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Retinal, NPY- and 5 ht- inputs to the aged suprachiasmatic nucleus in common marmosets (Callithrix jacchus)
Accepted Manuscript Title: RETINAL, NPY- AND 5-HT- INPUTS TO THE AGED SUPRACHIASMATIC NUCLEUS IN COMMON MARMOSETS (Callithrix jacchus) Authors: Rovena C.G.J. Engelberth, Kayo D. de Azevedo Silva, Felipe Porto Fiuza, Joacil Germano Soares, Miriam S.M.O. Costa, Ruthnaldo R. de Melo Lima, Expedito Silva do Nascimento J´unior, Jos´e R. dos Santos, Jos´e R.L.P Cavalcanti, Jeferson S. Cavalcante PII: DOI: Reference:
S0168-0102(17)30154-2 http://dx.doi.org/doi:10.1016/j.neures.2017.03.005 NSR 4024
To appear in:
Neuroscience Research
Received date: Revised date: Accepted date:
5-8-2016 2-11-2016 9-3-2017
Please cite this article as: Engelberth, Rovena C.G.J., de Azevedo Silva, Kayo D., Fiuza, Felipe Porto, Soares, Joacil Germano, Costa, Miriam S.M.O., de Melo Lima, Ruthnaldo R., do Nascimento, Expedito Silva, dos Santos, Jos´e R., Cavalcanti, Jos´e R.L.P, Cavalcante, Jeferson S., RETINAL, NPY- AND 5-HT- INPUTS TO THE AGED SUPRACHIASMATIC NUCLEUS IN COMMON MARMOSETS (Callithrix jacchus).Neuroscience Research http://dx.doi.org/10.1016/j.neures.2017.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RETINAL, NPY- AND 5-HT- INPUTS TO THE AGED SUPRACHIASMATIC NUCLEUS IN COMMON MARMOSETS (Callithrix jacchus).
Rovena C. G. J. Engelberth1, Kayo D. de Azevedo Silva1, Felipe Porto Fiuza1, Joacil Germano Soares2, Miriam S. M. O. Costa2, Ruthnaldo R. de Melo Lima2, Expedito Silva do Nascimento Júnior2, José R. dos Santos3, José R. L. P Cavalcanti4, Jeferson S. Cavalcante1
1- Laboratory of Neurochemical Studies, Physiology Department, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970, Natal, RN, Brazil. 2- Laboratory of Neuroanatomy, Morphology Department, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil. 3- Department of Cellular Biology, Federal University of Sergipe, Aracaju, SE, Brazil 4- Laboratory of Experimental Neurology, Department of Biomedical Sciences, Health Science Center, University of State of Rio Grande do Norte, Mossoró, RN, Brazil
*Correspondence:
Rovena Clara G.J. Engelberth
Phone: +55 84 3215 3409 Fax: +55 84 3211 9206 E-mail: [email protected]
Highlights Engelberth et al., 2016
Aging leads to changes in retinal input and neurochemical expression of marmoset SCN
The area of CTb terminal boutons in SCN is reduced in aged marmosets
An age-related loss in NPY-IR and 5-HT-IR is reported in marmoset SCN
ABSTRACT The circadian timing system (CTS) anticipates optimal physiological patterns in response to environmental fluctuations, such as light-dark cycle. Since age-related disruption of circadian synchronization is linked to several pathological conditions, we characterized alterations of neurochemical constituents and retinal projections to the major pacemaker of CTS, the suprachiasmatic nucleus (SCN), in adult and aged marmosets. We used intraocular injections of neural tracer Cholera toxin b (CTb) to report age-related reductions in CTb, neuropeptide Y and serotonin immunoreactivities. Considering these projections arise in SCN from nuclei that relay environmental information to entrain the circadian clock, we provide important anatomical correlates to age-associated physiological deficits.
