appears to be due to a reduced capacity of the 5-HT1A receptor to activate G proteins, in that 5-HT1A receptor-stimulated [35S]GTPγS binding is attenuated in the raphe nuclei (Hensler, 2002). In the present study, we found that a reduction in serotonin transporter sites in the dorsal raphe at 17–19 months of age apparently does not alter 5-HT1A receptor-stimulated [35S]GTP γS binding. A decrease in the serotonin transporter sites would be expected to lead to an increase in the extracellular levels of serotonin and a desensitization of 5-HT1A receptors in the raphe nuclei. We are uncertain as to why we did not observe a change in 5-HT1A receptor function at the level of receptor-G protein interaction in the dorsal raphe of old hamsters, but suggest that the modest (19%) decrease in serotonin transporter observed in old versus young hamsters may not be sufficient to alter 5-HT1A receptor function in the dorsal raphe nucleus. In contrast to the absence of an age-related difference in 5-HT1A receptor-stimulated [35S]GTPγS binding in the midbrain raphe nuclei, there was an age-related increase

in 5-HT1A receptor-stimulated [35S]GTPγS binding in the CA1 region of the hippocampus with no change in the number of 5-HT1A receptor binding sites. These data, which suggest that the capacity of the 5-HT1A receptor to activate G proteins is increased in the CA1 region of the hippocampus of old animals, are interesting because of the importance of this region in memory and learning. Selective lesions of the CA1 region in humans can cause learning and memory disturbances (Zola-Morgan et al., 1986). Aging is also associated with deficits in these processes, particularly spatial memory, in humans (Moffat et al., 2001). In rats and hamsters, many age-related changes in the CA1 region of the hippocampus have been observed. In conjunction with the findings that stimulation of post-synaptic 5-HT1A receptors in the CA1 region impairs spatial learning in rats (Carli et al., 1992, 1995), our results suggest that enhanced 5-HT1A receptor function at the level of receptor-G protein interaction in the CA1 region may contribute to the loss of spatial memory that occurs with aging. In the present study, the dentate gyrus also exhibited age-related differences in 5-HT1A receptors. In this region, in contrast to the CA1 region of the hippocampus, there was a marked decrease in 5-HT1A receptor binding. Similar observations have been made in rats, where aging results in a selective and marked decline in the binding of the agonist radioligand [3H]8-OH-DPAT to 5-HT1A receptor in the dentate gyrus, whereas the CA regions are unaffected (Nyakas et al., 1997). Corticosterone and stress also selectively regulate 5-HT1A receptor expression in the dentate gyrus but not CA regions (Mendelson and McEwen, 1992; Meijer and De Kloet, 1994). The higher basal plasma corticosterone level and the prolonged plasma corticosterone stress response associated with aging may be a major factor underlying the age-dependent loss of 5-HT1A receptors in the dentate gyrus (see Nyakas et al., 1997 and references therein). Although the density of 5-HT1A receptor sites in the dentate gyrus of old hamsters is reduced, 5-HT1A receptor-stimulated [35S]GTPγS binding was not altered in this region of the hippocampus. Taken together these data suggest that there is an age-related compensatory increase in the capacity of the remaining 5-HT1A receptors to activate G proteins in the dentate gyrus. The enhanced 5-HT1A receptor function at the level of receptor-G protein interaction in the dentate gyrus may contribute to age-related deficits in memory. In conclusion, the decrease in serotonin transporter sites in the dorsal raphe nucleus of old hamsters was not associated with a change in 5-HT1A receptor number or function at the level of interaction with G proteins, indicating that aging differentially affects the serotonin transporter and 5-HT1A receptors in the raphe. In contrast to the dorsal raphe, which did not exhibit age-related changes in 5-HT1A receptor number or 5-HT1A receptor activation of G proteins, age-related increases in the

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capacity of the 5-HT1A receptor to activate G proteins were observed in the CA1 region of the hippocampus. In the dentate gyrus, however, age-related decreases in 5-HT1A receptor number were not accompanied by a change in 5-HT1A receptor activation of G proteins, suggesting a compensatory increase in the capacity of 5HT1A receptors to activate G proteins in this brain region with age. Our findings are consistent with the proposal that aging is a complex process, rather than simple, progressive degeneration; a process that involves heterogeneous changes that vary with region, and range from deterioration to preservation and even to functional compensation (Barnes, 1994).

Acknowledgements The authors would like to thank Mrs. Teri Burke, Ms. Mari Valdez, Ms. Deborah Wheeler and Ms. Karrie Greer for excellent technical assistance. This research was supported by AG 13418 (MJD) and US PHS grant MH 52369 (JGH).

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