Marilyn J. Duncan

Comparison of the effects of aging on 5-HT7 and 5-HT1A receptors in discrete regions of the circadian timing system in hamsters

The circadian timekeeping system exhibits many functional changes with aging, including a loss of sensitivity to time cues such as systemic injections of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In order to elucidate the neurochemical mechanisms responsible for this age-related loss of sensitivity of the circadian pacemaker to serotonin agonists, the present study used quantitative autoradiography to determine whether aging decreases serotonin receptor populations in male Syrian hamsters. Four neuroanatomical regions that regulate circadian timekeeping were studied (the suprachiasmatic nuclei [SCN], the lateral geniculate nuclei [LGN], and the median raphe nucleus [MRN] and dorsal raphe nucleus [DRN]). The specific binding of [3H]8-OH-DPAT to serotonin7 (5-HT7) and serotonin1A (5-HT1A) receptors was investigated by competitive inhibition with ritanserin and pindolol, respectively. The results showed that the SCN, IGL, MRN, and DRN of the male Syrian hamster exhibited specific binding of [3H]8-OH-DPAT to both the 5-HT7 and 5-HT1A receptors, and that the latter receptor subtype is more abundant in all of these regions. At 17-19 months of age, a 50% decrease in 5-HT7 receptors was found in the DRN but not in any other regions. No significant age-related changes in 5-HT1A receptors were observed in any regions examined. The finding that a marked decrease in 5-HT7 receptors occurs in the DRN at the age previously characterized by loss of sensitivity to 8-OH-DPAT suggests that this region and this receptor subtype play important roles in 8-OH-DPAT induction of circadian phase shifts in vivo and that they constitute an important locus of aging in the circadian timing system.

Aging selectively suppresses vasoactive intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the Syrian hamster.

Aging leads to many changes in the expression of circadian rhythms, including reduced amplitude, altered relationship to the environmental illumination cycle, and reduced sensitivity to phase resetting signals. Neuropeptide synthesizing neurons in the suprachiasmatic nucleus (SCN), the principal circadian pacemaker in mammals, play a role in regulating pacemaker function and in coupling the pacemaker to overt circadian rhythms. Aging may alter the activity of neuropeptide neurons in the SCN, which could be reflected in changes in mRNA expression. Therefore, this study investigated whether aging alters the level or rhythm of expression of neuropeptide mRNAs in the SCN of male Syrian hamsters, a well established model for the study of age-related changes in circadian rhythms. Three age groups of hamsters (young [3--5 months old], middle-aged [12--15 months old] and old [19--22 months old] were sacrificed at five times of day. Their brains were dissected and sections through the suprachiasmatic nucleus were prepared and used for in situ hybridization for mRNAs for vasoactive intestinal peptide (VIP), arginine vasopressin (AVP) and somatostatin (SS). Aging selectively decreased the SCN expression of VIP mRNA without affecting AVP mRNA or SS mRNA. Also, only AVP mRNA expression exhibited a robust 24-h rhythm, in contrast to previous findings in other species that VIP mRNA and SS mRNA, as well as AVP mRNA, exhibit 24-h rhythms in the SCN. The present findings suggest that age-related reductions in VIP mRNA expression may contribute to the alterations in entrainment and attenuated sensitivity to phase resetting signals that are characteristic of aging. Furthermore, the results demonstrate that neuropeptide gene expression in the SCN is differentially regulated by aging and varies among species.

Aging and SB-269970-a, a selective 5-HT7 receptor antagonist, attenuate circadian phase advances induced by microinjections of serotonergic drugs in the hamster dorsal raphe nucleus

Aging leads to many changes in the circadian timekeeping system, including reduced sensitivity to phase-resetting signals such as systemic administration of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In previous studies, we observed an age-related decrease in 5-HT7 receptor binding sites, one of the receptor subtypes that is activated by 8-OH-DPAT, in the dorsal raphe nucleus. In this study, we tested the hypotheses that (1) aging reduces circadian phase shifts induced by local administration of 8-OH-DPAT (30 microM, i.e., 1.97 ng) or 5-carboxamidotryptamine (5-CT, 100 nM, i.e., 6.39 pg), another serotonin agonist, into the dorsal raphe and (2) 5-HT7 receptors mediate the phase shifts induced by administration of 5-CT and 8-OH-DPAT into the dorsal raphe. Young (3-5 months), middle-aged (12-13 months) and old hamsters (17-19 months) were surgically implanted with chronic guide cannulae aimed at the dorsal raphe, and were housed in cages equipped with running wheels. Aging significantly inhibited (P<0.01) the phase advances in running-wheel rhythms induced by 8-OH-DPAT microinjected during the midsubjective day. 5-CT induced phase advances tended to decrease with aging, but this effect was not significant (P<0.12). Microinjection of the selective 5-HT7 receptor antagonist, SB-269970-A (50-5000 nM, i.e., 0.39-390 pg), 15 min before microinjection of 5-CT or 8-OH-DPAT into the dorsal raphe of young hamsters, significantly inhibited phase shifts. In conjunction with our previous study, these findings indicate that an age-related reduction in 5-HT7 receptors in the dorsal raphe nucleus is an important neurochemical mechanism leading to aging deficits in the circadian timekeeping system.

κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: relationship to Ca2+ mobilization, development, and the antiproliferative effect of opioids

To assess the role of kappa-opioid receptors in astrocyte development, the effect of kappa-agonists on the growth of astroglia derived from 1-2-day-old mouse cerebra was examined in vitro. kappa-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of kappa-receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6-7, as many as 50% of the astrocytes displayed kappa-receptor (KA8) immunoreactivity or exhibited increases in [Ca2+]i in response to kappa-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the kappa-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were kappa-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca2+]i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that kappa-receptor activation increases astroglial [Ca2+]i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express kappa-opioid receptors in vitro, and suggest that the activation of kappa-receptors mobilizes [Ca2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing kappa-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.

Localization of serotonin5A receptors in discrete regions of the circadian timing system in the Syrian hamster

Endogenous serotonin and serotonergic drugs influence many aspects of circadian rhythms, including phase shifts, onset of locomotor activity, and period length and integrity of rhythms during exposure to constant light. The receptor subtype(s) mediating all of these circadian effects of serotonin has (have) not been identified. Immunoreactivity for the serotonin(5A) (5-HT(5A)) receptor has recently been identified in the rat suprachiasmatic nucleus (SCN). In this study, we investigated the distribution of the 5-HT(5A) receptors in four neural components of the circadian timing system (the SCN, the intergeniculate leaflet, and the median and dorsal raphe nuclei), in the Syrian hamster. Single and dual immunohistochemistry were conducted using an affinity-purified rabbit antibody generated against a peptide sequence unique to the 5-HT(5A) receptor, guinea pig anti-5-HT antisera and guinea pig anti-GABA antisera. For single labeling, immunoreactivity was visualized using DAB-nickel as the chromagen. All four regions showed strong, yet distinct, immunoreactivity for the 5-HT(5A) receptor. No specific labeling was present in the absorption or omission controls. For double labeling, immunoreactivity was visualized using immunofluorescence with Cy5- and FITC-labeled second antibodies followed by confocal microscopy. In the raphe nuclei, 5-HT-immunoreactivity and 5-HT(5A)-immunoreactivity were co-localized in cell bodies and axons. GABA-immunoreactive fibers surrounded some of the 5-HT(5A) receptor-immunoreactive cell bodies in the raphe nuclei. In conclusion, the 5-HT(5A) receptors are localized within several important neuroanatomical substrates of the circadian timekeeping system, and within the raphe nuclei, appear to be present on serotonin neurons. These findings suggest that some of the circadian effects of 5-HT may be mediated by the 5-HT(5A) receptor, which may function as a presynaptic autoreceptor.

Developmental appearance and age related changes in specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female Syrian hamsters.

In Syrian hamsters, the circadian timing system is sensitive to melatonin during gestation but is not responsive in the adult. In order to further understand this developmental change in melatonin responsiveness, in vitro autoradiography was used to assess the presence of specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei of female hamsters of selected embryonic (E) and postnatal (PN) ages (e.g. E13, E14, E15, PN1, PN2, PN12, PN25, PN112-133). Specific 2-[125I]iodomelatonin binding sites were seen in the suprachiasmatic nuclei of some of the E14 hamsters and all the perinatal hamsters (E15, PN1 and PN2) but not in older hamsters. In contrast, specific 2-[125I]iodomelatonin binding sites were seen in the pars tuberalis of all hamsters (with the exception of one), regardless of age. The transient expression of specific 2-[125I]iodomelatonin binding sites in the suprachiasmatic nuclei suggests that melatonin may have some special functions restricted to early development. The specific 2-[125I]iodomelatonin binding sites in the embryonic suprachiasmatic nuclei may represent the substrate for maternal melatonin to set the phase of the developing circadian timing system.

Photoperiodic exposure and time of day modulate the expression of arginine vasopressin mRNA and vasoactive intestinal peptide mRNA in the suprachiasmatic nuclei of Siberian hamsters.

In hamsters, changes in ambient photoperiod lead to alterations in the circadian rhythm of pineal melatonin secretion and subsequent changes in reproductive function. The present study examined whether photoperiod also alters 24-h rhythms in neuropeptide mRNA levels in the SCN of Siberian hamsters. In situ hybridization and quantitative autoradiography were used to assess messenger RNA levels for vasopressin (AVP) and vasoactive intestinal peptide (VIP) in the SCN of hamsters sacrificed at six times of day following exposure to long (16 h light/day) or short (10 h light/day) photoperiod for 2 weeks. Both AVP mRNA and VIP mRNA in the SCN were significantly affected by time of day and photoperiodic exposure. The 24-h profiles of AVP mRNA and VIP mRNA showed different relationships to the light: dark cycle, suggesting that these profiles are differentially regulated. In general, short photoperiod tended to suppress AVP mRNA and VIP mRNA in the SCN; this effect on AVP mRNA was significant at two times of day. These results complement and extend previous findings of 24-h h profiles in neuropeptide mRNA expression in the rat SCN by showing that these 24-h profiles are also characteristic of the Siberian hamster SCN and that they can be modulated by photoperiod.

