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NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress
General and Comparative Endocrinology xxx (2017) xxx–xxx
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NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress Elizabeth Heigrujam 1, Ishfaq Ali 1, Shobha Bhargava ⇑ Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India
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Article history: Received 1 April 2016 Revised 6 January 2017 Accepted 7 January 2017 Available online xxxx Keywords: Neuropeptide Y Salinity stress E. cyanophlyctis Brain Immunohistochemistry
a b s t r a c t Most of the amphibians breed in temporary ponds vulnerable to occasional desiccation, thus, leaving their larvae exposed to stressful fluctuations in various environmental parameters including salinity. These animals possess a well suited central adaptive mechanism to adapt to these alterations. Neuropeptide Y (NPY), a 36 amino acid neurotransmitter, has been reported to antagonize various neuropsychological consequences of stress within the mammalian brain. Osmotic regulation of NPY in the hypothalamo-neurohypophysial pathway of mammalian brain is also known. Although the molecule possesses an extensive distribution in the brain of amphibians, its functional association is not well understood. We have investigated the endogenous response of NPY-ergic system to osmotically stressful conditions in the brain of Indian skipper frog-Euphlyctis cyanophlyctis tadpoles. Using Immunohistochemistry, we observed an up-regulation of NPY immunoreactivity (NPY-ir) in the brain of tadpoles exposed to stressful salt concentrations. A significant increase of NPY-ir occurred in the pallium and septum regions of telencephalon; preoptic area, epithalamic, thalamic and hypothalamic parts of diencephalon. Most of the regions are implicated in the modulation of stress and anxiety related brain functions and have also been shown to respond to the salinity stress in mammals. In addition, NPY producing neurons in pre-optic and hypothalamic parts show a close co-existence with the vasopressin-ergic neurons. Thus, our study suggests a possible role of NPY in stabilizing the neuro-endocrinological consequences of osmotic stress in an amphibian brain. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Euphlyctis cyanophlyctis, a dicrogloccid frog, inhabits diverse habitats in south Asia and is known to tolerate a wide range of salt water concentrations (Khan, 1997; Hopkins and Brodie, 2015). The animal breeds in temporary ponds, vulnerable to occasional desiccation and frequent fluctuations in different environmental
Abbreviations: ADH, vasopressin; Al, amygdala lateralis; Am, amygdala medialis; CNS, central nervous system; CRH, corticotropin-releasing hormone; DP, dorsal pallium; IR, infundibular recess; LP, lateral pallium; ME, median eminence; MP, medial pallium; NCER, nucleus cerebella; NGS, normal goat serum; NHD, nucleus habenularis dorsalis; NHV, nucleus habenularis ventralis; NID, nucleus infundibularis dorsalis; NIV, nucleus infundibularis ventralis; NMS, nucleus medialis septi; NPC, nucleus posterocentralis thalami; NPL, nucleus posterolateralis thalami; NPO, nucleus preopticus; NPY, neuropeptide Y; NT, motor nucleus of the trigeminal nerve; OT, optic tectum; PR, preoptic recess; Pi, pars intermedia hypophysis; PN, pars nervosa hypophysis; PVN, paraventricular nucleus; RA, raphe nucleus; SGS, stratum griseum superficiale tecti; SON, supraoptic nuclei. ⇑ Corresponding author. E-mail address: [email protected] (S. Bhargava). 1 Equal contribution of the authors.
parameters including salinity (Khan, 2014; Griffiths, 1997; Brady and Griffiths, 2000). The larval stage persists for 3 months during which they face a steep rise and fall in the salt concentrations (Khan, 2014). In order to provide stability, a series of physiological, neuro-endocrinological, and biochemical responses integrate to maintain homeostasis (Hoar, 1988; Hopkins et al., 2016; McCormick and Bradshaw, 2006). This is mainly achieved by a well coordinated orchestration of various interrelated hormonal systems including hypothalamo–neurohypophysial system (see review by Uchiyama and Konno, 2006; Uchiyama et al., 2014). Since CNS along with the olfactory system intermediates the environmental stimulus and various response mechanisms inside the body, it is of paramount importance to find out the brain areas involved in the modulation of these changes. Here, we have tried to evaluate the neuro-anatomical response of endogenous neuropeptide Y (NPY) – a 36 amino acid neuromodulator known to resilience stress in the brain of mammals– during osmotic stress in the brain E. cyanophlyctis tadpoles. Neuropeptide Y (NPY) has been known as an important component of stress antagonising peptides in the brain of mammals
http://dx.doi.org/10.1016/j.ygcen.2017.01.015 0016-6480/Ó 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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(Heilig, 2004; Morales-Medina et al., 2011). The peptide possesses a remarkable conservation in its structure (up to 93%) and function throughout the vertebrates (Colmers and Wahlestedt, 1993; Larhammar et al., 1993; Larsson et al., 2008). Based mainly on its neuro-anatomical localisation, various roles have been assigned to the peptide (Wu et al., 2011; Beck et al., 2014; Ali et al., 2016). Although the peptide has also been shown to possess an extensive distribution throughout the brain of amphibians (D’Aniello et al., 1996; Ebersole et al., 2001; Mousley et al., 2006; Tuinhof et al., 1994), its functional relevance remains largely unknown. Role of NPY in mediating the physiological consequences of osmotic stress is well established in mammals. Hooi et al., in 1989 showed the involvement of NPY in the fluid homeostasis in rat, following the close anatomical relationship between nerve terminals containing NPY and vasopressin in the hypothalamic paraventricular (PVN) and supra-optic nucleus (SON) of rat brain. Co-existence of NPY and vasopressin expressing neurons and an up-regulation of NPY in the magnocellular hypothalamoneurohypophysial system after salt loading in rat has also been reported (Larsen et al., 1992). An increase in the NPY-ir in the supra-optic neurons of magnocellular hypothalamus in vasopressin deficient homozygous Brattleboro (di/di) rats, against the control long evans rats, is also shown (Bundzikova et al., 2008, 2010). Despite all these evidences about NPY response to salt adaptation in terrestrial animals, no such study has been reported in the animals living in aquatic habitats where they experience a wide range of changes in the salt concentration (Bentley, 2002; Shoemaker and Nagy, 1977). Since these animals are exposed to osmotic fluctuations in their natural habitat, it is interesting to look for the underlying neurological mechanisms mediating the central responses to osmotic stress. Herein, we investigate the relative neuroanatomical response of NPY in the developing brain of the tadpoles of Euphlyctis cyanophlyctis exposed to various grades of salinities.
Animals were kept in the potential hyperosmotic solutions for 4 h which was done in accordance to the earlier reports of stress induced peptide release in the tadpoles of same stage (Yao et al., 2004; Luo et al., 2000; Ali et al., 2016). After 4 h, the tadpoles in each solution were anesthetized using 2-phenoxyethanol, brains were dissected and fixed in Bouin’s fixative for 24 h. This was followed by the dehydration of the tissue with increased grades-10% (2 h), 20% (2 h) and 30% (overnight at 4 °C) of sucrose in phosphate buffer saline (PBS, 0.01 M, pH 7.4) solution for cryo-protection. The tissues were later embedded in Shandon cryomatrix and cut on a cryostat (Lieca CM 1520) in the transverse plane at a thickness of 20 lm. Sections were mounted on poly-L-lysine coated slides and stored in 20 °C until immunohistochemical staining.
