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Serotonin transporter localization in the hamster suprachiasmatic nucleus
Brain Research 893 (2001) 77–83 www.elsevier.com / locate / bres
Research report
Serotonin transporter localization in the hamster suprachiasmatic nucleus Roseann Legutko, Robert L. Gannon* Department of Biology, Dowling College, Oakdale, Long Island, NY 11769 -1999, USA Accepted 28 November 2000
Abstract Pacemaker cells within the hamster suprachiasmatic nucleus generate circadian rhythms. The suprachiasmatic nucleus is heavily innervated by serotonin axons originating in the median raphe nuclei. Consequently, serotonergic agonists and antagonists or agents that alter levels of serotonin in the synapse following transmission can modulate many aspects of circadian rhythmicity. Examples of the latter are some antidepressants and the stimulant amphetamine that bind to the serotonin transporter and block serotonin reuptake. It has been hypothesized that circadian rhythm dysfunction may be involved in depression, and that the efficacy of certain antidepressants in treating depression may involve an alteration of serotonin levels and certain circadian rhythm parameters. However, although the hamster is the behavioral model of choice for the study of circadian rhythms, the identification of serotonin transporters in this species has not been reported. Therefore, in this report we describe the distribution of the serotonin transporter in the hamster suprachiasmatic nucleus using immunohistochemical techniques. Our results demonstrate a dense labeling of the serotonin transporter throughout the ventral and medial regions of the suprachiasmatic nucleus, a pattern that overlaps the distribution of serotonergic afferents in this nucleus. Amphetamines and certain antidepressants may serve as substrates for this transporter and elicit chronopharmacological activity by elevating serotonin levels in the suprachiasmatic nucleus. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Biological rhythm; Circadian rhythm; Reuptake; Antidepressant; Amphetamine
1. Introduction The suprachiasmatic nucleus (SCN) of the hypothalamus is known to be the site of the master pacemaker that controls circadian activity rhythms in mammalian species [22]. The phase of the SCN circadian pacemaker is entrained to the environmental light–dark cycle by afferent retinal input via the retinohypothalamic tract [9], and this input can be modified by afferent transmission from geniculate nuclei of the thalamus [6] and midbrain raphe nuclei [7,11,12]. Afferent fibers from these three regions in the hamster brain employ excitatory amino acids (retina) [5,13], neuropeptide Y and enkephalin (geniculate) [8,16] and serotonin (raphe) [11,14] as their transmitters, thus a *Corresponding author. Tel.: 11-631-244-3339; fax: 11-631-5635140. E-mail address: [email protected] (R.L. Gannon).
wide array of pharmacological compounds will affect the phase of circadian rhythms. Included among these chronobiotic compounds are drugs-of-abuse such as amphetamine and some clinically prescribed antidepressants [10,18]. Amphetamine and certain antidepressants are known to interact with serotonin transmission in other brain regions as well as the SCN [23,26]; therefore an understanding of serotonin’s role in circadian rhythms has considerable clinical relevance. The hamster SCN receives a dense innervation of serotonergic fibers originating from the median raphe nuclei [11]. Exogenously applied serotonergic agonists will typically inhibit light-induced phase adjustments of circadian activity rhythms by activating both presynaptic and postsynaptic serotonin receptors within the SCN [15]. Activation of presynaptic 5HT 1B receptors on retinohypothalamic terminals within the SCN will block light-induced phase shifts of hamster activity rhythms,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03290-X
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R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83
presumably by inhibiting glutamate release from the terminals [20]. Activation of postsynaptic 5HT 1A,7 receptors within the SCN will also inhibit light-induced modulation of hamster circadian activity rhythms through as yet unknown mechanisms [29]. Therefore, any modulation of serotonin levels within the hamster SCN will likely have an effect on the photic responsiveness of the circadian pacemaker. Serotonin is removed from the synaptic cleft by reuptake through the serotonin transporter (SERT). Certain antidepressants such as fluoxetine block the SERT and increase levels of serotonin in the synaptic cleft [26]. Amphetamine and its derivatives also block reuptake and they have the additional effect of causing the SERT to release previously sequestered serotonin back into the synaptic cleft [3,25]. Therefore, both fluoxetine and amphetamines increase extracellular serotonin levels, and amphetamine inhibits light-induced phase shifts of hamster activity rhythms [18]. Surprisingly, the effect of fluoxetine on light-induced phase shifts in hamsters has not been reported. Nevertheless, there is sufficient evidence to suggest that altering SERT function in the hamster SCN will affect the ability of light to adjust circadian activity rhythms. Although SERT distribution has been characterized in the rat SCN [1], it has not been similarly described in the hamster. Therefore, the present study describes the distribution of SERT immunoreactivity within the hamster SCN.
