- Email: [email protected]
Differential distribution of afferents containing serotonin and neuropeptide Y within the marmoset suprachiasmatic nucleus
Brain Research 927 (2002) 200–203 www.elsevier.com / locate / bres
Short communication
Differential distribution of afferents containing serotonin and neuropeptide Y within the marmoset suprachiasmatic nucleus a a b a, Jeferson S. Cavalcante , Adilson S. Alves , Miriam S.M.O. Costa , Luiz R.G. Britto * a
˜ Paulo, Av. Prof. Lineu Prestes 1524, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao ˜ Paulo, S.P., Brazil 05508 -900 Sao b Laboratory of Chronobiology, Department of Morphology, Biosciences Center, Federal University of Rio Grande do Norte, 59072 -970 Natal RN, Brazil Accepted 12 November 2001
Abstract Neuropeptide Y-containing fibers / terminals were immunohistochemically detected in the ventral portion of the marmoset suprachiasmatic nucleus, approximately matching the distribution of its retinal afferents. On the other hand, serotonergic fibers / terminals were found mostly in central and dorsal areas of the suprachiasmatic nucleus, almost completely sparing its ventral portion. These data may represent a morphological substrate for differential actions of serotonin and neuropeptide Y in the control of circadian rhythmicity in marmosets. 2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Subcortical visual pathways Keywords: Circadian rhythm; Intergeniculate leaflet; Neuropeptide; Serotonergic system; Suprachiasmatic nucleus
Three chemically-identified afferent pathways appear to reach the mammalian suprachiasmatic nucleus (SCN), which are the retino–hypothalamic (RHT), geniculo– hypothalamic (GHT), and the raphe–SCN pathways [18,20,21,24,28]. The RHT uses the excitatory amino acid glutamate as its main neurotransmitter [8,18] and appears to adjust the circadian clock to the environmental light– dark cycle [11,17]. The GHT originates from the intergeniculate leaflet (IGL), which in primates appears to be part of the pregeniculate nucleus [4–6,13]. Many IGL neurons produce neuropeptide Y (NPY) and their axons and terminals exhibit a pattern of distribution into the SCN that is approximately coincident with the pattern of distribution of the RHT fibers [1–3,10,12,16,22, 23,26,27,32]. Nevertheless, it should be mentioned that some primate species appear to lack NPY input to the SCN
*Corresponding author. Tel.: 155-11-3818-7242; fax: 155-11-38187426. E-mail address: [email protected] (L.R.G. Britto).
[4]. The IGL acts as a secondary mechanism modulating the response of circadian rhythms to light or light offset [24]. The raphe–SCN pathway originates from the median raphe nucleus [19] and also appears to modulate the effects of light on circadian rhythmicity [25], which is in keeping with the localization of 5-HT processes in the SCN zone where RHT processes are distributed [1,9,20,30,31]. In this study we analyzed the distribution of NPY and 5-HT-containing axons and presumptive terminals within the marmoset SCN in relation to its retinal innervation by the RHT. Furthermore, we analyzed the relationship of serotonergic and NPY-positive fibers / terminals to SCN perikarya. The SCN neurons were identified by calbindin (CB) immunostaining, as previous studies have revealed that most of the neurons throughout the marmoset SCN contain CB-like immunoreactivity [5]. Four adult male marmosets from the Primatology Center of the Federal University of Rio Grande do Norte, Natal (Brazil), were used in this study. The subjects were ˜ Paulo, anesthetized with sodium thiopental (Abbott, Sao Brazil, 40 mg / kg, i.p.) and each received an unilateral
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03312-1
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