Abbreviations: 5-HT, serotonin; CTb, cholera toxin subunit b; CTS,Circadian Timing System; DAB, diaminobenzedine; GHT, geniculohypothalamic tract; IGL, intergeniculate leaflet;-IR, Immunoreactivity; NPY, neuropeptide Y; LD, Light-Dark; OD, optical density; PBS, phosphate buffered saline; RHT, retinohypothalamic tract; SCN, suprachiasmatic nucleus; VIP, vasoactive intestinal polypeptide; VP, vasopressin
Keywords: Suprachiasmatic nucleus; aging; serotonin; neuropeptide Y; retinal projections; Cholera toxin subunit b
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Optimal physiology requires the anticipation of environmental cycles, allowing an appropriate adjustment of endogenous necessity to ecological demands. The circadian timing system (CTS), a network of central brain structures and peripheral oscillators, offers such adjustments. The mammalian CTS is under control of one major pacemaker located in anterior hypothalamus, the suprachiasmatic nucleus (SCN; Morin, 2013). This central pacemaker is a pair of nuclei, located dorsal to the optic chiasm and lateral to the third ventricle, subdivided into core and shell regions. While the core corresponds to the previously described ventrolateral portion, characterized by expression of vasoactive intestinal polypeptide (VIP), the shell region corresponds to the dorsomedial portion of SCN, distinguished by presence of vasopressin (VP) immunoreactive (-IR) neurons (Morin, 2007). The SCN is synchronized by photic stimuli of light-dark (LD) cycle from two major pathways: a direct retino-hypothalamic tract (RHT), originated from the melanopsin-containing retinal ganglion cells (Hattar et al., 2002); and an indirect pathway through the geniculo-hypothalamic tract (GHT), in which the retinal input arises to thalamic intergeniculate leaflet (IGL) prior to modulation of SCN function (Harrington et al., 1987). Additionally, IGL also relays non-photic information (e.g. food intake and behavioral arousal) to SCN, mainly through neuropeptide Y (NPY) action. A third modulatory pathway to SCN is represented by serotonin (-5HT) projections from the median raphe nucleus, involved in entrainment of the master pacemaker to activity/exercise non-photic stimuli (for review see Dibner et al., 2010). Aging is a complex biological process protected or aggravated by environmental factors. As a consequence of aging, the circadian synchronization is disrupted leading to several physiological deficits (Engelberth et al., 2013; Farajnia et al., 2014). For instance, a longitudinal study with a male marmoset reported, at the old age, a significant phase delay of activity rhythm to the Light-Dark cycle, as well as reduction on total daily activity, fragmentation and loss of photic synchronization (Gonçalves et al., 2016). Moreover, it was suggested the aging of circadian clock is associated with incidence of cardiovascular diseases, metabolic syndrome and neurodegenerative disorders (Kondratova; Kondratov, 2012). Among the evidences to support this hypothesis are the reported age-related changes in expression of SCN neurotransmitters, such as reduction of VIP, VP, neurotensin and 5-HT receptors (Duncan et al., 2010for review see Engelberth et al., 2013). Furthermore, morphological changes, such like 2
decreases of cell counts, also have been reported in SCN of aged marmosets (Engelberth et al., 2014) and rats (Tsukahara et al., 2005). Despite the aforementioned data, the relationship between biological aging and neurochemical alterations in SCN is not yet fully understood. Considering age-related deficits are reported in retinal inputs to several brain nuclei, as well as in expression of IGL and raphe neurochemical constituents (Lupi et al., 2012; Fiuza et al., 2016), our aim here is to describe potential age-related changes of the major modulatory pathways to SCN entrainment. In this context, here we use intraocular injection of the anterograde neural tracer cholera toxin Subunit b (CTb) and immunohistochemistry for CTb, NPY and 5-HT to compare the expression of main SCN terminal fields originated from RHT, GHT and median raphe nucleus between young and aged marmosets. Male marmosets from the Primatology Center of Federal University of Rio Grande do Norte, Natal, Brazil, were divided into two groups: adult and aged (3-6 and 10-12 years old respectively, n=4 per group). They were maintained at 22°C, 50% humidity in a12:12h LD cycle. Food and water were available ad libitum. The use of animals was approved by the Brazilian Environmental Protection Agency (IBAMA register 1/24/92/0039-00) and all procedures were in accordance with Brazilian law number 11.794/2008 for animal experimental use. All experiments were approved by local ethics committee for animal use (CEUA-UFRN number 026/2010). The animals were given tramadol hydrochloride and xylazine as preanaesthetic medication, both at the dose of 5mg/kg intramuscularly and maintained on inhalation anaesthesia with isoflurane and 100% oxygen administered by a mask. Then, 80μl of CTb neural tracer (1mg/ml; List Biological Laboratories, Inc., Campbell, CA), in a solution containing 10% dimethylsulfoxide, was injected into the vitreous chamber of the left eye with a glass micropipette attached to a 100μl Hamilton syringe. After 5 days post-injection, the marmosets were deeply anesthetized and transcardially perfused with a 300 mL NaCl solution (0.9 %; 32 °C) followed by 600 mL paraformaldehyde (4 %) in a 0.1 M phosphate-buffered saline (PBS), pH 7.4. These procedures were performed between 5:30 and 6:30 p.m. Following perfusion, the brains were removed, post-fixed with paraformaldehyde (4 %) overnight, and immersed in a solution containing 30 % sucrose with 0.1 M PBS, pH 7. 4 for 3 days. Brains were cut into six series of coronal sections (30 μm) collected at a 180-μm interval. 3