2. Materials and methods For this study five adult male Syrian hamsters, Mesocricetus auratus, were purchased from Charles River Laboratories (Kingston, NY) and were |150 g when used. Hamsters were individually housed under a 14:10-h light-
:dark cycle with food and water provided ad libitum. Mid-way through the dark cycle at zeitgeber time 17 (lights-off is zeitgeber 12), hamsters were anesthetized under dim red light (,1 lx) with ketamine (200 mg / kg), acepromazine (2 mg / kg), and xylazine (20 mg / kg) before being transcardially perfused with saline and fixative. The perfusion formulations consisted of 0.9% heparinized saline (100 ml) followed by 4% paraformaldehyde prepared in 0.1 M phosphate buffer (125 ml, pH 7.4). Brains were removed following perfusions and stored overnight in 4% buffered paraformaldehyde before being cryoprotected in a solution of 30% sucrose. Brains were stored at 48C for |1 month before use. Frontal sections of hamster brains were cut 25-mm thick using a cryostat (Shandon Lipshaw, Pittsburgh, PA) and placed free-floating in 24-well tissue culture plates for immunohistochemistry. A 0.1-M phosphate buffer containing 0.12% gelatin and 0.25% Triton X-100 was used during each step of the staining procedure. All steps were performed at room temperature while rocking. Sections were incubated overnight in 1% hydrogen peroxide, washed, incubated with 25% ammonium chloride for 1 h, washed again, then incubated overnight with rabbit antiSERT polyclonal antibody (1:10,000; Oncogene Research Products, Cambridge, MA). Following removal of the primary antibody, sections were washed again then sequentially incubated for 1 h with biotinylated secondary antibody and ABC reagent from the Vectastain anti-rabbit peroxidase kit according to kit instructions (Vector, Burlingame, CA). Staining of sections was performed using Sigma Fast metal enhanced diaminobenzidine tablets (Sigma, St. Louis, MO). Finally, sections were washed with isotonic saline, mounted, dehydrated in alcohols and coverslipped. For controls, primary antibody was omitted in one
Fig. 1. Example of control used to demonstrate SERT specificity in the hamster brain. Dense labeling in the SCN (left panel) was absent when the primary antibody was omitted from the staining procedure (right panel). Scale bar represents 200 mm.
R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83
experiment, and primary antibody was pre-absorbed with control peptide (5 mg / ml, Oncogene Research Products) overnight at 48C before use in a separate experiment. Brightfield images were digitally captured using the Pixera Professional CCD camera (Pixera, Los Gatos, CA) and printed using a Hewlett Packard PhotoSmart photo printer. Images were not adjusted following capture in any way.
3. Results SERT immunoreactivity was widespread throughout the hamster brain. Staining was prominent in fibers throughout the diencephalon and telencephalon, with a dense plexus of fibers and neuropil staining concentrated in the SCN. The specificity of the staining patterns as well as control results indicate that the SERT antibody used in this study also
79
cross-reacts with hamster SERT sites. There was no staining in the control study where the SERT antibody was omitted (Fig. 1), while pre-incubation with control peptide resulted in a dark reaction product with no specific staining pattern (not shown). SERT immunoreactivity was detected in the entire rostral-caudal extent of the SCN (Figs. 2 and 3). In rostral regions, SERT immunoreactivity was limited to a thin strip of the ventral SCN that thickened in area while progressing toward the middle region of the nucleus (Figs. 2A, and 3A,B). In the mid-SCN regions, SERT immunoreactivity was pronounced along the ventral and medial aspects of the SCN with a slight outward progression of immunoreactivity in the dorsal regions. SERT immunoreactivity in the ventral SCN was widest (extended the greatest distance laterally) within this region (Figs. 2B,C, and 3C,D). The lateral areas of the mid-SCN contained only sparse staining. In more caudal regions of the SCN, SERT immuno-
Fig. 2. SERT immunoreactivity in a single hamster brain. Labeling is pronounced in the rostral (A), middle (B,C), and caudal (D) SCN. OC, optic chiasm; V, third ventricle. Scale bar represents 200 mm.