intraocular injection of 60 ml of a 1% solution of cholera toxin subunit B (CTb; List Labs., Campbell, CA, USA) in distilled water. The anterograde transport of CTb has been previously described to be a reliable method to label the RHT [7]. After 5–7 days, the animals were deeply anesthetized and perfused transcardially with phosphatebuffered saline and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were postfixed for 2–4 h, and then cryoprotected with 30% sucrose in PB. Coronal, 30-mm sections of the brains were then obtained on a sliding freezing microtome. Sections through the marmoset hypothalamus were stained for CTb, NPY, 5HT, and CB by means of conventional double-labeling immunofluorescence techniques. The antibodies used were a goat anti-CTb (List Labs.), rabbit anti-NPY (Accurate, Westbury, NY, USA) and anti-5-HT (Protos Biotech, New York, NY, USA), and a mouse anti-CB (Sigma, St. Louis, MO, USA). The antibodies were diluted 1:1,000 in PB with 0.3% Triton X-100. Secondary rhodamine and fluorescein-labeled anti-goat, anti-rabbit, and anti-mouse donkey antibodies (Jackson Labs., Westgrove, PA, USA) were used to reveal immunoreactivity. The secondary antibodies were used at 1:100 in PB with 0.3% Triton X-100. Incubation times were 12 h for the appropriate combination of primary antibodies and 2 h for the appropriate combinations of secondary antibodies, always at room temperature (ca. 24 8C). Controls for the specificity of staining were the omission of the primary antibodies from the procedure and their substitution for normal sera from the same species. Staining was completely abolished under these conditions. Preadsorption controls have been previously conducted and revealed that the commercial
201
antibodies employed in this study produce reliable and specific staining in the marmoset brain [5–7]. The material was first analyzed on a routine microscope equipped for epifluorescence and then on a confocal microscope. Optical 1-mm sections were generally used to obtain digital images of the stained material. The images were only minimally processed for brightness and contrast. The pattern of immunoreactivity for all antigens studied here was very consistent among all four marmoset brains. The boundaries of the SCN were identified in the present experiments by the distribution of CB-positive perikarya, as these cells have been shown to delineate the cytoarchitectonically-defined SCN [5–7]. CTb-labeled fibers and presumptive terminals were mainly observed into the ventral portion of the marmoset SCN, as expected from previous data [7]. NPY-positive varicose fibers and presumptive terminals were observed to overlap with the CTb-positive processes in the ventral portion of the SCN (Fig. 1), throughout its rostrocaudal extension. Only a few NPY-positive fibers could be detected outside the main retinorecipient area of the marmoset SCN. Surprisingly, the 5-HT varicose fibers and presumptive terminals appear to exhibit a distribution that is almost complementary to that found for CTb and NPY. This pattern was also observed throughout the rostrocaudal extension of the marmoset SCN, with 5-HT fibers basically sparing the ventral, retinorecipient area of the SCN (Fig. 1). The 5-HT fibers and presumptive terminals appear to be mainly distributed throughout the remainder of the SCN. The analysis of the relationship of NPY-positive and 5-HTpositive fibers / terminals and the SCN somata revealed that both types of fibers exhibit varicosities in close relation to
Fig. 1. Digital images of coronal sections through the marmoset hypothalamus. These images illustrate the pattern of distribution of NPY and 5-HT fibers / terminals (green) within the suprachiasmatic nucleus (SCN), in relation to its retinal afferents. The retino–hypothalamic tract (red) was labeled with intraocular injections of CTb. Note the almost complete absence of 5-HT-positive fibers in the ventral SCN, where most of the retinal afferents terminate. The dashed line on the image at the left depicts the approximate boundaries of the marmoset SCN, as defined by the distribution of CB-positive neurons [5]. Overlaying of CTb and NPY-stained fibers and processes produced the apparent co-localization pattern observed in the left image. CTb and NPY fibers appeared, however, clearly distinct when viewed at high magnification. OC, Optic chiasm; 3V, third ventricle. Scale bar: 70 mm.