2. Material and methods
2.3. Characterization of antibody
2.1. Animal collection and tissue processing
The monoclonal primary antibody against NPY used in the present study was obtained from Sigma Aldrich. The peptide has been reported to possess a 93% homology throughout the vertebrates (Colmers and Wahlestedt, 1993; Larhammar et al., 1993; Larsson et al., 2008; Crespi et al., 2004). The anti-NPY antibody used in the study was against the immunogen synthetic neuropeptide Y (NPY, Porcine-N9528) was produced in rabbit and used at a dilution of 1:5000. Prior to its usage, the antibody was characterized for its specificity in the frog brain. Various control procedures were performed to test its cross-reactivity. Sections were incubated with diluted antibodies pre-adsorbed with the peptide to determine the specificity. Omission of the primary or secondary antibodies from the reaction mixture also showed no immunoreactions.
Lower developmental stages (stage 35–37) of Euphlyctis cyanophlyctis tadpoles were collected from the fields of Taleghar located in the Ghat regions of Sahyadri hills in Maharashtra, India. Animals were kept in glass chambers of 30 45 10 cm3 and were fed with ad libitum boiled spinach until stage 38 (Gosner, 1960). Pre-metamorphic stage (38–39) was used for study mainly because there are no major developmental instincts, anatomical or morphological changes in this the stage. And the physiological processes of the animal, at this stage, are also considered to be stabilised (Gosner, 1960). The animals were treated in accordance to the ethical guidelines for animal usage established by Savitribai Phule Pune University, Pune, India. In the natural conditions the frog breeds in fresh water with an average salinity of 0.013% (130 ppm). Since, the larval forms in Euphlyctis cyanophlyctis persists for 3 months, the salinity of these temporary ponds, due to desiccation and accumulation of salts, in summer shoots up to 0.2% (2000 ppm) or even higher. In the laboratory, the animals were acclimatised to tap water with 100 ppm salt concentration. The animals were further acclimatised to room temperature and 12 h day/night cycle. Tadpoles (n = 5) each were taken and exposed to increased gradients of salt concentrations viz, 0.25% and 0.50% based on threshold tolerance of the animal derived earlier. 5n tadpoles already acclimatised to the tap water were fixed and served as control.
2.2. Immunohistochemistry Sections were used to check down the possible alterations in expression of NPY in normal and stressed animals following the standard immunohistochemical protocol (Shewale et al., 2014; Ali et al., 2016). Briefly, the steps included the 0.3% hydrogen peroxide in methanol treatment for 1 h which was followed by hour long incubation with blocking agent containing 0.5% BSA (Bovine serum albumin) and 0.5% gelatin in PBS. PBS washes were given after every step. Sections were afterwards incubated for 1 h in NGS (1:40 dilution, Vectastain) and immediately kept for an overnight (16 h) incubation at 4 °C with Rabbit monoclonal antibody against NPY (1:5000) containing 0.5% BSA and gelatine. On the second day, the addition of secondary antibody- biotinylated goat anti-mouse IgG antibody at room temperature (1:200, Vectastain)- and ABC reagent (Vectastain, ABC Kit, 1:100) followed after consecutive buffer (PBS) washes. Sections were incubated with 3,3 diaminobenzidine tetra hydrochloride (DAB) solution (0.05 M, pH 7.2 Tris buffer and 0.02% H2O2) for 8–10 min and then subsequently washed in distilled water, dehydrated in alcohol, cleared in xylene, mounted in distyrene plasticizer xylene (Merck, India) and photographed for further observation.
2.4. Morphometry Digital images of NPY immunoreactivity were obtained on a Nikon Inverted Microscope equipped with a Nikon DS-Ri2 Microscope camera including Nikon Basic Research software (NIS elements) Sections through the same region of the brain using neuro-anatomical landmarks were used for analysis (Neary and Northcutt, 1983; Wada et al., 1980). For cell counting, all visible cell bodies stained within the defined brain regions were counted manually (using Image J software), keeping the same counting area for control. Data from each brain region in an animal was calculated by taking the average counts from five brain slices. Further,
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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the average counts from the left and right sides of brain at specific brain regions of interest were also considered.
2.5. Statistical analysis In each treatment different areas of the brain and their cell counts were plotted as box plots. The comparative analysis in the cell counts of normal and salinity treated groups was tested using Kruskal–Wallis test. A post hoc analysis using Mann–Whitney U test with Bonferroni correction was performed to check the difference between the controls and experimental samples. All statistical analysis were done in freeware PAST version 2.14 (Hammer et al., 2001).
3. Results Assay for relative expression of NPY in the brain of Euphlyctis cyanophlyctis tadpoles exposed to different gradients of salt concentrations revealed an up-regulation of NPY-ir in various regions of the animal brain. Antisera directed against the NPY antigen was checked for its specificity prior to any experimentation. Omission of primary or secondary antibody from the protocol did not develop any immunoreaction (Fig. 1B). Pre-adsorption of primary antibody with pure NPY peptide also abolished immunostaining (Fig. 1A). In response to salinity stress, a significant increase in the expression of NPY immunoreactivity was shown by various regions of the tadpole brain including telencephalon, diencephalon and some parts of mesencephalon. The neuro-anotomical terminology and identification of different regions was adopted from the stereotaxic atlas of various developmental stages of frog brain (Lopez and Gonzalez, 2002; Mathieu et al., 2001; Neary and Northcutt, 1983; Wada et al., 1980). Although there was an evident increase in the NPY-ir among the control and 0.50% saline groups; some of the corresponding regions of the control and 0.25% salinity grades exhibited almost same immunostaining. Most of the immunoreactive material comprised of the axonal processes. In some regions, cells were round to oval shaped with beaded fibers. Given below is the differential account of NPY-ir observed in different regions of frog brain exposed to increasing concentrations of salt medium. In the telencephalon, salinity induced increase in the NPY-ir perikarya was seen in the pallium and septum regions. (Fig. 2 A–C). A significant increase was observed in the immunoreactive perikarya of the stressed animals as compared to controls. In the pallium region, NPY-ir cells increased in the dorsal pallium (DP) (Number of cells: n = 21) and medial pallium (MP) (n = 24) in the tadpole brains exposed to 0.50% salinity (Fig. 2C). Control brains showed weak immunoreaction in the pallium region (n = 22). There was however a slight increase in the cell number in 0.25% saline groups (n = 30). In the septum region, although it showed some variation in the staining pattern among the three groups, the difference was not significant. Amygdala also seemed to respond to the salinity by increasing cell number in the amygdala medialis (Am), however, the difference was not significant. In the diencephalon, Nucleus preopticus (NPO) possessed immunoreaction in all the three grades of animals, the only difference we encountered was on the dorsal side of anterior NPO. There were only a few or no cells present in this region of the control animals (Fig. 3A). The animals exposed to 0.25% salinity also possessed a few cells in these regions (Fig. 3B). However, a robust increase in the number of cells was observed in these regions of tadpole brains exposed to hyper saline (0.50%) conditions (Fig. 3C). Cells were oval and darkly stained with beaded fibers projected towards the dorsal side of the brain.