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and granular making identification of individual fibers and varicosities difficult (Fig. 4B). In less dense regions such as the cingulate cortex, SERT immunoreactivity in individual fibers with associated thickenings or varicosities was clearly evident (Fig. 4A). SERT immunoreactivity of fibers in the cingulate cortex is included in this study to clearly demonstrate SERT labeling of axons in the hamster brain and to allow for the comparison of immunolabeling patterns between this study in hamsters and that seen in other species [1,2,28].
4. Discussion
Fig. 3. Schematic representation of SERT immunoreactivity in a separate hamster brain. This pattern is characteristic to each hamster brain used in this study. (A,B) Rostral SCN; (C,D) middle SCN; (E,F) caudal SCN. 3V, third ventricle; OC, optic chiasm.
reactivity was mostly confined to medial areas of the nucleus, although there was a slight flare in staining patterns seen in both dorsal and ventral aspects of the SCN at this level (Figs. 2D, and 3E,F). High-power examination of SERT immunoreactivity within the SCN revealed some clearly defined fibers, although staining of the neuropil was typically very dense
SERT immunoreactivity is readily detected within the hamster brain using a polyclonal antisera directed against the rat SERT. In the hamster, SERT immunoreactivity labels axons spread throughout the diencephalon and telencephalon, with the appearance of punctate varicosities visible along many axons. This pattern of staining is similar to that demonstrated in other studies using rats [1,28]. SERT immunoreactivity within the hamster SCN is distributed in a pattern similar to that for serotonin fibers projecting to the SCN from the median raphe nuclei [11]. It is reasonable to assume then that the SERTs detected in this study are localized on serotonin fibers projecting from the median raphe. However, a recent study in the rat nucleus accumbens demonstrated two populations of serotonin fibers, those with and without SERT immunolabeling [2]. Therefore, there remains a slight possibility that in the hamster the SERT is located on non-serotonergic axons or only a subset of serotonergic axons within the SCN. The presence of SERTs in the hamster SCN has recently been inferred from binding studies using [ 3 H]paroxetine [4], although that study did not discuss the distribution of SERTs within the SCN. Together, then these results demonstrate a significant population of SERTs in the hamster SCN. SERT activity in the hamster and rat SCN would explain the results observed when amphetamines are used to modify circadian activity rhythms. In the hamster, methamphetamine inhibits both light-induced phase advances and delays, similar to that found after application of the serotonin agonist 8-OH-DPAT [29]. Methamphetamine has also been shown to increase serotonin release in the rat SCN both in vivo [19] and in vitro [18]. Finally, methamphetamine inhibits light-induced Fos expression in the rat SCN [18], similar to that seen upon application of serotonin agonists [27]. Therefore, it is likely that amphetamines affect circadian activity in the rat and hamster by elevating levels of serotonin in the SCN upon serving as a substrate for the SERTs located in the hamster and rat SCN. Tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs) also block the activity of SERTs and increase the level of serotonin in the synapse. How-
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Fig. 4. (A) SERT immunoreactivity in the hamster cingulate cortex. In this region, axonal staining is less dense and individual fibers, some with associated varicosities, are clearly evident. (B) SERT immunoreactivity in the hamster SCN. Staining is more dense making the individual identification of axonal fibers more difficult, although some fibers are seen coursing through the optic chiasm (arrowhead). OC, optic chiasm; SCN, suprachiasmatic nuclei. Scale bar represents 50 mm.