202
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
CB-positive perikarya and occasionally their dendrites, although in different areas of the SCN (Fig. 2). Our results reveal a differential distribution of 5-HT and NPY fibers / terminals into the marmoset SCN. These data disclosed a distribution of 5-HT processes within SCN that is very different from the pattern found in other species studied so far. Indeed, 5-HT fibers have been mainly described in a ventral or ventromedial position into the retinorecipient SCN in rodents, cats, and monkeys, matching or at least overlapping in part the distribution of NPY fibers [1,9,20,30,31]. The distribution of NPY-positive processes within the marmoset SCN, on the other hand, appeared to be similar to the distribution found in other species [2,3,20,32], with the NPY fibers reaching mainly the ventrally-located SCN neurons. The ventral group of SCN neurons is known to receive two of the main afferent pathways of the SCN, from the retina and the IGL, respectively through the RHT and the GHT. Some of these neurons are characterized by their content of vasoactive intestinal polypeptide and the peptide histidine–isoleucine [1,20,28], and appear to mainly project to the midline thalamus, some hypothalamus regions, and the basal forebrain [15]. The centrally and dorsally-located SCN neurons may represent the SCN neurons known to contain vasopressin and neurophysin [1,20,28]. This set of neurons appears to project, among other structures, to the hypothalamic dorsomedial nucleus, subparaventricular zone,
and the paraventricular nucleus [15,33,34]. Therefore, 5HT and NPY could potentially control different aspects of marmoset SCN function by acting on different sets of efferent neurons. The finding that both 5-HT and NPY processes are found in close correlation to the marmoset SCN perikarya indicate that a significant portion of 5-HT and NPY actions are postsynaptic. Indeed, there are reports of postynaptic 5-HT 7 and Y1 / Y5 receptors within the SCN [14,29]. However, it should be mentioned that functional presynaptic receptors both for 5-HT and NPY have also been described into the SCN [14,29]. The distribution of 5-HT processes found in the present experiments apparently precludes a presynaptic 5-HT action upon RHT axons in the marmoset. The differential distribution of 5-HT and NPY fibers / terminals in relation to the RHT found in this study indicates that the GHT and raphe–SCN pathways are possibly involved in different aspects of the functional organization of circadian rhythms in the marmoset. The distribution of those pathways has to be studied in additional species to verify if that arrangement represents an unique characteristic of the marmoset brain. Functional studies have also to be conducted in this species to evaluate the possible photic and non-photic influences of the GHT and the raphe–SCN pathway on SCN-dependent rhythms [20,25]. These studies will surely help to clarify the role of serotonergic and peptidergic systems in the organization of the circadian timing system.
Acknowledgements This study was supported by FAPESP, CNPq, and PRONEX / MCT (L.R.G.B.). J.S.C. and A.S.A. were the recipients of fellowships from FAPESP and CNPq, respectively. M.S.M.O.C. was supported by PPPG-UFRN.
References
Fig. 2. High-power digital images of coronal sections through the marmoset hypothalamus, illustrating sites of close apposition of NPY and 5-HT varicosities on CB-positive SCN somata and their dendrites. Scale bar: 5 mm.
[1] J.P. Card, R.Y. Moore, The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience 13 (1984) 415–431. [2] J.P. Card, R.Y. Moore, Neuropeptide Y localization in the rat suprachiasmatic nucleus and periventricular hypothalamus, Neurosci. Lett. 88 (1988) 241–246. [3] J.P. Card, R.Y. Moore, Organization of lateral geniculate–hypothalamic connections in the rat, J. Comp. Neurol. 284 (1989) 135–147. [4] N. Chevassus-au-Louis, H.M. Cooper, Is there a geniculohypothalamic tract in primates? A comparative immunohistochemical study in the circadian system of strepsirhine and haplorhine species, Brain Res. 805 (1998) 213–219. [5] M.S.M.O. Costa, L.R.G. Britto, Calbindin immunoreactivity delineates the circadian visual centers of the brain of the common marmoset (Callithrix jacchus), Brain Res. Bull. 43 (1997) 369–373. [6] M.S.M.O. Costa, L.F. Moreira, V. Alones, J. Lu, U.R. Santee, J.S.