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Epithalamus displayed a robust increase in the NPY-ir cells and fibers. Both the habenularis dorsalis (NHD) and habenularis ventralis (NHV) showed strongly immunopositive cluster of cells crowded together on the ventricular side. These cells possessed long and elongated fibers, which overlaped the whole region in 50% saline tadpoles (Fig. 4C). The reaction intensity was relatively weak in the control and 0.25% salinity exposed tadpoles (Fig. 4A–C). Thalamus possessed the most evident difference in the control and stressed tadpoles. The cell number and immunostaining showed a significant contrast in the thalamus (Fig. 5A–C). NPY-ir cells with finely visible axons were observed in the nucleus posterocentral thalami (NPC) and nucleus posterolateralis thalami (NPL) of all the three grades of tadpoles (Fig. 5, A–C). However, these regions in the salinity treated tadpoles possessed more number of cells (n = 34) than the control tadpoles (n = 23). Number of the cells in the 0.25% salinity grade were mostly in between the two extreme gradients (n = 30). The cells were oval to round shaped and remained confined only to the ventral side of NPC and NPL regions. These cells further possessed long fibers going towards the lateral side (Fig. 5A–C). There was a considerable difference in the NPY-ir in the hypothalamus of control and stressed tadpoles. Exposure to the hypersaline medium increased the NPY-ir perikarya in the infundibular regions. In the anterior part of the infundibulum, there was an increase in the cells and fibers of both nucleus infundibularis ventralis (NIV) and nucleus infundibularis dorsalis (NID) regions of 0.50% saline treated tadpoles. In the posterior region also infundibulum showed more immunoreactive cells (n = 18) in the 0.50% saline tadpole (Fig. 6C) than both 0.25% saline (n = 12) and control (n = 12) animals which both possessed same number of cells (Fig. 6A, B). Cells in these regions were oval shaped with long axons moving away from the infundibular recess (IR). Although immunoreactive cells and fibers were also present in the tegmentum, Striatum griseum periventricularis tecti (SGS) of the mesencephalon and the central gray (RA), median eminence (ME) and nucleus cerebella (NCER) area of rhombencephalon, there was no considerable difference in the control and salinity exposed tadpoles. Intermediate lobe (Pi) and pars nervosa (PN) of the pituitary exhibited darkly stained fibers in all the three grades without any difference in the staining pattern between the normal and salinity treated animals. 4. Discussion Studies on the role of neuropeptides in the stress physiology of mammals have unambiguously proved that the stress related behaviours are centrally regulated by an alternating action of stress and anti-stress neuropeptides (see review Kormos and Gaszner, 2013; Morales-Medina et al., 2011). In the current work, we have demonstrated salinity stress induced changes in the expression of NPY-ir in the brain of tadpole E. Cyanophlyctis. Our study revealed an up-regulation in NPY-ir in the brain of E. cyanophlyctis tadpoles exposed to challenging salinities. Neuropeptide Y-ir perikarya increased most significantly in the pallium, septum, and preoptic area of the telencephalon and epithalamus, thalamus and hypothalamus of diencephalon. Amygdala and striatum griseum regions also exhibited a modest difference in the brain of stressed tadpoles as compared to controls. Although the other regions of the hindbrain including the tectal lamina, pituitary, raphe nucleus, median eminence and nucleus cerebella also showed immunopositive perikarya, there was no observable difference in the cell number or the dendrite staining in the control and experimental groups. The NPY distributions throughout the brain of E. cyanophlyctis tadpoles was very much similar to the localisation of NPY in other
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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Fig. 1. Transverse sections through the pallium region (MP, DP) of telencephalon of E. Cyanophlyctis tadpole brain displaying loss of immunoreactions when the antisera was preadsorbed with NPY peptide (A) and after the primary antibody was omitted from the reaction mixture. Normal immunoreactive cells after the application of anti-NPY to the sections of same region can be seen (C).
Fig. 2. Transverse section showing relative distribution of NPY peptide in the tadpole’s brain of frog E. cyanophlyctis in control and saline groups. (NPY-ir cells are shown by the arrow heads). NPY-ir cells and associated fibers are seen in the pallium (dorsal Pallium-DP and medial Pallium-MP). An increase in the immunostaining of NPY-ir perikarya can be observed in the experimental tadpoles as compared to the tadpoles fixed in normal conditions. The control section (A) has weakly stained cells lower in number (n = 22) than that of the 0.50% salinity treated tadpoles (n = 45) (C). 0.25% salinity treated tadpoles (B) also possess more cells (n = 30) than the control groups. Kruskal–Wallis value for Pallium is H = 9.915; P = 0.00703. Scale Bar, A–C, 50 lm. For abbreviation see list.
amphibians studied earlier (D’Aniello et al., 1996; Ebersole et al., 2001; Mousley et al., 2006; Tuinhof et al., 1994), However we could not observe NPY-ir in the motor nucleus of trigeminal nerve (NT) of E. Cyanophlyctis.
Most of the regions, wherein the NPY-ir displayed a significant increase following the osmotically stressful conditions, are also known to harbour CRH-containing neurons within the brain of anurans (Yao et al., 2004). These regions form the stress axis of
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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Fig. 3. Transverse section showing distribution of NPY in the tadpole’s brain of frog E. cyanophlyctis exposed to different salinities (NPY-ir cells are shown by arrow heads). Sections through the anterior part of the NPO in the dorsal side reveal a major increase following the exposure to higher salinities. There are only a few cells (n = 1) in the control groups (A). Cell number increases slightly (n = 2) in the 0.25% salinity grades (B). However when the tadpoles are exposed to 0.50% salt concentration of the water medium (C), there is a robust increase in the number of cells (n = 11). Kruskal–Wallis value for Pallium is H = 9.42; P = 0.009005. Scale Bar, A–C, 50 lm. For abbreviation see list.
Fig. 4. Immunohistochemical photomicrographs through the epithalamus of the tadpole brain of E. cyanophlyctis showing relative NPY-ir when exposed to different salinity grades (NPY-ir cells are shown by the arrow heads). A dense cluster of cells with their fibers projecting towards the periphery can be seen in the 0.50% salinity treated tadpoles (C). However control (A) and 0.25% grade possess comparatively less immunostaining. Both the hebenular areas (NHV and NHD) possess a robust staining in 0.50% salinity treated tadpoles as compared to control. Kruskal–Wallis value for pallium, septum medialis and nucleus preoptics are H = 9.42; P = 0.009005. Scale Bar, A–C, 50 lm. For abbreviation see list.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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Fig. 5. Transverse section showing relative NPY-ir in the thalamus of the tadpole brain of E. cyanophlyctis exposed to different salinities (NPY-ir cells are shown by arrow heads). Given in the figure are the nucleus posterocentralis thalami (NPC) and nucleus posterolateralis thalami of (NPL) Thalamus. Sections reveal an increase in the NPY-ir with the increase in the salinity of the medium. NPC and NPL regions of the control possess a cluster of cells scattered throughout the ventral region of NPC (n = 19) and NPL possesses only a few cells (n = 4). There are more cells (n = 20,10) in the 0.25% salinity treated animals(B). However 0.50% salinity treated animals show the highest cell count (n = 30,4) in the respective areas (C). Kruskal–Wallis value for pallium, septum medialis and nucleus preoptics are H = 9.555; P = 0.006036. Scale Bar, A–C, 50 lm. For abbreviation see list.