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ever, a significant effect of these compounds on circadian activity rhythms has been difficult to demonstrate. The tricyclic antidepressant imipramine did not affect circadian period or light-induced phase shifts in the hamster [24], although it does induce Fos expression in the rat SCN [17], and exhibits a circadian rhythmicity in binding in the rat SCN [30]. These studies are complicated by the action of imipramine on other monoamine transmitters. The SSRI fluoxetine however is more specific for the SERT. Fluoxetine has been reported to shorten circadian period in mice [21], but to have no effect on circadian period in either rat [31] or hamster [10]. Fluoxetine will also induce Fos expression in the rat SCN similar to 8-OH-DPAT [17]. Unfortunately, the effect of fluoxetine on light-induced phase shifts in hamsters, rats, or mice has not been reported. It would be predicted that chronic or perhaps even acute administration of fluoxetine would inhibit lightinduced changes in circadian-related events similar to serotonin. In conclusion, this report describes the distribution of the SERT in the hamster SCN. Since SERTs are targets for clinically used antidepressants and drugs of abuse, SERT distribution and activity has considerable clinical relevance. The density of SERTs in the hamster SCN would predict that drugs interacting with SERTs would affect circadian rhythms, and that this interaction may contribute to the behavioral effects of many compounds. However, the small amount of relevant literature indicates that the relationship between SERT-interacting drugs and circadian rhythms remains to be determined.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
Acknowledgements
[18]
This study was supported by NSF IBN-9728574 (R.L.G.). R. Legutko is an undergraduate student in the Biology Department at Dowling College.
[19]
References [1] S. Amir, B. Robinson, T. Ratovitski, M.A. Rea, J. Stewart, R. Simantov, A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet, Neuroscience 84 (1998) 1059–1073. [2] P. Brown, M.E. Molliver, Dual serotonin (5-HT) projections to the nucleus accumbens core and shell: relation of the 5-HT transporter to amphetamine-induced neurotoxicity, J. Neurosci. 20 (2000) 1952–1963. [3] D. Crespi, T. Mennini, M. Gobbi, Carrier-dependent and Ca (21)dependent 5-HT and dopamine release induced by (1)-amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (1)-fenfluramine, Br. J. Pharmacol. 121 (1997) 1735–1743. [4] M.J. Duncan, C.J. Crafton, D.L. Wheeler, Aging regulates 5-HT1B receptors and serotonin reuptake sites in the SCN, Brain Res. 856 (2000) 213–219. [5] F.J. Ebling, The role of glutamate in the photic regulation of the suprachiasmatic nucleus, Prog. Neurobiol. 50 (1996) 109–132. [6] K. Edelstein, S. Amir, The role of the intergeniculate leaflet in
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entrainment of circadian rhythms to a skeleton photoperiod, J. Neurosci. 19 (1999) 372–380. J.D. Glass, L.A. DiNardo, J.C. Ehlen, Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting, Brain Res. 859 (2000) 224–232. M.E. Harrington, D.W. Nance, B. Rusak, Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract, Brain Res. Bull. 15 (1985) 465–472. R.F. Johnson, L.P. Morin, R.Y. Moore, Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin, Brain Res. 462 (1988) 301–312. H. Klemfuss, D.F. Kripke, Antidepressant and depressogenic drugs lack consistent effects on hamster circadian rhythms, Psychiatry Res. 53 (1994) 173–184. E.L. Meyer-Bernstein, L.P. Morin, Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation, J. Neurosci. 