Three series of sections were incubated overnight with the following primary antibodies in a dilution containing 2 % normal donkey serum with PBS (0.1 M) and 0.5 % Triton X-100: Goat-CTb (1:5000; List Biological Labs, Campbell, CA, USA), Rabbit-NPY (1:1000; Sigma-Aldrich, St Louis, MO, USA) or Rabbit-5-HT (1:5000; Sigma-Aldrich, St Louis, MO, USA). After rinsing, sections were incubated with biotinylated secondary antibodies anti-Goat and anti-Rabbit (1:1000; Jackson ImmunoResearch Labs, Westgrove, PA, USA) diluted with PBS (0.1M) and 0.5 % Triton X-100. Then, sections were incubated in a 2 % avidin-biotin solution (ABC Elite kit, Vector Labs, Burlingame, CA, USA), with NaCl addition, for 120 min. The sections were placed with a 2.5 % solution of diaminobenzidine (DAB) diluted with PBS (0.1M) for 5 min. The final reaction was performed by adding a 0.01% H2O2 solution for 1 min, to reveal marked areas in brown colors resulting from DAB oxidation. After DAB revelation, sections were washed in PBS (0.1M) four times and stored overnight at 4 °C. All sections were submitted simultaneously to these procedures in order to minimize background differences and ensure the same conditions to development of chromogen. Sections were mounted in gelatinized slides, dried, dehydrated in graded ethanol solutions, cleared in xylene, and cover slipped with DPX embedding matrix. Digital pictures of biological tissues were taken by a CCD camera (Nikon DXM-1200) connected to a light microscope (Olympus BX-41). To quantify 5-HT, NPY, and CTb immunoreactivities, five sections representing SCN from each brain were analyzed bilaterally through optical densitometry (OD). For these measurements, the sections were gray-scaled and the mean value of pixels in the region of interest was subtracted from a non-stained control area of the same tissue. We also performed a detailed analysis of fibers/terminals and distribution of synaptic buttons on SCN extension using a 40x and 100x objectives. The area of boutons was measured using the public domain image analysis software Image J and Canvas X (ACD Systems, Victoria, British Columbia, Canada). To confirm whether there are differences between the animal groups, the nonparametric Mann-Whitney test was applied. These data are expressed as median (interquartile range q3-q1). The level of significance was set at p< 0.05. Data analysis was performed using GraphPad Prism software version 6.0. We observed a strong fiber labeling of CTb-IR in adult SCN together with clearly delimited terminal boutons (Fig. 1). In aged SCN, however, the projection fibers were not present (Fig. 1). Nevertheless, the presence of boutons confirms the 4
arrival of retinal projections to the aged SCN, evidencing a vast reduction in retinal inputs when compared to adults. Quantitative analysis through Mann-Whitney test reveals a significant decrease in SCN bouton area of aged marmosets when compared to adult ones [adult: 14.60 (19.61-10.35); aged: 7.77 (10.33-5.77); p = 0.0001; Fig. 4A].
Figure 1(1.5 column) We found a higher concentration of fibers and terminals NPY-IR, similar to neuropil, in adult ventrolateral SCN when compared to aged ones (Fig. 2). The MannWhitney test revealed a significant effect of age on alterations in NPY OD between age groups [adult: 49994 (51252- 48719); aged: 22802 (24470-19989); p= 0.0286; Fig. 4B]
Figure 2 (single column) For immunostaining to 5-HT, it is possible to qualitatively observe a decrease in dorsomedial SCN labeling of aged marmosets compared to adult animals (Fig. 3). Mann-Whitney test revealed a significant decrease in 5-HT OD at the aged dorsomedial SCN compared to adult ones [adult marmoset: 448 (528-403); aged marmoset: 316 (344-240); p = 0.0286; Fig. 4C]. Figure 3 (single column)
Figure 4 (single column)
Using a sensitive neuronal tracer, we show a reduction in photic input to the central pacemaker as an outcome of the aging process in marmosets. Correspondingly, the retinal projections to both SCN and IGL are reduced in aged mice and rats (Lupi et al., 2012; Fiuza et al., 2016). It is noteworthy that age-related impairments of circadian response to light occur in both entrainment and re-entrainment to photic cues (Benloucif et al., 2006). Indeed, SCN immunoreactivity to immediate early gene c-Fos decreases after light stimulation in aged hamsters (Zhang et al., 1996) and in both aged and seasonal cycle-accelerated primates (Aujard et al., 2001). In this context, our 5