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17] [18] [19]
[20]
Cavalcante, P.R.A. Moraes, L.R.G. Britto, M. Menaker, Characterization of the circadian system in a brazilian species of monkey (Callithrix jacchus): immunohistochemical analysis and retinal projections, Biol. Rhythm Res. 29 (1998) 510–520. M.S.M.O. Costa, U.R. Santee, J.S. Cavalcante, P.R.A. Moraes, N.P. Santos, L.R.G. Britto, Retinohypothalamic projections in the common marmoset (Callithrix jacchus): a study using cholera toxin, J. Comp. Neurol. 415 (1999) 393–403. M.J. De Vries, B.N. Cardozo, J. Van Der Want, A. Wolf, J.H. Meijer, Glutamate immunoreactivity in terminals of the retinohypothalamic tract of the brown Norwegian rat, Brain Res. 612 (1993) 231–237. N. Goel, T.M. Lee, L. Smale, Suprachiasmatic nucleus and intergeniculate leaflet in the diurnal rodent Octodon degus: retinal projections and immunocytochemical characterization, Neuroscience 92 (1999) 1491–1509. M.E. Harrington, D.M. Nance, B. Rusak, Double-labeling of neuropeptide Y-immunoreactive neurons which project from the geniculate to the suprachiasmatic nuclei, Brain Res. 410 (1987) 275–282. R.F. Johnson, R.Y. Moore, L.P. Morin, Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract, Brain Res. 460 (1988) 297–313. 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. J.H. Kaas, M.F. Huerta, The subcortical visual system of primates, Comp. Primate Biol. 4 (1988) 327–391. P.J. Larsen, P. Kristensen, Distribution of neuropeptide Y receptor expression in the rat suprachiasmatic nucleus, Mol. Brain Res. 60 (1998) 69–76. R.K. Leak, R.Y. Moore, Topographic organization of suprachiasmatic nucleus projection neurons, J. Comp. Neurol. 433 (2001) 312–334. P.W. Mantyh, J.A. Kemp, The distribution of putative neurotransmitters in the lateral geniculate nucleus of the rat, Brain Res. 288 (1983) 344–348. J.H. Meijer, Physiological basis for photic entrainment, Eur. J. Morphol. 28 (1990) 308–316. J.H. Meijer, W.J. Rietveld, Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev. 69 (1989) 671–707. E.L. Meyer-Bernstein, L.P. Morin, Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythms modulation, J. Neurosci. 16 (1996) 2097–2111. J.D. Miller, L.P. Morin, W.J. Schwartz, R.Y. Moore, New insights into the mammalian circadian clock, Sleep 19 (1996) 641–667.
203
[21] M.M. Moga, R.Y. Moore, Organization of neural inputs to the suprachiasmatic nucleus in the rat, J. Comp. Neurol. 389 (1997) 508–534. [22] R.Y. Moore, J.P. Card, Intergeniculate leaflet: an anatomically and functionally distinct subdivision of the lateral geniculate complex, J. Comp. Neurol. 344 (1994) 403–430. [23] R.Y. Moore, E.L. Gustafson, J.P. Card, Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide, molluscan cardioexcitatory peptide and neuropeptide Y, Cell Tissue Res. 236 (1984) 41–46. [24] L.P. Morin, The circadian visual system, Brain Res. Rev. 67 (1994) 102–127. [25] L.P. Morin, Serotonin and the regulation of mammalian circadian rhythmicity, Ann. Med. 31 (1999) 12–33. [26] 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. [27] G.E. Pickard, The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection, J. Comp. Neurol. 211 (1982) 65–83. [28] S. Reuss, Components and connections of the circadian timing system in mammals, Cell Tissue Res. 285 (1996) 353–378. [29] B.N. Smith, P.J. Sollars, F.E. Dudek, G.E. Pickard, Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT 1B and 5-HT 7 receptors, J. Biol. Rhythms 16 (2001) 25–38. [30] H.W.M. Steinbusch, Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals, Neuroscience 6 (1981) 557–618. [31] S. Ueda, M. Kawata, Y. Sano, Identification of serotonin and vasopressin immunoreactivities in the suprachiasmatic nucleus of four mammalian species, Cell Tissue Res. 234 (1983) 237–248. [32] S. Ueda, M. Kawata, Y. Sano, Identification of neuropeptide Y immunoreactivity in the suprachiasmatic nucleus and the lateral geniculate nucleus of some mammals, Neurosci. Lett. 68 (1986) 7–10. [33] A.G. Watts, L.W. Swanson, The efferent projections of the suprachiasmatic nucleus II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat, J. Comp. Neurol. 258 (1987) 230–252. [34] A.G. Watts, L.W. Swanson, G. Sanchez-Watts, Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat, J. Comp. Neurol. 258 (1987) 204–229.