16 (1996) 2097–2111. E.L. Meyer-Bernstein, L.P. Morin, Electrical stimulation of the median or dorsal raphe nuclei reduces light-induced FOS protein in the suprachiasmatic nucleus and causes circadian activity rhythm phase shifts, Neuroscience 92 (1999) 267–279. E.M. Mintz, C.L. Marvel, C.F. Gillespie, K.M. Price, H.E. Albers, Activation of NMDA receptors in the suprachiasmatic nucleus produces light-like phase shifts of the circadian clock in vivo, J. Neurosci. 19 (1999) 5124–5130. R.Y. Moore, A.E. Halaris, B.A. Jones, Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol. 180 (1978) 417–438. L.P. Morin, Serotonin and the regulation of mammalian circadian rhythmicity, Ann. Med. 31 (1999) 12–33. L.P. Morin, J. Blanchard, Organization of the hamster intergeniculate leaflet: NPY and ENK projections to the suprachiasmatic nucleus, intergeniculate leaflet and posterior limitans nucleus, Vis. Neurosci. 12 (1995) 57–67. U.L. Mullins, G. Gianutsos, A.S. Eison, Effects of antidepressants on 5-HT 7 receptor regulation in the rat hypothalamus, Neuropsychopharmacology 21 (1999) 352–367. M. Ono, A. Watanabe, Y. Matsumoto, T. Fukushima, Y. Nishikawa, T. Moriya, S. Shibata, S. Watanabe, Methamphetamine modifies the photic entraining responses in the rodent suprachiasmatic nucleus via serotonin release, Neuroscience 72 (1996) 213–224. N. Ozaki, D. Nakahara, Y. Kasahara, T. Nagatsu, The effect of methamphetamine on serotonin and its metabolite in the suprachiasmatic nucleus: a microdialysis study, J. Neural Transm. Gen. Sect. 86 (1991) 175–179. G.E. Pickard, B.N. Smith, M. Belenky, M.A. Rea, F.E. Dudek, P.J. Sollars, 5-HT 1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus, J. Neurosci. 19 (1999) 4034– 4045. B. Possidente, A.R. Lumia, S. McEldowney, M. Rapp, Fluoxetine shortens circadian period for wheel running activity in mice, Brain Res. Bull. 28 (1992) 629–631. M.R. Ralph, R.G. Foster, F.C. Davis, M. Menaker, Transplanted suprachiasmatic nucleus determines circadian period, Science 247 (1990) 975–978. S. Ramamoorthy, R.D. Blakely, Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants, Science 285 (1999) 763–766. R. Refinetti, M. Menaker, Effects of imipramine on circadian rhythms in the golden hamster, Pharmacol. Biochem. Behav. 45 (1993) 27–33. G. Rudnick, S.C. Wall, p-Chloramphetamine induces serotonin release through serotonin transporters, Biochemistry 31 (1992) 6710–6718. P. Schloss, D.C. Williams, The serotonin transporter: a primary target for antidepressant drugs, J. Psychopharmacol. 12 (1998) 115–121.
R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83 [27] M. Selim, J.D. Glass, U.E. Hauser, M.A. Rea, Serotonergic inhibition of light-induced fos protein expression and extracellular glutamate in the suprachiasmatic nuclei, Brain Res. 621 (1993) 181–188. [28] C. Sur, H. Betz, P. Schloss, Immunocytochemical detection of the serotonin transporter in rat brain, Neuroscience 73 (1996) 217–231. [29] E.T. Weber, R.L. Gannon, M.A. Rea, Local administration of serotonin agonists blocks light-induced phase advances of the circadian activity rhythm in the hamster, J. Biol. Rhythms 13 (1998) 209–218.
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[30] A. Wirz-Justice, K. Krauchi, T. Morimasa, R. Willener, H. Feer, Circadian rhythm of [ 3 H]imipramine binding in the rat suprachiasmatic nuclei, Eur. J. Pharmacol. 87 (1983) 331–333. [31] F. Wollnik, Effects of chronic administration and withdrawal of antidepressant agents on circadian activity rhythms in rats, Pharmacol. Biochem. Behav. 43 (1992) 549–561.