Fig. 6. Transverse sections through the brain of E. Cyanophlyctis depicting the comparative NPY immunostaining in control and salinity treated animals (NPY-ir cells are shown by arrow heads). Sections through the infundibulum display variation in the cell number in the animals exposed to different salinities. Nucleus infundibular ventralis (NIV) region of the 0.50% salinity grade possess more number of cells(C) the 0.25% salinity treated animals (B). There are only few cells visible in the parallel sections of control tadpoles (A). Kruskal–Wallis value for NIV is H = 10.22 and P = 0.006036. The same superscripts over the bars represent the data which are not significantly different. Scale Bar A–C 100 lm. For abbreviation see list.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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the brain which has been found to be conserved throughout the vertebrates (Dedovic et al., 2009; Alldredge, 2010). Salinity stress also induced an increase in the NPY-ir in the pallium and amygdala, which are well known to play a crucial role in the stress and anxiety modulation in mammals (Kubo et al., 2004; Saha et al., 2000; Cullinan et al., 1995). Furthermore, we observed a significant increase in NPY-ir in the NPO of salinity-treated tadpoles, a region which is homologous to PVN in the rat hypothalamus, a principle site for neuroendocrine stress responses (Yao et al., 2004; Ziegler and Herman, 2002). Similarly, Yao et al. (2004) observed an increased expression of anti-stress peptides in the NPO of juvenile frogs, and suggested its role in stress modulation of amphibians. Presence of NPY-ir in the regions associated with stress regulation and its salinity induced increase in our study establishes the role of NPY in modulating the consequences of osmotic stress in amphibians. Furthermore, Epithalamus (NHV, NHD), Thalamus (NPC, NPL) and hypothalamus (NIV, NID) are also involved in the behavioural responses to the stress, pain and anxiety in mammals. (Wirtshafter et al., 1994; Heldt et al., 2006; Dedovic et al., 2009). Therefore, increase in NPY immunoreactivity in these areas also supports its role as an anti-stress neurotransmitter in anuran brain. Salinity-induced increase in the NPY-ir in magnocellular neurons of hypothalamic supraoptic (SON) and periventicular (PVN) nuclei of rats have been reported by Larsen, et al. in 1992. Increase in the NPY-ir in the supra-optic neurons of magnocellular hypothalamus in vasopressin deficient homozygous Brattleboro (di/di) rats, against the control long evans rats has also been reported (Bundzikova et al., 2008, 2010). Hooi et al. (1989) demonstrated a close anatomical relationship between nerve terminals containing NPY and vasopressin (ADH) in the hypothalamic paraventricular (PVN) and supraoptic nuclei of mammalian brain. The overall distribution of NPY-ir in our study also correlates with the distribution of vasopressin-ergic cell bodies in the amphibian brain (see review Moore and Lowry, 1998) suggesting its involvement in the regulation of endogenous salt adaptation. Our results also display an increase of NPY-ir in the infundibular (NIV, NID) and pre-optic nuclei which are analogous to the magnocellular neurons in mammals. Furthermore, our observations draw a strong support from the fact that injections of NPY into the SON increased plasma concentrations of arginine vasopressin (ADH)-an endogenous hormone which maintains body osmolality-in the rat (Hooi et al., 1989). Thus, based on our findings we suggest that NPY may contribute to the pathways known to mediate the autonomic and endocrine responses to osmotic changes in the environment. Acknowledgments Authors acknowledged the financial Grant from Department of Science and Technology (DST), Government of India, New Delhi, India. Authors are grateful to Dr. N.K. Subhedar (IISER Pune, India) for his valuable suggestions. Ishfaq Ali thanks DST for providing junior research fellowship. We thank Amul sakharkar for proofreading the manuscript and Swapnil Shewale for statistical analysis. References Ali, I., Bhargava, S., 2016. Neuropeptide Y in the brain of Euphlyctis cyanophlyctis tadpoles responds to hypoxic stress. Gen. Comp. Endocrinol. Alldredge, B., 2010. Pathogenic involvement of neuropeptides in anxiety and depression. Neuropeptides 44, 215–224. Beck, B., Pourié, G., 2014. Ghrelin, neuropeptide Y, and other feeding-regulatory peptides active in the hippocampus: role in learning and memory. Nutr. Rev. 71 (8), 541–561. http://dx.doi.org/10.1111/nure.12045. Bentley, P.J., 2002. The amphibia; endocrines and osmoregulation, a comparative account in vertebrates second ed. of vol. 1. (Chapter 6). In: Bradshaw, S.D.,
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Burggren, W., Heller, H.C., Ishii, S., Langer, H., Neuweiler, G., Randall, D.J. (Eds.), Zoophysiology, 39. Springer-Verlag, Berlin, Heidelberg, pp. 155–186. Brady, L.D., Griffiths, R.A., 2000. Developmental responses to pond desiccation in tadpoles of the British anuran amphibians (Bufo bufo, B. calamita and Rana temporaria). J. Zool. 252 (1), 61–69. Bundzikova, J., Pirnik, Z., Zelena, D., Mikkelsen, J.D., Kiss, A., 2008. Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats. Cell. Mol. Neurobiol. 28 (8). Bundzikova, J., Pirnik, Z., Zelena, D., Mikkelsen, J.D., Kiss, A., 2010. The alpha2adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons in the long evans and brattleboro rats. J. Physiol. Pharmacol. 61 (4). Colmers, W., Wahlestedt, C., 1993. The biology of neuropeptide Y and related peptides. Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of corticotropin releasing factor, neuropeptide Y and corticosterone in the regulation of food intake In Xenopus laevis. J. Neuroendocr. 16 (3), 279–288. Cullinan, W.E., Herman, J.P., Battaglia, D.F., Akil, H., Watson, S.J., 1995. Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64, 477–505. D’Aniello, B., Vallarino, M., Pinelli, C., Fiorentino, M., Rastogi, R., 1996. Neuropeptide Y: localisation in the brain and pituitary of the developing frog (Rana esculenta). Cell Tissue Res. 285, 253–259. Dedovic, K., Duchesne, A., Andrews, J., Engert, V., Pruessner, J.C., 2009. The brain and the stress axis: the neural correlates of cortisol regulation in response to stress. Neuroimage 47, 864–871. Ebersole, T.J., Conlon, J.M., Goetz, F.W., Boyd, S.K., 2001. Characterization and distribution of neuropeptide Y in the brain of a caecilian amphibian. Peptides 22 (3), 325–334. Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16 (3), 183–190. Griffiths, R.A., 1997. Temporary ponds as amphibian habitats. Aquat. Conserv. Mar. Freshwater Ecosyst. 7 (2), 119–126. Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electronica 4 (1), 9. Heilig, M., 2004. NPY in stress, anxiety and depression. Neuropeptides 38, 213–224. Heldt, S.A., Ressler, K.J., 2006. Lesions of the habenula produce stress-and dopamine-dependent alterations in prepulse inhibition and locomotion. Brain Res. 1073, 229–239. Hoar, W.S., 1988. The physiology of smelting salmonoids. In: Hoar, W.S., Randall, D. J. (Eds.), Fish Physiology. Academic Press, San Diego, pp. 275–343. Hooi, S.C., Richardson, G.S., McDonald, J.K., Allen, J.M., Martin, J.B., Koenig, J.I., 1989. Neuropeptide Y (NPY) and vasopressin (AVP) in the hypothalamoneurohypophysial axis of salt-loaded or Brattleboro rats. Brain Res. 486, 21– 220. Hopkins, G.R., Brodie Jr, E.D., Neuman-Lee, L.A., Mohammadi, S., Brusch IV, G.A., Hopkins, Z.M., French, S.S., 2016. Physiological responses to salinity vary with proximity to the ocean in a coastal amphibian. Physiol. Biochem. Zool. 89 (4). Hopkins, G.R., Brodie Jr, E.D., 2015. Occurrence of amphibians in saline habitats: a review and evolutionary perspective. Herpetological Monogr. 