Short communication
Differential distribution of afferents containing serotonin and neuropeptide Y within the marmoset suprachiasmatic nucleus a a b a, Jeferson S. Cavalcante , Adilson S. Alves , Miriam S.M.O. Costa , Luiz R.G. Britto * a
˜ Paulo, Av. Prof. Lineu Prestes 1524, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao ˜ Paulo, S.P., Brazil 05508 -900 Sao b Laboratory of Chronobiology, Department of Morphology, Biosciences Center, Federal University of Rio Grande do Norte, 59072 -970 Natal RN, Brazil Accepted 12 November 2001
Abstract Neuropeptide Y-containing fibers / terminals were immunohistochemically detected in the ventral portion of the marmoset suprachiasmatic nucleus, approximately matching the distribution of its retinal afferents. On the other hand, serotonergic fibers / terminals were found mostly in central and dorsal areas of the suprachiasmatic nucleus, almost completely sparing its ventral portion. These data may represent a morphological substrate for differential actions of serotonin and neuropeptide Y in the control of circadian rhythmicity in marmosets. 2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Subcortical visual pathways Keywords: Circadian rhythm; Intergeniculate leaflet; Neuropeptide; Serotonergic system; Suprachiasmatic nucleus
Three chemically-identified afferent pathways appear to reach the mammalian suprachiasmatic nucleus (SCN), which are the retino–hypothalamic (RHT), geniculo– hypothalamic (GHT), and the raphe–SCN pathways [18,20,21,24,28]. The RHT uses the excitatory amino acid glutamate as its main neurotransmitter [8,18] and appears to adjust the circadian clock to the environmental light– dark cycle [11,17]. The GHT originates from the intergeniculate leaflet (IGL), which in primates appears to be part of the pregeniculate nucleus [4–6,13]. Many IGL neurons produce neuropeptide Y (NPY) and their axons and terminals exhibit a pattern of distribution into the SCN that is approximately coincident with the pattern of distribution of the RHT fibers [1–3,10,12,16,22, 23,26,27,32]. Nevertheless, it should be mentioned that some primate species appear to lack NPY input to the SCN
*Corresponding author. Tel.: 155-11-3818-7242; fax: 155-11-38187426. E-mail address: [email protected] (L.R.G. Britto).
[4]. The IGL acts as a secondary mechanism modulating the response of circadian rhythms to light or light offset [24]. The raphe–SCN pathway originates from the median raphe nucleus [19] and also appears to modulate the effects of light on circadian rhythmicity [25], which is in keeping with the localization of 5-HT processes in the SCN zone where RHT processes are distributed [1,9,20,30,31]. In this study we analyzed the distribution of NPY and 5-HT-containing axons and presumptive terminals within the marmoset SCN in relation to its retinal innervation by the RHT. Furthermore, we analyzed the relationship of serotonergic and NPY-positive fibers / terminals to SCN perikarya. The SCN neurons were identified by calbindin (CB) immunostaining, as previous studies have revealed that most of the neurons throughout the marmoset SCN contain CB-like immunoreactivity [5]. Four adult male marmosets from the Primatology Center of the Federal University of Rio Grande do Norte, Natal (Brazil), were used in this study. The subjects were ˜ Paulo, anesthetized with sodium thiopental (Abbott, Sao Brazil, 40 mg / kg, i.p.) and each received an unilateral
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03312-1
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