Research report
Serotonin transporter localization in the hamster suprachiasmatic nucleus Roseann Legutko, Robert L. Gannon* Department of Biology, Dowling College, Oakdale, Long Island, NY 11769 -1999, USA Accepted 28 November 2000
Abstract Pacemaker cells within the hamster suprachiasmatic nucleus generate circadian rhythms. The suprachiasmatic nucleus is heavily innervated by serotonin axons originating in the median raphe nuclei. Consequently, serotonergic agonists and antagonists or agents that alter levels of serotonin in the synapse following transmission can modulate many aspects of circadian rhythmicity. Examples of the latter are some antidepressants and the stimulant amphetamine that bind to the serotonin transporter and block serotonin reuptake. It has been hypothesized that circadian rhythm dysfunction may be involved in depression, and that the efficacy of certain antidepressants in treating depression may involve an alteration of serotonin levels and certain circadian rhythm parameters. However, although the hamster is the behavioral model of choice for the study of circadian rhythms, the identification of serotonin transporters in this species has not been reported. Therefore, in this report we describe the distribution of the serotonin transporter in the hamster suprachiasmatic nucleus using immunohistochemical techniques. Our results demonstrate a dense labeling of the serotonin transporter throughout the ventral and medial regions of the suprachiasmatic nucleus, a pattern that overlaps the distribution of serotonergic afferents in this nucleus. Amphetamines and certain antidepressants may serve as substrates for this transporter and elicit chronopharmacological activity by elevating serotonin levels in the suprachiasmatic nucleus. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Biological rhythm; Circadian rhythm; Reuptake; Antidepressant; Amphetamine
1. Introduction The suprachiasmatic nucleus (SCN) of the hypothalamus is known to be the site of the master pacemaker that controls circadian activity rhythms in mammalian species [22]. The phase of the SCN circadian pacemaker is entrained to the environmental light–dark cycle by afferent retinal input via the retinohypothalamic tract [9], and this input can be modified by afferent transmission from geniculate nuclei of the thalamus [6] and midbrain raphe nuclei [7,11,12]. Afferent fibers from these three regions in the hamster brain employ excitatory amino acids (retina) [5,13], neuropeptide Y and enkephalin (geniculate) [8,16] and serotonin (raphe) [11,14] as their transmitters, thus a *Corresponding author. Tel.: 11-631-244-3339; fax: 11-631-5635140. E-mail address: [email protected] (R.L. Gannon).
wide array of pharmacological compounds will affect the phase of circadian rhythms. Included among these chronobiotic compounds are drugs-of-abuse such as amphetamine and some clinically prescribed antidepressants [10,18]. Amphetamine and certain antidepressants are known to interact with serotonin transmission in other brain regions as well as the SCN [23,26]; therefore an understanding of serotonin’s role in circadian rhythms has considerable clinical relevance. The hamster SCN receives a dense innervation of serotonergic fibers originating from the median raphe nuclei [11]. Exogenously applied serotonergic agonists will typically inhibit light-induced phase adjustments of circadian activity rhythms by activating both presynaptic and postsynaptic serotonin receptors within the SCN [15]. Activation of presynaptic 5HT 1B receptors on retinohypothalamic terminals within the SCN will block light-induced phase shifts of hamster activity rhythms,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03290-X
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R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83
presumably by inhibiting glutamate release from the terminals [20]. Activation of postsynaptic 5HT 1A,7 receptors within the SCN will also inhibit light-induced modulation of hamster circadian activity rhythms through as yet unknown mechanisms [29]. Therefore, any modulation of serotonin levels within the hamster SCN will likely have an effect on the photic responsiveness of the circadian pacemaker. Serotonin is removed from the synaptic cleft by reuptake through the serotonin transporter (SERT). Certain antidepressants such as fluoxetine block the SERT and increase levels of serotonin in the synaptic cleft [26]. Amphetamine and its derivatives also block reuptake and they have the additional effect of causing the SERT to release previously sequestered serotonin back into the synaptic cleft [3,25]. Therefore, both fluoxetine and amphetamines increase extracellular serotonin levels, and amphetamine inhibits light-induced phase shifts of hamster activity rhythms [18]. Surprisingly, the effect of fluoxetine on light-induced phase shifts in hamsters has not been reported. Nevertheless, there is sufficient evidence to suggest that altering SERT function in the hamster SCN will affect the ability of light to adjust circadian activity rhythms. Although SERT distribution has been characterized in the rat SCN [1], it has not been similarly described in the hamster. Therefore, the present study describes the distribution of SERT immunoreactivity within the hamster SCN.