data corroborates these studies reinforcing the age-related loss in retinal connections to central brain structures of CTS as an important factor to light responsiveness. Conversely, the retinal projection to SCN remains unaltered in elderly golden hamsters (Zhang et al., 1998). Collectively, these data point to species-specific agerelated changes in retinal afferents to CTS (Lupi et al., 2012; Fiuza et al., 2016). Through OD analysis we report a NPY decrease in SCN of aged marmosets when compared to adult ones. The NPY-IR terminals are found in ventrolateral portion of SCN in hamsters (Card and Moore, 1984), rats (Mantyh and Kemp, 1983) and marmosets (Cavalcante et al. 2002). The main source of NPY-IR fibers in SCN is GHT, which arises from IGL cells in rodents (Moore and Card, 1994) and from its homologous structure in primates, the pregeniculate nucleus (Lima et al., 2012). Interestingly, NPY seems to antagonize six of the nine hallmarks of aging proposed by López-Ótin et al (2013), namely loss of proteostasis, stem cell exhaustion, altered intercellular communication, deregulated nutrient sensing, cellular senescence and mitochondrial dysfunction (Botelho and Cavadas, 2015). In addition, caloric restriction is an intervention capable of enhancing lifespan in several animal models and is associated with an increased expression of NPY in hypothalamic areas such as arcuate nucleus (Minor et al., 2009). Since caloric restriction is also related to an improvement of autophagy, Aveleira et al. (2015) investigated whether this improvement is due to NPY action and reported this neuropeptide in fact stimulates the autophagic flux in hypothalamic neurons, providing an insight for development of anti-aging interventions. In the present work, using a primate model we corroborate previous reports of age-related reductions of NPY and its receptors in several brain areas, such hippocampus cortex, hypothalamus and IGL (Veyrat-Durebex et al., 2013; Fiuza et al., 2016). Thus, we contribute to identification of area-specific aging impacts on NPY expression, which could be explored in the development of the aforementioned anti-aging interventions. Considering this neuropeptide mediates photic and non-photic responses to central clock, the age-related reduction of NPY-IR is likely related to impairments of IGL-SCN communication, implicating in deficits to circadian entrainment. We found a significant 5-HT-IR reduction in SCN of aged marmosets when compared to adult ones. Similarly, serotonergic projections to SCN and 5-HT concentration in striatum and occpital cortex are also diminished in aged rats (Van Luijtelaar et al., 1989; Stemmelin et al., 2000). In addition, expression of serotonergic 6
receptors is changed in neural components of circadian system of aged hamsters (Duncan et al., 2010). In mammals, changes associated with senescence of the serotonergic system are known risk factors for diseases such as diabetes (Mattson et al., 2004) and alterations in sleep, sexual behavior and mood (Meltzer et al., 1998). Moreover, the serotonergic network is impaired in several neuropsychological conditions which affect aged population, like clinical depression, anxiety disorders, Parkinson and Alzheimer diseases (Fidalgo et al., 2013). Considering that age-related disruptions of circadian homeostatic function are linked to sleep, memory and mood disorders (Kondratova and Kondratov, 2012) the identification of serotonergic changes in central nervous system might be a key point to understand and antagonize the aging process. Overall, the present work characterizes important age-related impairments of CTb, NPY and 5-HT immunoreactivities in SCN, which represents the major afferent pathways of central clock entrainment to environmental cycles, providing new insights to understand the impact of aging in marmoset nervous system.
Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors’ Contribution Dr. Rovena Clara G. J. Engelberth, Dr. Jeferson Souza Cavalcante and Dr. Carolina V. de M. Azevedo defined experimental design, performed experiments and analyzed histological sections; besides, they prepared the first version of the paper; Dr. Jose Ronaldo dos Santos and Dr. José Rodolfo L. P. Cavalcanti revised the final version of the paper; Felipe Porto Fiuza, Kayo Diogenes de A. Silva, Dr. Judney C. Cavalcante, Dr. Miriam Stela M. O. Costa, and Dr. Expedito S. Nascimento Jr worked on immunohistochemical processing and funded part of the research.
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Acknowledgements The study was supported by Brazilian funding agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Apoio à Pesquisa no Rio Grande do Norte (FAPERN).
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Figure Captions Figure 1- Digitalized photomicrographs of coronal brain sections processed for CTb immunohistochemistry showing the SCN of adult and aged Callithrix jacchus. CTb-immunostained fibers are present in adult SCN (A, B and C) whereas only terminal boutons are found in aged SCN (A’, B’ and C’). The delimited areas in A, A’, B and B’ representes the field shown at higher magnifications in B, B’, C and C’ respectively. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 100 µm; B and B’ = 40 µm; C and C’ = 20 µm
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Figure 2- Digitalized photomicrographs of coronal brain sections processed for NPY immunohistochemistry showing the SCN of adult and aged Callithrix jacchus (A and A’). A detailed visualization of the delimited area in A and A’ is presented at higher magnifications in B and B’. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 30 µm; B and B’ = 15 µm.
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Figure 3- Digitalized photomicrographs of coronal brain sections processed for 5-HT immunohistochemistry showing the SCN of adult and aged Callithrix jacchus (A and A’). A detailed visualization of the delimited area in A and A’ is presented at higher magnifications in B and B’. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 30 µm; B and B’ = 15 µm.