intraocular injection of 60 ml of a 1% solution of cholera toxin subunit B (CTb; List Labs., Campbell, CA, USA) in distilled water. The anterograde transport of CTb has been previously described to be a reliable method to label the RHT [7]. After 5–7 days, the animals were deeply anesthetized and perfused transcardially with phosphatebuffered saline and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were postfixed for 2–4 h, and then cryoprotected with 30% sucrose in PB. Coronal, 30-mm sections of the brains were then obtained on a sliding freezing microtome. Sections through the marmoset hypothalamus were stained for CTb, NPY, 5HT, and CB by means of conventional double-labeling immunofluorescence techniques. The antibodies used were a goat anti-CTb (List Labs.), rabbit anti-NPY (Accurate, Westbury, NY, USA) and anti-5-HT (Protos Biotech, New York, NY, USA), and a mouse anti-CB (Sigma, St. Louis, MO, USA). The antibodies were diluted 1:1,000 in PB with 0.3% Triton X-100. Secondary rhodamine and fluorescein-labeled anti-goat, anti-rabbit, and anti-mouse donkey antibodies (Jackson Labs., Westgrove, PA, USA) were used to reveal immunoreactivity. The secondary antibodies were used at 1:100 in PB with 0.3% Triton X-100. Incubation times were 12 h for the appropriate combination of primary antibodies and 2 h for the appropriate combinations of secondary antibodies, always at room temperature (ca. 24 8C). Controls for the specificity of staining were the omission of the primary antibodies from the procedure and their substitution for normal sera from the same species. Staining was completely abolished under these conditions. Preadsorption controls have been previously conducted and revealed that the commercial
201
antibodies employed in this study produce reliable and specific staining in the marmoset brain [5–7]. The material was first analyzed on a routine microscope equipped for epifluorescence and then on a confocal microscope. Optical 1-mm sections were generally used to obtain digital images of the stained material. The images were only minimally processed for brightness and contrast. The pattern of immunoreactivity for all antigens studied here was very consistent among all four marmoset brains. The boundaries of the SCN were identified in the present experiments by the distribution of CB-positive perikarya, as these cells have been shown to delineate the cytoarchitectonically-defined SCN [5–7]. CTb-labeled fibers and presumptive terminals were mainly observed into the ventral portion of the marmoset SCN, as expected from previous data [7]. NPY-positive varicose fibers and presumptive terminals were observed to overlap with the CTb-positive processes in the ventral portion of the SCN (Fig. 1), throughout its rostrocaudal extension. Only a few NPY-positive fibers could be detected outside the main retinorecipient area of the marmoset SCN. Surprisingly, the 5-HT varicose fibers and presumptive terminals appear to exhibit a distribution that is almost complementary to that found for CTb and NPY. This pattern was also observed throughout the rostrocaudal extension of the marmoset SCN, with 5-HT fibers basically sparing the ventral, retinorecipient area of the SCN (Fig. 1). The 5-HT fibers and presumptive terminals appear to be mainly distributed throughout the remainder of the SCN. The analysis of the relationship of NPY-positive and 5-HTpositive fibers / terminals and the SCN somata revealed that both types of fibers exhibit varicosities in close relation to
Fig. 1. Digital images of coronal sections through the marmoset hypothalamus. These images illustrate the pattern of distribution of NPY and 5-HT fibers / terminals (green) within the suprachiasmatic nucleus (SCN), in relation to its retinal afferents. The retino–hypothalamic tract (red) was labeled with intraocular injections of CTb. Note the almost complete absence of 5-HT-positive fibers in the ventral SCN, where most of the retinal afferents terminate. The dashed line on the image at the left depicts the approximate boundaries of the marmoset SCN, as defined by the distribution of CB-positive neurons [5]. Overlaying of CTb and NPY-stained fibers and processes produced the apparent co-localization pattern observed in the left image. CTb and NPY fibers appeared, however, clearly distinct when viewed at high magnification. OC, Optic chiasm; 3V, third ventricle. Scale bar: 70 mm.