2. Materials and methods For this study five adult male Syrian hamsters, Mesocricetus auratus, were purchased from Charles River Laboratories (Kingston, NY) and were |150 g when used. Hamsters were individually housed under a 14:10-h light-
:dark cycle with food and water provided ad libitum. Mid-way through the dark cycle at zeitgeber time 17 (lights-off is zeitgeber 12), hamsters were anesthetized under dim red light (,1 lx) with ketamine (200 mg / kg), acepromazine (2 mg / kg), and xylazine (20 mg / kg) before being transcardially perfused with saline and fixative. The perfusion formulations consisted of 0.9% heparinized saline (100 ml) followed by 4% paraformaldehyde prepared in 0.1 M phosphate buffer (125 ml, pH 7.4). Brains were removed following perfusions and stored overnight in 4% buffered paraformaldehyde before being cryoprotected in a solution of 30% sucrose. Brains were stored at 48C for |1 month before use. Frontal sections of hamster brains were cut 25-mm thick using a cryostat (Shandon Lipshaw, Pittsburgh, PA) and placed free-floating in 24-well tissue culture plates for immunohistochemistry. A 0.1-M phosphate buffer containing 0.12% gelatin and 0.25% Triton X-100 was used during each step of the staining procedure. All steps were performed at room temperature while rocking. Sections were incubated overnight in 1% hydrogen peroxide, washed, incubated with 25% ammonium chloride for 1 h, washed again, then incubated overnight with rabbit antiSERT polyclonal antibody (1:10,000; Oncogene Research Products, Cambridge, MA). Following removal of the primary antibody, sections were washed again then sequentially incubated for 1 h with biotinylated secondary antibody and ABC reagent from the Vectastain anti-rabbit peroxidase kit according to kit instructions (Vector, Burlingame, CA). Staining of sections was performed using Sigma Fast metal enhanced diaminobenzidine tablets (Sigma, St. Louis, MO). Finally, sections were washed with isotonic saline, mounted, dehydrated in alcohols and coverslipped. For controls, primary antibody was omitted in one
Fig. 1. Example of control used to demonstrate SERT specificity in the hamster brain. Dense labeling in the SCN (left panel) was absent when the primary antibody was omitted from the staining procedure (right panel). Scale bar represents 200 mm.
R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83
experiment, and primary antibody was pre-absorbed with control peptide (5 mg / ml, Oncogene Research Products) overnight at 48C before use in a separate experiment. Brightfield images were digitally captured using the Pixera Professional CCD camera (Pixera, Los Gatos, CA) and printed using a Hewlett Packard PhotoSmart photo printer. Images were not adjusted following capture in any way.
3. Results SERT immunoreactivity was widespread throughout the hamster brain. Staining was prominent in fibers throughout the diencephalon and telencephalon, with a dense plexus of fibers and neuropil staining concentrated in the SCN. The specificity of the staining patterns as well as control results indicate that the SERT antibody used in this study also
79
cross-reacts with hamster SERT sites. There was no staining in the control study where the SERT antibody was omitted (Fig. 1), while pre-incubation with control peptide resulted in a dark reaction product with no specific staining pattern (not shown). SERT immunoreactivity was detected in the entire rostral-caudal extent of the SCN (Figs. 2 and 3). In rostral regions, SERT immunoreactivity was limited to a thin strip of the ventral SCN that thickened in area while progressing toward the middle region of the nucleus (Figs. 2A, and 3A,B). In the mid-SCN regions, SERT immunoreactivity was pronounced along the ventral and medial aspects of the SCN with a slight outward progression of immunoreactivity in the dorsal regions. SERT immunoreactivity in the ventral SCN was widest (extended the greatest distance laterally) within this region (Figs. 2B,C, and 3C,D). The lateral areas of the mid-SCN contained only sparse staining. In more caudal regions of the SCN, SERT immuno-
Fig. 2. SERT immunoreactivity in a single hamster brain. Labeling is pronounced in the rostral (A), middle (B,C), and caudal (D) SCN. OC, optic chiasm; V, third ventricle. Scale bar represents 200 mm.
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R. Legutko, R.L. Gannon / Brain Research 893 (2001) 77 – 83
and granular making identification of individual fibers and varicosities difficult (Fig. 4B). In less dense regions such as the cingulate cortex, SERT immunoreactivity in individual fibers with associated thickenings or varicosities was clearly evident (Fig. 4A). SERT immunoreactivity of fibers in the cingulate cortex is included in this study to clearly demonstrate SERT labeling of axons in the hamster brain and to allow for the comparison of immunolabeling patterns between this study in hamsters and that seen in other species [1,2,28].