29 (1), 1–27. Khan, M.S., 1997. A new subspecies of common skittering frog Euphlyctis. Pak. J. Zool. 29 (2), 107–112. Khan, M.S., 2014. The filter feeding mechanism in tadpoles. Bull. Chicago Herpetological Soc. 49 (8), 116–119. Kormos, V., Gaszner, B., 2013. Role of neuropeptides in anxiety, stress, and depression: from animals to humans. Neuropeptides 47, 401–419. Kubo, T., Okatani, H., Nishigori, Y., Hagiwara, Y., Fukumori, R., Goshima, Y., 2004. Involvement of the medial amygdaloid nucleus in restraint stress-induced pressor responses in rats. Neurosci. Lett. 354 (1), 84–86. Larhammar, D., Söderberg, C., Blomquist, A.G., 1993. Evolution of the neuropeptide Y family of peptides. In the Biology of Neuropeptide Y and Related Peptides. Humana Press, pp. 1–41. Larsen, P.J., Sheikh, S.P., Mikkelsen, J.D., 1992. Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway. Brain Res. 573, 181–189. Larsson, T.A., Olsson, F., Sundstrom, G., Lundin, L.G., Brenner, S., Venkatesh, B., Larhammar, D., 2008. Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions. BMC Evol. Biol. 8 (1), 1. Lopez, J.M., Gonzalez, A., 2002. Ontogeny of NADPH diaphorase/nitric oxide synthase reactivity in the brain of Xenopus laevis. J. Comp. Neurol. 445, 59–77. Luo, Y., Kaur, C., Ling, E., 2000. Hypobaric hypoxia induces Fos and neuronal nitric oxide synthase expression in the paraventricular and supraoptic nucleus in rats. Neurosci. Lett. 296 (2–3), 145–148. Mathieu, M., Yon, L., Charifou, I., Trabucchi, M., Vallarino, M., Pinelli, C., Fournier, A., Rastogi, R.K., Vaudry, H., 2001. Ontogeny of pituitary adenylate cyclaseactivating polypeptide (PACAP) in the frog (Rana ridibunda) tadpole brain: immunohistochemical localization and biochemical characterization. J. Comp. Neurol. 431 (1), 11–27. McCormick, S.D., Bradshaw, D., 2006. Hormonal control of salt and water balance in vertebrates. Gen. Comp. Endocrinol. 147 (1), 3–8. Moore, F.L., Lowry, C.A., 1998. Comparative neuroanatomy of vasotocin and vasopressin in amphibians and other vertebrates. Comp. Biochem. Physiol. C: Pharmacol. Toxicol. Endocrinol. 119 (3), 251–260. Morales-Medina, J.C., Dumont, Y., Quirion, R., 2011. A possible role of neuropeptide Y in depression and stress. Brain Res. 1314, 194–205.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
8
E. Heigrujam et al. / General and Comparative Endocrinology xxx (2017) xxx–xxx
Mousley, A., Polese, G., Marks, N.J., Eisthen, H.L., 2006. Terminal nerve-derived neuropeptide y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum). J. Neurosci. 26 (29), 7707–7717. Neary, T.J., Northcutt, R.G., 1983. Nuclear organization of the bullfrog diencephalon. J. Comp. Neurol. 213, 262–278. Saha, S., Batten, T.F.C., Henderson, Z., 2000. A GABA-ergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study. Neuroscience 99, 613–626. Shewale, S.A., Gaupale, T.C., Bhargava, S., 2014. Temperature dependent changes in cocaine- and amphetamine regulated transcript (CART) peptide in the brain of tadpole, Sylvirana temporalis. Gen. Comp. Endocrinol. 220, 61–69. Shoemaker, V.H., Nagy, K.A., 1977. Osmoregulation in amphibians and reptiles. Annu. Rev. Physiol. 39, 449–471. Tuinhof, R., Gonzalez, A., Smeets, W.J.A.J., Roubos, E.W., 1994. Neuropeptide Y in the developing and adult brain of the South African clawed toad Xenopus laevis. J. Chem. Neuroanat. 7, 271–283. Uchiyama, M., Maejima, S., Wong, M.K.S., et al., 2014. Changes in plasma angiotensin II, aldosterone, arginine vasotocin, corticosterone, and electrolyte
concentrations during acclimation to dry condition and seawater in the crabeating frog. Gen. Comp. Endocrinol. 195, 40–46. Uchiyama, M., Konno, N., 2006. Review: hormonal regulation of ion and water transport in anuran amphibians. Gen. Comp. Endocrinol. 147, 54–61. Wada, M., Urano, A., Gorbman, A., 1980. Stereotaxic atlas for diencephalon nuclei of the frog Rana pipiens. Arch. Histol. Jpn. 43, 157–173. Wirtshafter, D., Asin, K.E., Pitzer, M.R., 1994. Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula. Brain Res. 633, 21–26. Wu, G., Feder, A., Wegener, G., Bailey, C., Saxena, S., Charney, D., Mathé, A.A., 2011. Central functions of neuropeptide Y in mood and anxiety disorders. Expert Opin. Ther. Targets 15, 1317–1331. Yao, M., Westphal, N.J., Denver, R.J., 2004. Distribution and acute stressorinduced activation of corticotrophin-releasing hormone neurones in the central nervous system of Xenopus laevis. J. Neuroendocrinol. 16, 880– 893. Ziegler, D.R., Herman, J.P., 2002. Neurocircuitry of stress integration: anatomical pathways regulating the hypothalamo–pituitary–adrenocortical axis of the rat. Integr. Comp. Biol. 42, 541–551.
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NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress Elizabeth Heigrujam 1, Ishfaq Ali 1, Shobha Bhargava ⇑ Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India
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Article history: Received 1 April 2016 Revised 6 January 2017 Accepted 7 January 2017 Available online xxxx Keywords: Neuropeptide Y Salinity stress E. cyanophlyctis Brain Immunohistochemistry
a b s t r a c t Most of the amphibians breed in temporary ponds vulnerable to occasional desiccation, thus, leaving their larvae exposed to stressful fluctuations in various environmental parameters including salinity. These animals possess a well suited central adaptive mechanism to adapt to these alterations. Neuropeptide Y (NPY), a 36 amino acid neurotransmitter, has been reported to antagonize various neuropsychological consequences of stress within the mammalian brain. Osmotic regulation of NPY in the hypothalamo-neurohypophysial pathway of mammalian brain is also known. Although the molecule possesses an extensive distribution in the brain of amphibians, its functional association is not well understood. We have investigated the endogenous response of NPY-ergic system to osmotically stressful conditions in the brain of Indian skipper frog-Euphlyctis cyanophlyctis tadpoles. Using Immunohistochemistry, we observed an up-regulation of NPY immunoreactivity (NPY-ir) in the brain of tadpoles exposed to stressful salt concentrations. A significant increase of NPY-ir occurred in the pallium and septum regions of telencephalon; preoptic area, epithalamic, thalamic and hypothalamic parts of diencephalon. Most of the regions are implicated in the modulation of stress and anxiety related brain functions and have also been shown to respond to the salinity stress in mammals. In addition, NPY producing neurons in pre-optic and hypothalamic parts show a close co-existence with the vasopressin-ergic neurons. Thus, our study suggests a possible role of NPY in stabilizing the neuro-endocrinological consequences of osmotic stress in an amphibian brain. Ó 2017 Elsevier Inc. All rights reserved.