202
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
CB-positive perikarya and occasionally their dendrites, although in different areas of the SCN (Fig. 2). Our results reveal a differential distribution of 5-HT and NPY fibers / terminals into the marmoset SCN. These data disclosed a distribution of 5-HT processes within SCN that is very different from the pattern found in other species studied so far. Indeed, 5-HT fibers have been mainly described in a ventral or ventromedial position into the retinorecipient SCN in rodents, cats, and monkeys, matching or at least overlapping in part the distribution of NPY fibers [1,9,20,30,31]. The distribution of NPY-positive processes within the marmoset SCN, on the other hand, appeared to be similar to the distribution found in other species [2,3,20,32], with the NPY fibers reaching mainly the ventrally-located SCN neurons. The ventral group of SCN neurons is known to receive two of the main afferent pathways of the SCN, from the retina and the IGL, respectively through the RHT and the GHT. Some of these neurons are characterized by their content of vasoactive intestinal polypeptide and the peptide histidine–isoleucine [1,20,28], and appear to mainly project to the midline thalamus, some hypothalamus regions, and the basal forebrain [15]. The centrally and dorsally-located SCN neurons may represent the SCN neurons known to contain vasopressin and neurophysin [1,20,28]. This set of neurons appears to project, among other structures, to the hypothalamic dorsomedial nucleus, subparaventricular zone,
and the paraventricular nucleus [15,33,34]. Therefore, 5HT and NPY could potentially control different aspects of marmoset SCN function by acting on different sets of efferent neurons. The finding that both 5-HT and NPY processes are found in close correlation to the marmoset SCN perikarya indicate that a significant portion of 5-HT and NPY actions are postsynaptic. Indeed, there are reports of postynaptic 5-HT 7 and Y1 / Y5 receptors within the SCN [14,29]. However, it should be mentioned that functional presynaptic receptors both for 5-HT and NPY have also been described into the SCN [14,29]. The distribution of 5-HT processes found in the present experiments apparently precludes a presynaptic 5-HT action upon RHT axons in the marmoset. The differential distribution of 5-HT and NPY fibers / terminals in relation to the RHT found in this study indicates that the GHT and raphe–SCN pathways are possibly involved in different aspects of the functional organization of circadian rhythms in the marmoset. The distribution of those pathways has to be studied in additional species to verify if that arrangement represents an unique characteristic of the marmoset brain. Functional studies have also to be conducted in this species to evaluate the possible photic and non-photic influences of the GHT and the raphe–SCN pathway on SCN-dependent rhythms [20,25]. These studies will surely help to clarify the role of serotonergic and peptidergic systems in the organization of the circadian timing system.
Acknowledgements This study was supported by FAPESP, CNPq, and PRONEX / MCT (L.R.G.B.). J.S.C. and A.S.A. were the recipients of fellowships from FAPESP and CNPq, respectively. M.S.M.O.C. was supported by PPPG-UFRN.
References
Fig. 2. High-power digital images of coronal sections through the marmoset hypothalamus, illustrating sites of close apposition of NPY and 5-HT varicosities on CB-positive SCN somata and their dendrites. Scale bar: 5 mm.
[1] J.P. Card, R.Y. Moore, The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience 13 (1984) 415–431. [2] J.P. Card, R.Y. Moore, Neuropeptide Y localization in the rat suprachiasmatic nucleus and periventricular hypothalamus, Neurosci. Lett. 88 (1988) 241–246. [3] J.P. Card, R.Y. Moore, Organization of lateral geniculate–hypothalamic connections in the rat, J. Comp. Neurol. 284 (1989) 135–147. [4] N. Chevassus-au-Louis, H.M. Cooper, Is there a geniculohypothalamic tract in primates? A comparative immunohistochemical study in the circadian system of strepsirhine and haplorhine species, Brain Res. 805 (1998) 213–219. [5] M.S.M.O. Costa, L.R.G. Britto, Calbindin immunoreactivity delineates the circadian visual centers of the brain of the common marmoset (Callithrix jacchus), Brain Res. Bull. 43 (1997) 369–373. [6] M.S.M.O. Costa, L.F. Moreira, V. Alones, J. Lu, U.R. Santee, J.S.