4. Discussion
Fig. 3. Schematic representation of SERT immunoreactivity in a separate hamster brain. This pattern is characteristic to each hamster brain used in this study. (A,B) Rostral SCN; (C,D) middle SCN; (E,F) caudal SCN. 3V, third ventricle; OC, optic chiasm.
reactivity was mostly confined to medial areas of the nucleus, although there was a slight flare in staining patterns seen in both dorsal and ventral aspects of the SCN at this level (Figs. 2D, and 3E,F). High-power examination of SERT immunoreactivity within the SCN revealed some clearly defined fibers, although staining of the neuropil was typically very dense
SERT immunoreactivity is readily detected within the hamster brain using a polyclonal antisera directed against the rat SERT. In the hamster, SERT immunoreactivity labels axons spread throughout the diencephalon and telencephalon, with the appearance of punctate varicosities visible along many axons. This pattern of staining is similar to that demonstrated in other studies using rats [1,28]. SERT immunoreactivity within the hamster SCN is distributed in a pattern similar to that for serotonin fibers projecting to the SCN from the median raphe nuclei [11]. It is reasonable to assume then that the SERTs detected in this study are localized on serotonin fibers projecting from the median raphe. However, a recent study in the rat nucleus accumbens demonstrated two populations of serotonin fibers, those with and without SERT immunolabeling [2]. Therefore, there remains a slight possibility that in the hamster the SERT is located on non-serotonergic axons or only a subset of serotonergic axons within the SCN. The presence of SERTs in the hamster SCN has recently been inferred from binding studies using [ 3 H]paroxetine [4], although that study did not discuss the distribution of SERTs within the SCN. Together, then these results demonstrate a significant population of SERTs in the hamster SCN. SERT activity in the hamster and rat SCN would explain the results observed when amphetamines are used to modify circadian activity rhythms. In the hamster, methamphetamine inhibits both light-induced phase advances and delays, similar to that found after application of the serotonin agonist 8-OH-DPAT [29]. Methamphetamine has also been shown to increase serotonin release in the rat SCN both in vivo [19] and in vitro [18]. Finally, methamphetamine inhibits light-induced Fos expression in the rat SCN [18], similar to that seen upon application of serotonin agonists [27]. Therefore, it is likely that amphetamines affect circadian activity in the rat and hamster by elevating levels of serotonin in the SCN upon serving as a substrate for the SERTs located in the hamster and rat SCN. Tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs) also block the activity of SERTs and increase the level of serotonin in the synapse. How-
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Fig. 4. (A) SERT immunoreactivity in the hamster cingulate cortex. In this region, axonal staining is less dense and individual fibers, some with associated varicosities, are clearly evident. (B) SERT immunoreactivity in the hamster SCN. Staining is more dense making the individual identification of axonal fibers more difficult, although some fibers are seen coursing through the optic chiasm (arrowhead). OC, optic chiasm; SCN, suprachiasmatic nuclei. Scale bar represents 50 mm.
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ever, a significant effect of these compounds on circadian activity rhythms has been difficult to demonstrate. The tricyclic antidepressant imipramine did not affect circadian period or light-induced phase shifts in the hamster [24], although it does induce Fos expression in the rat SCN [17], and exhibits a circadian rhythmicity in binding in the rat SCN [30]. These studies are complicated by the action of imipramine on other monoamine transmitters. The SSRI fluoxetine however is more specific for the SERT. Fluoxetine has been reported to shorten circadian period in mice [21], but to have no effect on circadian period in either rat [31] or hamster [10]. Fluoxetine will also induce Fos expression in the rat SCN similar to 8-OH-DPAT [17]. Unfortunately, the effect of fluoxetine on light-induced phase shifts in hamsters, rats, or mice has not been reported. It would be predicted that chronic or perhaps even acute administration of fluoxetine would inhibit lightinduced changes in circadian-related events similar to serotonin. In conclusion, this report describes the distribution of the SERT in the hamster SCN. Since SERTs are targets for clinically used antidepressants and drugs of abuse, SERT distribution and activity has considerable clinical relevance. The density of SERTs in the hamster SCN would predict that drugs interacting with SERTs would affect circadian rhythms, and that this interaction may contribute to the behavioral effects of many compounds. However, the small amount of relevant literature indicates that the relationship between SERT-interacting drugs and circadian rhythms remains to be determined.
[7]
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[9]
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[11]
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Acknowledgements
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This study was supported by NSF IBN-9728574 (R.L.G.). R. Legutko is an undergraduate student in the Biology Department at Dowling College.
[19]
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