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Figure 4- Mann-Whitney test shows a significant effect of age in reduction of: A) terminal boutons area in CTb-immunostained SCN of aged marmosets: 7.77 (10.33-5.77) when compared to adults: 14.60 (19.61-10.35; p = 0.0001; B) NPY-IR of SCN in aged
marmosets: 22802 (24470-19989) when compared to adults: 49994 (51252- 48719, p = 0.0286). 5-HT-IR of SCN in aged marmosets: 316 (344-240) when compared to adults: 448 (528-403, p = 0.0286). All data expressed as median (interquartile range q3-q1).
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S0168-0102(17)30154-2 http://dx.doi.org/doi:10.1016/j.neures.2017.03.005 NSR 4024
To appear in:
Neuroscience Research
Received date: Revised date: Accepted date:
5-8-2016 2-11-2016 9-3-2017
Please cite this article as: Engelberth, Rovena C.G.J., de Azevedo Silva, Kayo D., Fiuza, Felipe Porto, Soares, Joacil Germano, Costa, Miriam S.M.O., de Melo Lima, Ruthnaldo R., do Nascimento, Expedito Silva, dos Santos, Jos´e R., Cavalcanti, Jos´e R.L.P, Cavalcante, Jeferson S., RETINAL, NPY- AND 5-HT- INPUTS TO THE AGED SUPRACHIASMATIC NUCLEUS IN COMMON MARMOSETS (Callithrix jacchus).Neuroscience Research http://dx.doi.org/10.1016/j.neures.2017.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
RETINAL, NPY- AND 5-HT- INPUTS TO THE AGED SUPRACHIASMATIC NUCLEUS IN COMMON MARMOSETS (Callithrix jacchus).
Rovena C. G. J. Engelberth1, Kayo D. de Azevedo Silva1, Felipe Porto Fiuza1, Joacil Germano Soares2, Miriam S. M. O. Costa2, Ruthnaldo R. de Melo Lima2, Expedito Silva do Nascimento Júnior2, José R. dos Santos3, José R. L. P Cavalcanti4, Jeferson S. Cavalcante1
1- Laboratory of Neurochemical Studies, Physiology Department, Biosciences Center, Federal University of Rio Grande do Norte, 59072-970, Natal, RN, Brazil. 2- Laboratory of Neuroanatomy, Morphology Department, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil. 3- Department of Cellular Biology, Federal University of Sergipe, Aracaju, SE, Brazil 4- Laboratory of Experimental Neurology, Department of Biomedical Sciences, Health Science Center, University of State of Rio Grande do Norte, Mossoró, RN, Brazil
*Correspondence:
Rovena Clara G.J. Engelberth
Phone: +55 84 3215 3409 Fax: +55 84 3211 9206 E-mail: [email protected]
Highlights Engelberth et al., 2016
Aging leads to changes in retinal input and neurochemical expression of marmoset SCN
The area of CTb terminal boutons in SCN is reduced in aged marmosets
An age-related loss in NPY-IR and 5-HT-IR is reported in marmoset SCN
ABSTRACT The circadian timing system (CTS) anticipates optimal physiological patterns in response to environmental fluctuations, such as light-dark cycle. Since age-related disruption of circadian synchronization is linked to several pathological conditions, we characterized alterations of neurochemical constituents and retinal projections to the major pacemaker of CTS, the suprachiasmatic nucleus (SCN), in adult and aged marmosets. We used intraocular injections of neural tracer Cholera toxin b (CTb) to report age-related reductions in CTb, neuropeptide Y and serotonin immunoreactivities. Considering these projections arise in SCN from nuclei that relay environmental information to entrain the circadian clock, we provide important anatomical correlates to age-associated physiological deficits.