Aging, estradiol and time of day differentially affect serotonin transporter binding in the central nervous system of female rats

Estrogen-related changes in serotonergic neuronal transmission, including changes in the number of serotonin transporter (SERT) binding sites, have been cited as a possible cause for changes in mood, memory and sleep that occur during the menopausal transition. However, both aging and estradiol regulate SERT binding sites in the brain. The goal of this experiment was to determine how aging and estrogen interact to regulate SERT levels in the forebrain of young and reproductively senescent female Sprague-Dawley rats using [3H]paroxetine. The density of specific [3H]paroxetine binding in various brain regions was compared in young (2-4 months) and reproductively senescent (10-12 months) female rats at three times of day. In most brain regions examined, estrogen and aging independently increased the number of [3H]paroxetine binding sites. The only region that displayed a reduction in [3H]paroxetine binding with age was the suprachiasmatic nucleus (SCN). Time of day influenced [3H]paroxetine binding in the SCN and the paraventricular thalamus (PVT), two regions known to be involved in the regulation of circadian rhythms. Aging and/or estrogen also altered the pattern of binding in these regions. Thus, based on the results of this study, we conclude that aging and estrogen both act to regulate SERT binding sites in the forebrain of female rats, and that this regulation is region specific.

Influence of aging on Bmal1 and Per2 expression in extra-SCN oscillators in hamster brain.

Deletion of the core clock gene, Bmal1, ablates circadian rhythms and accelerates aging, leading to cognitive deficits and tissue atrophy (e.g., skeletal muscle) (Kondratov et al., 2006, Kondratova et al., 2010). Although normal aging has been shown to attenuate Bmal1 expression in the master circadian pacemaker in the suprachiasmatic nucleus (SCN), relatively little is known about age-related changes in Bmal1 expression in other tissues, where Bmal1 may have multiple functions. This study tested the hypothesis that aging reduces Bmal1 expression in extra-SCN oscillators including brain substrates for memory and in skeletal muscle. Brains and gastrocnemius muscles were collected from young (3-5 months) and old hamsters (17-21 months) euthanized at four times of day. Bmal1 mRNA expression was determined by conducting in situ hybridization on brain sections or real-time PCR on muscle samples. The results showed age-related attenuation of Bmal1 expression in many brain regions, and included loss of diurnal rhythms in the hippocampal CA2 and CA3 subfields, but no change in muscle. In situ hybridization for Per2 mRNA was also conducted and showed age-related reduction of diurnal rhythm amplitude selectively in the hippocampal CA1 and DG subfields. In conclusion, aging has tissue-dependent effects on Bmal1 expression in extra-SCN oscillators. These finding on normal aging will provide a reference for comparing potential changes in Bmal1 and Per2 expression in age-related pathologies. In conjunction with previous reports, the results suggest the possibility that attenuation of clock gene expression in some brain regions (the hippocampus, cingulate cortex and SCN) may contribute to age-related cognitive deficits.

Aging regulates 5-HT1B receptors and serotonin reuptake sites in the SCN

Middle age is associated with changes in circadian rhythms (e.g., alterations in the timing of the circadian wheel running rhythm) which resemble changes induced by selective destruction of the serotonergic input to the suprachiasmatic nucleus (SCN), the principal mammalian circadian pacemaker. We hypothesized that serotonergic neurotransmission in the SCN is decreased in middle-aged hamsters, as compared to young adults. This hypothesis was tested indirectly by investigating the effect of aging on two markers of serotonin neurotransmission, 5-HT(1B) receptors and serotonin reuptake sites, which are regulated by serotonin. Previous studies have shown that experimentally induced decreases in serotonergic neurotransmission increase 5-HT(1B) receptors but decrease serotonin reuptake sites. Quantitative autoradiography was conducted using [125I]iodocyanopindolol ([125I]ICYP) and [3H]paroxetine, selective radioligands for the 5-HT(1B) receptors and the serotonin reuptake sites, respectively. Consistent with the hypothesis, specific ([125I]ICYP binding was significantly elevated in the SCN of middle-aged hamsters, as compared to young hamsters. The results also showed that serotonin reuptake sites in the SCN were significantly increased in both middle-aged and old hamsters, as compared to young controls. This result could not have been caused by decreased serotonin release. Alternatively, increased serotonin reuptake, which would reduce serotonin levels in the synaptic cleft, may cause or contribute to the increase in 5-HT(1B) receptor binding in the SCN in middle aged animals. These results show that the SCN exhibits changes in serotonergic function during middle age, which has been characterized by changes in the expression of circadian rhythms. Because these changes occur during middle age, they probably reflect the aging process, rather than senescence or disease.