1. Introduction Euphlyctis cyanophlyctis, a dicrogloccid frog, inhabits diverse habitats in south Asia and is known to tolerate a wide range of salt water concentrations (Khan, 1997; Hopkins and Brodie, 2015). The animal breeds in temporary ponds, vulnerable to occasional desiccation and frequent fluctuations in different environmental
Abbreviations: ADH, vasopressin; Al, amygdala lateralis; Am, amygdala medialis; CNS, central nervous system; CRH, corticotropin-releasing hormone; DP, dorsal pallium; IR, infundibular recess; LP, lateral pallium; ME, median eminence; MP, medial pallium; NCER, nucleus cerebella; NGS, normal goat serum; NHD, nucleus habenularis dorsalis; NHV, nucleus habenularis ventralis; NID, nucleus infundibularis dorsalis; NIV, nucleus infundibularis ventralis; NMS, nucleus medialis septi; NPC, nucleus posterocentralis thalami; NPL, nucleus posterolateralis thalami; NPO, nucleus preopticus; NPY, neuropeptide Y; NT, motor nucleus of the trigeminal nerve; OT, optic tectum; PR, preoptic recess; Pi, pars intermedia hypophysis; PN, pars nervosa hypophysis; PVN, paraventricular nucleus; RA, raphe nucleus; SGS, stratum griseum superficiale tecti; SON, supraoptic nuclei. ⇑ Corresponding author. E-mail address: [email protected] (S. Bhargava). 1 Equal contribution of the authors.
parameters including salinity (Khan, 2014; Griffiths, 1997; Brady and Griffiths, 2000). The larval stage persists for 3 months during which they face a steep rise and fall in the salt concentrations (Khan, 2014). In order to provide stability, a series of physiological, neuro-endocrinological, and biochemical responses integrate to maintain homeostasis (Hoar, 1988; Hopkins et al., 2016; McCormick and Bradshaw, 2006). This is mainly achieved by a well coordinated orchestration of various interrelated hormonal systems including hypothalamo–neurohypophysial system (see review by Uchiyama and Konno, 2006; Uchiyama et al., 2014). Since CNS along with the olfactory system intermediates the environmental stimulus and various response mechanisms inside the body, it is of paramount importance to find out the brain areas involved in the modulation of these changes. Here, we have tried to evaluate the neuro-anatomical response of endogenous neuropeptide Y (NPY) – a 36 amino acid neuromodulator known to resilience stress in the brain of mammals– during osmotic stress in the brain E. cyanophlyctis tadpoles. Neuropeptide Y (NPY) has been known as an important component of stress antagonising peptides in the brain of mammals
http://dx.doi.org/10.1016/j.ygcen.2017.01.015 0016-6480/Ó 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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(Heilig, 2004; Morales-Medina et al., 2011). The peptide possesses a remarkable conservation in its structure (up to 93%) and function throughout the vertebrates (Colmers and Wahlestedt, 1993; Larhammar et al., 1993; Larsson et al., 2008). Based mainly on its neuro-anatomical localisation, various roles have been assigned to the peptide (Wu et al., 2011; Beck et al., 2014; Ali et al., 2016). Although the peptide has also been shown to possess an extensive distribution throughout the brain of amphibians (D’Aniello et al., 1996; Ebersole et al., 2001; Mousley et al., 2006; Tuinhof et al., 1994), its functional relevance remains largely unknown. Role of NPY in mediating the physiological consequences of osmotic stress is well established in mammals. Hooi et al., in 1989 showed the involvement of NPY in the fluid homeostasis in rat, following the close anatomical relationship between nerve terminals containing NPY and vasopressin in the hypothalamic paraventricular (PVN) and supra-optic nucleus (SON) of rat brain. Co-existence of NPY and vasopressin expressing neurons and an up-regulation of NPY in the magnocellular hypothalamoneurohypophysial system after salt loading in rat has also been reported (Larsen et al., 1992). An increase in the NPY-ir in the supra-optic neurons of magnocellular hypothalamus in vasopressin deficient homozygous Brattleboro (di/di) rats, against the control long evans rats, is also shown (Bundzikova et al., 2008, 2010). Despite all these evidences about NPY response to salt adaptation in terrestrial animals, no such study has been reported in the animals living in aquatic habitats where they experience a wide range of changes in the salt concentration (Bentley, 2002; Shoemaker and Nagy, 1977). Since these animals are exposed to osmotic fluctuations in their natural habitat, it is interesting to look for the underlying neurological mechanisms mediating the central responses to osmotic stress. Herein, we investigate the relative neuroanatomical response of NPY in the developing brain of the tadpoles of Euphlyctis cyanophlyctis exposed to various grades of salinities.
Animals were kept in the potential hyperosmotic solutions for 4 h which was done in accordance to the earlier reports of stress induced peptide release in the tadpoles of same stage (Yao et al., 2004; Luo et al., 2000; Ali et al., 2016). After 4 h, the tadpoles in each solution were anesthetized using 2-phenoxyethanol, brains were dissected and fixed in Bouin’s fixative for 24 h. This was followed by the dehydration of the tissue with increased grades-10% (2 h), 20% (2 h) and 30% (overnight at 4 °C) of sucrose in phosphate buffer saline (PBS, 0.01 M, pH 7.4) solution for cryo-protection. The tissues were later embedded in Shandon cryomatrix and cut on a cryostat (Lieca CM 1520) in the transverse plane at a thickness of 20 lm. Sections were mounted on poly-L-lysine coated slides and stored in 20 °C until immunohistochemical staining.
2. Material and methods
2.3. Characterization of antibody
2.1. Animal collection and tissue processing
The monoclonal primary antibody against NPY used in the present study was obtained from Sigma Aldrich. The peptide has been reported to possess a 93% homology throughout the vertebrates (Colmers and Wahlestedt, 1993; Larhammar et al., 1993; Larsson et al., 2008; Crespi et al., 2004). The anti-NPY antibody used in the study was against the immunogen synthetic neuropeptide Y (NPY, Porcine-N9528) was produced in rabbit and used at a dilution of 1:5000. Prior to its usage, the antibody was characterized for its specificity in the frog brain. Various control procedures were performed to test its cross-reactivity. Sections were incubated with diluted antibodies pre-adsorbed with the peptide to determine the specificity. Omission of the primary or secondary antibodies from the reaction mixture also showed no immunoreactions.