Abbreviations: 5-HT, serotonin; CTb, cholera toxin subunit b; CTS,Circadian Timing System; DAB, diaminobenzedine; GHT, geniculohypothalamic tract; IGL, intergeniculate leaflet;-IR, Immunoreactivity; NPY, neuropeptide Y; LD, Light-Dark; OD, optical density; PBS, phosphate buffered saline; RHT, retinohypothalamic tract; SCN, suprachiasmatic nucleus; VIP, vasoactive intestinal polypeptide; VP, vasopressin
Keywords: Suprachiasmatic nucleus; aging; serotonin; neuropeptide Y; retinal projections; Cholera toxin subunit b
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Optimal physiology requires the anticipation of environmental cycles, allowing an appropriate adjustment of endogenous necessity to ecological demands. The circadian timing system (CTS), a network of central brain structures and peripheral oscillators, offers such adjustments. The mammalian CTS is under control of one major pacemaker located in anterior hypothalamus, the suprachiasmatic nucleus (SCN; Morin, 2013). This central pacemaker is a pair of nuclei, located dorsal to the optic chiasm and lateral to the third ventricle, subdivided into core and shell regions. While the core corresponds to the previously described ventrolateral portion, characterized by expression of vasoactive intestinal polypeptide (VIP), the shell region corresponds to the dorsomedial portion of SCN, distinguished by presence of vasopressin (VP) immunoreactive (-IR) neurons (Morin, 2007). The SCN is synchronized by photic stimuli of light-dark (LD) cycle from two major pathways: a direct retino-hypothalamic tract (RHT), originated from the melanopsin-containing retinal ganglion cells (Hattar et al., 2002); and an indirect pathway through the geniculo-hypothalamic tract (GHT), in which the retinal input arises to thalamic intergeniculate leaflet (IGL) prior to modulation of SCN function (Harrington et al., 1987). Additionally, IGL also relays non-photic information (e.g. food intake and behavioral arousal) to SCN, mainly through neuropeptide Y (NPY) action. A third modulatory pathway to SCN is represented by serotonin (-5HT) projections from the median raphe nucleus, involved in entrainment of the master pacemaker to activity/exercise non-photic stimuli (for review see Dibner et al., 2010). Aging is a complex biological process protected or aggravated by environmental factors. As a consequence of aging, the circadian synchronization is disrupted leading to several physiological deficits (Engelberth et al., 2013; Farajnia et al., 2014). For instance, a longitudinal study with a male marmoset reported, at the old age, a significant phase delay of activity rhythm to the Light-Dark cycle, as well as reduction on total daily activity, fragmentation and loss of photic synchronization (Gonçalves et al., 2016). Moreover, it was suggested the aging of circadian clock is associated with incidence of cardiovascular diseases, metabolic syndrome and neurodegenerative disorders (Kondratova; Kondratov, 2012). Among the evidences to support this hypothesis are the reported age-related changes in expression of SCN neurotransmitters, such as reduction of VIP, VP, neurotensin and 5-HT receptors (Duncan et al., 2010for review see Engelberth et al., 2013). Furthermore, morphological changes, such like 2
decreases of cell counts, also have been reported in SCN of aged marmosets (Engelberth et al., 2014) and rats (Tsukahara et al., 2005). Despite the aforementioned data, the relationship between biological aging and neurochemical alterations in SCN is not yet fully understood. Considering age-related deficits are reported in retinal inputs to several brain nuclei, as well as in expression of IGL and raphe neurochemical constituents (Lupi et al., 2012; Fiuza et al., 2016), our aim here is to describe potential age-related changes of the major modulatory pathways to SCN entrainment. In this context, here we use intraocular injection of the anterograde neural tracer cholera toxin Subunit b (CTb) and immunohistochemistry for CTb, NPY and 5-HT to compare the expression of main SCN terminal fields originated from RHT, GHT and median raphe nucleus between young and aged marmosets. Male marmosets from the Primatology Center of Federal University of Rio Grande do Norte, Natal, Brazil, were divided into two groups: adult and aged (3-6 and 10-12 years old respectively, n=4 per group). They were maintained at 22°C, 50% humidity in a12:12h LD cycle. Food and water were available ad libitum. The use of animals was approved by the Brazilian Environmental Protection Agency (IBAMA register 1/24/92/0039-00) and all procedures were in accordance with Brazilian law number 11.794/2008 for animal experimental use. All experiments were approved by local ethics committee for animal use (CEUA-UFRN number 026/2010). The animals were given tramadol hydrochloride and xylazine as preanaesthetic medication, both at the dose of 5mg/kg intramuscularly and maintained on inhalation anaesthesia with isoflurane and 100% oxygen administered by a mask. Then, 80μl of CTb neural tracer (1mg/ml; List Biological Laboratories, Inc., Campbell, CA), in a solution containing 10% dimethylsulfoxide, was injected into the vitreous chamber of the left eye with a glass micropipette attached to a 100μl Hamilton syringe. After 5 days post-injection, the marmosets were deeply anesthetized and transcardially perfused with a 300 mL NaCl solution (0.9 %; 32 °C) followed by 600 mL paraformaldehyde (4 %) in a 0.1 M phosphate-buffered saline (PBS), pH 7.4. These procedures were performed between 5:30 and 6:30 p.m. Following perfusion, the brains were removed, post-fixed with paraformaldehyde (4 %) overnight, and immersed in a solution containing 30 % sucrose with 0.1 M PBS, pH 7. 4 for 3 days. Brains were cut into six series of coronal sections (30 μm) collected at a 180-μm interval. 3