J.S. Cavalcante et al. / Brain Research 927 (2002) 200 – 203
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17] [18] [19]
[20]
Cavalcante, P.R.A. Moraes, L.R.G. Britto, M. Menaker, Characterization of the circadian system in a brazilian species of monkey (Callithrix jacchus): immunohistochemical analysis and retinal projections, Biol. Rhythm Res. 29 (1998) 510–520. M.S.M.O. Costa, U.R. Santee, J.S. Cavalcante, P.R.A. Moraes, N.P. Santos, L.R.G. Britto, Retinohypothalamic projections in the common marmoset (Callithrix jacchus): a study using cholera toxin, J. Comp. Neurol. 415 (1999) 393–403. M.J. De Vries, B.N. Cardozo, J. Van Der Want, A. Wolf, J.H. Meijer, Glutamate immunoreactivity in terminals of the retinohypothalamic tract of the brown Norwegian rat, Brain Res. 612 (1993) 231–237. N. Goel, T.M. Lee, L. Smale, Suprachiasmatic nucleus and intergeniculate leaflet in the diurnal rodent Octodon degus: retinal projections and immunocytochemical characterization, Neuroscience 92 (1999) 1491–1509. M.E. Harrington, D.M. Nance, B. Rusak, Double-labeling of neuropeptide Y-immunoreactive neurons which project from the geniculate to the suprachiasmatic nuclei, Brain Res. 410 (1987) 275–282. R.F. Johnson, R.Y. Moore, L.P. Morin, Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract, Brain Res. 460 (1988) 297–313. 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. J.H. Kaas, M.F. Huerta, The subcortical visual system of primates, Comp. Primate Biol. 4 (1988) 327–391. P.J. Larsen, P. Kristensen, Distribution of neuropeptide Y receptor expression in the rat suprachiasmatic nucleus, Mol. Brain Res. 60 (1998) 69–76. R.K. Leak, R.Y. Moore, Topographic organization of suprachiasmatic nucleus projection neurons, J. Comp. Neurol. 433 (2001) 312–334. P.W. Mantyh, J.A. Kemp, The distribution of putative neurotransmitters in the lateral geniculate nucleus of the rat, Brain Res. 288 (1983) 344–348. J.H. Meijer, Physiological basis for photic entrainment, Eur. J. Morphol. 28 (1990) 308–316. J.H. Meijer, W.J. Rietveld, Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev. 69 (1989) 671–707. E.L. Meyer-Bernstein, L.P. Morin, Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythms modulation, J. Neurosci. 16 (1996) 2097–2111. J.D. Miller, L.P. Morin, W.J. Schwartz, R.Y. Moore, New insights into the mammalian circadian clock, Sleep 19 (1996) 641–667.
203
[21] M.M. Moga, R.Y. Moore, Organization of neural inputs to the suprachiasmatic nucleus in the rat, J. Comp. Neurol. 389 (1997) 508–534. [22] R.Y. Moore, J.P. Card, Intergeniculate leaflet: an anatomically and functionally distinct subdivision of the lateral geniculate complex, J. Comp. Neurol. 344 (1994) 403–430. [23] R.Y. Moore, E.L. Gustafson, J.P. Card, Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide, molluscan cardioexcitatory peptide and neuropeptide Y, Cell Tissue Res. 236 (1984) 41–46. [24] L.P. Morin, The circadian visual system, Brain Res. Rev. 67 (1994) 102–127. [25] L.P. Morin, Serotonin and the regulation of mammalian circadian rhythmicity, Ann. Med. 31 (1999) 12–33. [26] 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. [27] G.E. Pickard, The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection, J. Comp. Neurol. 211 (1982) 65–83. [28] S. Reuss, Components and connections of the circadian timing system in mammals, Cell Tissue Res. 285 (1996) 353–378. [29] B.N. Smith, P.J. Sollars, F.E. Dudek, G.E. Pickard, Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT 1B and 5-HT 7 receptors, J. Biol. Rhythms 16 (2001) 25–38. [30] H.W.M. Steinbusch, Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals, Neuroscience 6 (1981) 557–618. [31] S. Ueda, M. Kawata, Y. Sano, Identification of serotonin and vasopressin immunoreactivities in the suprachiasmatic nucleus of four mammalian species, Cell Tissue Res. 234 (1983) 237–248. [32] S. Ueda, M. Kawata, Y. Sano, Identification of neuropeptide Y immunoreactivity in the suprachiasmatic nucleus and the lateral geniculate nucleus of some mammals, Neurosci. Lett. 68 (1986) 7–10. [33] A.G. Watts, L.W. Swanson, The efferent projections of the suprachiasmatic nucleus II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat, J. Comp. Neurol. 258 (1987) 230–252. [34] A.G. Watts, L.W. Swanson, G. Sanchez-Watts, Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat, J. Comp. Neurol. 258 (1987) 204–229.