Lower developmental stages (stage 35–37) of Euphlyctis cyanophlyctis tadpoles were collected from the fields of Taleghar located in the Ghat regions of Sahyadri hills in Maharashtra, India. Animals were kept in glass chambers of 30 45 10 cm3 and were fed with ad libitum boiled spinach until stage 38 (Gosner, 1960). Pre-metamorphic stage (38–39) was used for study mainly because there are no major developmental instincts, anatomical or morphological changes in this the stage. And the physiological processes of the animal, at this stage, are also considered to be stabilised (Gosner, 1960). The animals were treated in accordance to the ethical guidelines for animal usage established by Savitribai Phule Pune University, Pune, India. In the natural conditions the frog breeds in fresh water with an average salinity of 0.013% (130 ppm). Since, the larval forms in Euphlyctis cyanophlyctis persists for 3 months, the salinity of these temporary ponds, due to desiccation and accumulation of salts, in summer shoots up to 0.2% (2000 ppm) or even higher. In the laboratory, the animals were acclimatised to tap water with 100 ppm salt concentration. The animals were further acclimatised to room temperature and 12 h day/night cycle. Tadpoles (n = 5) each were taken and exposed to increased gradients of salt concentrations viz, 0.25% and 0.50% based on threshold tolerance of the animal derived earlier. 5n tadpoles already acclimatised to the tap water were fixed and served as control.
2.2. Immunohistochemistry Sections were used to check down the possible alterations in expression of NPY in normal and stressed animals following the standard immunohistochemical protocol (Shewale et al., 2014; Ali et al., 2016). Briefly, the steps included the 0.3% hydrogen peroxide in methanol treatment for 1 h which was followed by hour long incubation with blocking agent containing 0.5% BSA (Bovine serum albumin) and 0.5% gelatin in PBS. PBS washes were given after every step. Sections were afterwards incubated for 1 h in NGS (1:40 dilution, Vectastain) and immediately kept for an overnight (16 h) incubation at 4 °C with Rabbit monoclonal antibody against NPY (1:5000) containing 0.5% BSA and gelatine. On the second day, the addition of secondary antibody- biotinylated goat anti-mouse IgG antibody at room temperature (1:200, Vectastain)- and ABC reagent (Vectastain, ABC Kit, 1:100) followed after consecutive buffer (PBS) washes. Sections were incubated with 3,3 diaminobenzidine tetra hydrochloride (DAB) solution (0.05 M, pH 7.2 Tris buffer and 0.02% H2O2) for 8–10 min and then subsequently washed in distilled water, dehydrated in alcohol, cleared in xylene, mounted in distyrene plasticizer xylene (Merck, India) and photographed for further observation.
2.4. Morphometry Digital images of NPY immunoreactivity were obtained on a Nikon Inverted Microscope equipped with a Nikon DS-Ri2 Microscope camera including Nikon Basic Research software (NIS elements) Sections through the same region of the brain using neuro-anatomical landmarks were used for analysis (Neary and Northcutt, 1983; Wada et al., 1980). For cell counting, all visible cell bodies stained within the defined brain regions were counted manually (using Image J software), keeping the same counting area for control. Data from each brain region in an animal was calculated by taking the average counts from five brain slices. Further,
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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the average counts from the left and right sides of brain at specific brain regions of interest were also considered.
2.5. Statistical analysis In each treatment different areas of the brain and their cell counts were plotted as box plots. The comparative analysis in the cell counts of normal and salinity treated groups was tested using Kruskal–Wallis test. A post hoc analysis using Mann–Whitney U test with Bonferroni correction was performed to check the difference between the controls and experimental samples. All statistical analysis were done in freeware PAST version 2.14 (Hammer et al., 2001).
3. Results Assay for relative expression of NPY in the brain of Euphlyctis cyanophlyctis tadpoles exposed to different gradients of salt concentrations revealed an up-regulation of NPY-ir in various regions of the animal brain. Antisera directed against the NPY antigen was checked for its specificity prior to any experimentation. Omission of primary or secondary antibody from the protocol did not develop any immunoreaction (Fig. 1B). Pre-adsorption of primary antibody with pure NPY peptide also abolished immunostaining (Fig. 1A). In response to salinity stress, a significant increase in the expression of NPY immunoreactivity was shown by various regions of the tadpole brain including telencephalon, diencephalon and some parts of mesencephalon. The neuro-anotomical terminology and identification of different regions was adopted from the stereotaxic atlas of various developmental stages of frog brain (Lopez and Gonzalez, 2002; Mathieu et al., 2001; Neary and Northcutt, 1983; Wada et al., 1980). Although there was an evident increase in the NPY-ir among the control and 0.50% saline groups; some of the corresponding regions of the control and 0.25% salinity grades exhibited almost same immunostaining. Most of the immunoreactive material comprised of the axonal processes. In some regions, cells were round to oval shaped with beaded fibers. Given below is the differential account of NPY-ir observed in different regions of frog brain exposed to increasing concentrations of salt medium. In the telencephalon, salinity induced increase in the NPY-ir perikarya was seen in the pallium and septum regions. (Fig. 2 A–C). A significant increase was observed in the immunoreactive perikarya of the stressed animals as compared to controls. In the pallium region, NPY-ir cells increased in the dorsal pallium (DP) (Number of cells: n = 21) and medial pallium (MP) (n = 24) in the tadpole brains exposed to 0.50% salinity (Fig. 2C). Control brains showed weak immunoreaction in the pallium region (n = 22). There was however a slight increase in the cell number in 0.25% saline groups (n = 30). In the septum region, although it showed some variation in the staining pattern among the three groups, the difference was not significant. Amygdala also seemed to respond to the salinity by increasing cell number in the amygdala medialis (Am), however, the difference was not significant. In the diencephalon, Nucleus preopticus (NPO) possessed immunoreaction in all the three grades of animals, the only difference we encountered was on the dorsal side of anterior NPO. There were only a few or no cells present in this region of the control animals (Fig. 3A). The animals exposed to 0.25% salinity also possessed a few cells in these regions (Fig. 3B). However, a robust increase in the number of cells was observed in these regions of tadpole brains exposed to hyper saline (0.50%) conditions (Fig. 3C). Cells were oval and darkly stained with beaded fibers projected towards the dorsal side of the brain.