Three series of sections were incubated overnight with the following primary antibodies in a dilution containing 2 % normal donkey serum with PBS (0.1 M) and 0.5 % Triton X-100: Goat-CTb (1:5000; List Biological Labs, Campbell, CA, USA), Rabbit-NPY (1:1000; Sigma-Aldrich, St Louis, MO, USA) or Rabbit-5-HT (1:5000; Sigma-Aldrich, St Louis, MO, USA). After rinsing, sections were incubated with biotinylated secondary antibodies anti-Goat and anti-Rabbit (1:1000; Jackson ImmunoResearch Labs, Westgrove, PA, USA) diluted with PBS (0.1M) and 0.5 % Triton X-100. Then, sections were incubated in a 2 % avidin-biotin solution (ABC Elite kit, Vector Labs, Burlingame, CA, USA), with NaCl addition, for 120 min. The sections were placed with a 2.5 % solution of diaminobenzidine (DAB) diluted with PBS (0.1M) for 5 min. The final reaction was performed by adding a 0.01% H2O2 solution for 1 min, to reveal marked areas in brown colors resulting from DAB oxidation. After DAB revelation, sections were washed in PBS (0.1M) four times and stored overnight at 4 °C. All sections were submitted simultaneously to these procedures in order to minimize background differences and ensure the same conditions to development of chromogen. Sections were mounted in gelatinized slides, dried, dehydrated in graded ethanol solutions, cleared in xylene, and cover slipped with DPX embedding matrix. Digital pictures of biological tissues were taken by a CCD camera (Nikon DXM-1200) connected to a light microscope (Olympus BX-41). To quantify 5-HT, NPY, and CTb immunoreactivities, five sections representing SCN from each brain were analyzed bilaterally through optical densitometry (OD). For these measurements, the sections were gray-scaled and the mean value of pixels in the region of interest was subtracted from a non-stained control area of the same tissue. We also performed a detailed analysis of fibers/terminals and distribution of synaptic buttons on SCN extension using a 40x and 100x objectives. The area of boutons was measured using the public domain image analysis software Image J and Canvas X (ACD Systems, Victoria, British Columbia, Canada). To confirm whether there are differences between the animal groups, the nonparametric Mann-Whitney test was applied. These data are expressed as median (interquartile range q3-q1). The level of significance was set at p< 0.05. Data analysis was performed using GraphPad Prism software version 6.0. We observed a strong fiber labeling of CTb-IR in adult SCN together with clearly delimited terminal boutons (Fig. 1). In aged SCN, however, the projection fibers were not present (Fig. 1). Nevertheless, the presence of boutons confirms the 4
arrival of retinal projections to the aged SCN, evidencing a vast reduction in retinal inputs when compared to adults. Quantitative analysis through Mann-Whitney test reveals a significant decrease in SCN bouton area of aged marmosets when compared to adult ones [adult: 14.60 (19.61-10.35); aged: 7.77 (10.33-5.77); p = 0.0001; Fig. 4A].
Figure 1(1.5 column) We found a higher concentration of fibers and terminals NPY-IR, similar to neuropil, in adult ventrolateral SCN when compared to aged ones (Fig. 2). The MannWhitney test revealed a significant effect of age on alterations in NPY OD between age groups [adult: 49994 (51252- 48719); aged: 22802 (24470-19989); p= 0.0286; Fig. 4B]
Figure 2 (single column) For immunostaining to 5-HT, it is possible to qualitatively observe a decrease in dorsomedial SCN labeling of aged marmosets compared to adult animals (Fig. 3). Mann-Whitney test revealed a significant decrease in 5-HT OD at the aged dorsomedial SCN compared to adult ones [adult marmoset: 448 (528-403); aged marmoset: 316 (344-240); p = 0.0286; Fig. 4C]. Figure 3 (single column)
Figure 4 (single column)
Using a sensitive neuronal tracer, we show a reduction in photic input to the central pacemaker as an outcome of the aging process in marmosets. Correspondingly, the retinal projections to both SCN and IGL are reduced in aged mice and rats (Lupi et al., 2012; Fiuza et al., 2016). It is noteworthy that age-related impairments of circadian response to light occur in both entrainment and re-entrainment to photic cues (Benloucif et al., 2006). Indeed, SCN immunoreactivity to immediate early gene c-Fos decreases after light stimulation in aged hamsters (Zhang et al., 1996) and in both aged and seasonal cycle-accelerated primates (Aujard et al., 2001). In this context, our 5
data corroborates these studies reinforcing the age-related loss in retinal connections to central brain structures of CTS as an important factor to light responsiveness. Conversely, the retinal projection to SCN remains unaltered in elderly golden hamsters (Zhang et al., 1998). Collectively, these data point to species-specific agerelated changes in retinal afferents to CTS (Lupi et al., 2012; Fiuza et al., 2016). Through OD analysis we report a NPY decrease in SCN of aged marmosets when compared to adult ones. The NPY-IR terminals are found in ventrolateral portion of SCN in hamsters (Card and Moore, 1984), rats (Mantyh and Kemp, 1983) and marmosets (Cavalcante et al. 2002). The main source of NPY-IR fibers in SCN is GHT, which arises from IGL cells in rodents (Moore and Card, 1994) and from its homologous structure in primates, the pregeniculate nucleus (Lima et al., 2012). Interestingly, NPY seems to antagonize six of the nine hallmarks of aging proposed by López-Ótin et al (2013), namely loss of proteostasis, stem cell exhaustion, altered intercellular communication, deregulated nutrient sensing, cellular senescence and mitochondrial dysfunction (Botelho and Cavadas, 2015). In addition, caloric restriction is an intervention capable of enhancing lifespan in several animal models and is associated with an increased expression of NPY in hypothalamic areas such as arcuate nucleus (Minor et al., 2009). Since caloric restriction is also related to an improvement of autophagy, Aveleira et al. (2015) investigated whether this improvement is due to NPY action and reported this neuropeptide in fact stimulates the autophagic flux in hypothalamic neurons, providing an insight for development of anti-aging interventions. In the present work, using a primate model we corroborate previous reports of age-related reductions of NPY and its receptors in several brain areas, such hippocampus cortex, hypothalamus and IGL (Veyrat-Durebex et al., 2013; Fiuza et al., 2016). Thus, we contribute to identification of area-specific aging impacts on NPY expression, which could be explored in the development of the aforementioned anti-aging interventions. Considering this neuropeptide mediates photic and non-photic responses to central clock, the age-related reduction of NPY-IR is likely related to impairments of IGL-SCN communication, implicating in deficits to circadian entrainment. We found a significant 5-HT-IR reduction in SCN of aged marmosets when compared to adult ones. Similarly, serotonergic projections to SCN and 5-HT concentration in striatum and occpital cortex are also diminished in aged rats (Van Luijtelaar et al., 1989; Stemmelin et al., 2000). In addition, expression of serotonergic 6
receptors is changed in neural components of circadian system of aged hamsters (Duncan et al., 2010). In mammals, changes associated with senescence of the serotonergic system are known risk factors for diseases such as diabetes (Mattson et al., 2004) and alterations in sleep, sexual behavior and mood (Meltzer et al., 1998). Moreover, the serotonergic network is impaired in several neuropsychological conditions which affect aged population, like clinical depression, anxiety disorders, Parkinson and Alzheimer diseases (Fidalgo et al., 2013). Considering that age-related disruptions of circadian homeostatic function are linked to sleep, memory and mood disorders (Kondratova and Kondratov, 2012) the identification of serotonergic changes in central nervous system might be a key point to understand and antagonize the aging process. Overall, the present work characterizes important age-related impairments of CTb, NPY and 5-HT immunoreactivities in SCN, which represents the major afferent pathways of central clock entrainment to environmental cycles, providing new insights to understand the impact of aging in marmoset nervous system.
Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors’ Contribution Dr. Rovena Clara G. J. Engelberth, Dr. Jeferson Souza Cavalcante and Dr. Carolina V. de M. Azevedo defined experimental design, performed experiments and analyzed histological sections; besides, they prepared the first version of the paper; Dr. Jose Ronaldo dos Santos and Dr. José Rodolfo L. P. Cavalcanti revised the final version of the paper; Felipe Porto Fiuza, Kayo Diogenes de A. Silva, Dr. Judney C. Cavalcante, Dr. Miriam Stela M. O. Costa, and Dr. Expedito S. Nascimento Jr worked on immunohistochemical processing and funded part of the research.
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Acknowledgements The study was supported by Brazilian funding agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Apoio à Pesquisa no Rio Grande do Norte (FAPERN).
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Figure Captions Figure 1- Digitalized photomicrographs of coronal brain sections processed for CTb immunohistochemistry showing the SCN of adult and aged Callithrix jacchus. CTb-immunostained fibers are present in adult SCN (A, B and C) whereas only terminal boutons are found in aged SCN (A’, B’ and C’). The delimited areas in A, A’, B and B’ representes the field shown at higher magnifications in B, B’, C and C’ respectively. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 100 µm; B and B’ = 40 µm; C and C’ = 20 µm
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Figure 2- Digitalized photomicrographs of coronal brain sections processed for NPY immunohistochemistry showing the SCN of adult and aged Callithrix jacchus (A and A’). A detailed visualization of the delimited area in A and A’ is presented at higher magnifications in B and B’. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 30 µm; B and B’ = 15 µm.
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Figure 3- Digitalized photomicrographs of coronal brain sections processed for 5-HT immunohistochemistry showing the SCN of adult and aged Callithrix jacchus (A and A’). A detailed visualization of the delimited area in A and A’ is presented at higher magnifications in B and B’. 3v: third ventricle; oc: optic chiasm. Scale bar: A and A’= 30 µm; B and B’ = 15 µm.
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Figure 4- Mann-Whitney test shows a significant effect of age in reduction of: A) terminal boutons area in CTb-immunostained SCN of aged marmosets: 7.77 (10.33-5.77) when compared to adults: 14.60 (19.61-10.35; p = 0.0001; B) NPY-IR of SCN in aged
marmosets: 22802 (24470-19989) when compared to adults: 49994 (51252- 48719, p = 0.0286). 5-HT-IR of SCN in aged marmosets: 316 (344-240) when compared to adults: 448 (528-403, p = 0.0286). All data expressed as median (interquartile range q3-q1).
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