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Epithalamus displayed a robust increase in the NPY-ir cells and fibers. Both the habenularis dorsalis (NHD) and habenularis ventralis (NHV) showed strongly immunopositive cluster of cells crowded together on the ventricular side. These cells possessed long and elongated fibers, which overlaped the whole region in 50% saline tadpoles (Fig. 4C). The reaction intensity was relatively weak in the control and 0.25% salinity exposed tadpoles (Fig. 4A–C). Thalamus possessed the most evident difference in the control and stressed tadpoles. The cell number and immunostaining showed a significant contrast in the thalamus (Fig. 5A–C). NPY-ir cells with finely visible axons were observed in the nucleus posterocentral thalami (NPC) and nucleus posterolateralis thalami (NPL) of all the three grades of tadpoles (Fig. 5, A–C). However, these regions in the salinity treated tadpoles possessed more number of cells (n = 34) than the control tadpoles (n = 23). Number of the cells in the 0.25% salinity grade were mostly in between the two extreme gradients (n = 30). The cells were oval to round shaped and remained confined only to the ventral side of NPC and NPL regions. These cells further possessed long fibers going towards the lateral side (Fig. 5A–C). There was a considerable difference in the NPY-ir in the hypothalamus of control and stressed tadpoles. Exposure to the hypersaline medium increased the NPY-ir perikarya in the infundibular regions. In the anterior part of the infundibulum, there was an increase in the cells and fibers of both nucleus infundibularis ventralis (NIV) and nucleus infundibularis dorsalis (NID) regions of 0.50% saline treated tadpoles. In the posterior region also infundibulum showed more immunoreactive cells (n = 18) in the 0.50% saline tadpole (Fig. 6C) than both 0.25% saline (n = 12) and control (n = 12) animals which both possessed same number of cells (Fig. 6A, B). Cells in these regions were oval shaped with long axons moving away from the infundibular recess (IR). Although immunoreactive cells and fibers were also present in the tegmentum, Striatum griseum periventricularis tecti (SGS) of the mesencephalon and the central gray (RA), median eminence (ME) and nucleus cerebella (NCER) area of rhombencephalon, there was no considerable difference in the control and salinity exposed tadpoles. Intermediate lobe (Pi) and pars nervosa (PN) of the pituitary exhibited darkly stained fibers in all the three grades without any difference in the staining pattern between the normal and salinity treated animals. 4. Discussion Studies on the role of neuropeptides in the stress physiology of mammals have unambiguously proved that the stress related behaviours are centrally regulated by an alternating action of stress and anti-stress neuropeptides (see review Kormos and Gaszner, 2013; Morales-Medina et al., 2011). In the current work, we have demonstrated salinity stress induced changes in the expression of NPY-ir in the brain of tadpole E. Cyanophlyctis. Our study revealed an up-regulation in NPY-ir in the brain of E. cyanophlyctis tadpoles exposed to challenging salinities. Neuropeptide Y-ir perikarya increased most significantly in the pallium, septum, and preoptic area of the telencephalon and epithalamus, thalamus and hypothalamus of diencephalon. Amygdala and striatum griseum regions also exhibited a modest difference in the brain of stressed tadpoles as compared to controls. Although the other regions of the hindbrain including the tectal lamina, pituitary, raphe nucleus, median eminence and nucleus cerebella also showed immunopositive perikarya, there was no observable difference in the cell number or the dendrite staining in the control and experimental groups. The NPY distributions throughout the brain of E. cyanophlyctis tadpoles was very much similar to the localisation of NPY in other
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Fig. 1. Transverse sections through the pallium region (MP, DP) of telencephalon of E. Cyanophlyctis tadpole brain displaying loss of immunoreactions when the antisera was preadsorbed with NPY peptide (A) and after the primary antibody was omitted from the reaction mixture. Normal immunoreactive cells after the application of anti-NPY to the sections of same region can be seen (C).
Fig. 2. Transverse section showing relative distribution of NPY peptide in the tadpole’s brain of frog E. cyanophlyctis in control and saline groups. (NPY-ir cells are shown by the arrow heads). NPY-ir cells and associated fibers are seen in the pallium (dorsal Pallium-DP and medial Pallium-MP). An increase in the immunostaining of NPY-ir perikarya can be observed in the experimental tadpoles as compared to the tadpoles fixed in normal conditions. The control section (A) has weakly stained cells lower in number (n = 22) than that of the 0.50% salinity treated tadpoles (n = 45) (C). 0.25% salinity treated tadpoles (B) also possess more cells (n = 30) than the control groups. Kruskal–Wallis value for Pallium is H = 9.915; P = 0.00703. Scale Bar, A–C, 50 lm. For abbreviation see list.
amphibians studied earlier (D’Aniello et al., 1996; Ebersole et al., 2001; Mousley et al., 2006; Tuinhof et al., 1994), However we could not observe NPY-ir in the motor nucleus of trigeminal nerve (NT) of E. Cyanophlyctis.
Most of the regions, wherein the NPY-ir displayed a significant increase following the osmotically stressful conditions, are also known to harbour CRH-containing neurons within the brain of anurans (Yao et al., 2004). These regions form the stress axis of
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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Fig. 3. Transverse section showing distribution of NPY in the tadpole’s brain of frog E. cyanophlyctis exposed to different salinities (NPY-ir cells are shown by arrow heads). Sections through the anterior part of the NPO in the dorsal side reveal a major increase following the exposure to higher salinities. There are only a few cells (n = 1) in the control groups (A). Cell number increases slightly (n = 2) in the 0.25% salinity grades (B). However when the tadpoles are exposed to 0.50% salt concentration of the water medium (C), there is a robust increase in the number of cells (n = 11). Kruskal–Wallis value for Pallium is H = 9.42; P = 0.009005. Scale Bar, A–C, 50 lm. For abbreviation see list.
Fig. 4. Immunohistochemical photomicrographs through the epithalamus of the tadpole brain of E. cyanophlyctis showing relative NPY-ir when exposed to different salinity grades (NPY-ir cells are shown by the arrow heads). A dense cluster of cells with their fibers projecting towards the periphery can be seen in the 0.50% salinity treated tadpoles (C). However control (A) and 0.25% grade possess comparatively less immunostaining. Both the hebenular areas (NHV and NHD) possess a robust staining in 0.50% salinity treated tadpoles as compared to control. Kruskal–Wallis value for pallium, septum medialis and nucleus preoptics are H = 9.42; P = 0.009005. Scale Bar, A–C, 50 lm. For abbreviation see list.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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Fig. 5. Transverse section showing relative NPY-ir in the thalamus of the tadpole brain of E. cyanophlyctis exposed to different salinities (NPY-ir cells are shown by arrow heads). Given in the figure are the nucleus posterocentralis thalami (NPC) and nucleus posterolateralis thalami of (NPL) Thalamus. Sections reveal an increase in the NPY-ir with the increase in the salinity of the medium. NPC and NPL regions of the control possess a cluster of cells scattered throughout the ventral region of NPC (n = 19) and NPL possesses only a few cells (n = 4). There are more cells (n = 20,10) in the 0.25% salinity treated animals(B). However 0.50% salinity treated animals show the highest cell count (n = 30,4) in the respective areas (C). Kruskal–Wallis value for pallium, septum medialis and nucleus preoptics are H = 9.555; P = 0.006036. Scale Bar, A–C, 50 lm. For abbreviation see list.
Fig. 6. Transverse sections through the brain of E. Cyanophlyctis depicting the comparative NPY immunostaining in control and salinity treated animals (NPY-ir cells are shown by arrow heads). Sections through the infundibulum display variation in the cell number in the animals exposed to different salinities. Nucleus infundibular ventralis (NIV) region of the 0.50% salinity grade possess more number of cells(C) the 0.25% salinity treated animals (B). There are only few cells visible in the parallel sections of control tadpoles (A). Kruskal–Wallis value for NIV is H = 10.22 and P = 0.006036. The same superscripts over the bars represent the data which are not significantly different. Scale Bar A–C 100 lm. For abbreviation see list.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
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the brain which has been found to be conserved throughout the vertebrates (Dedovic et al., 2009; Alldredge, 2010). Salinity stress also induced an increase in the NPY-ir in the pallium and amygdala, which are well known to play a crucial role in the stress and anxiety modulation in mammals (Kubo et al., 2004; Saha et al., 2000; Cullinan et al., 1995). Furthermore, we observed a significant increase in NPY-ir in the NPO of salinity-treated tadpoles, a region which is homologous to PVN in the rat hypothalamus, a principle site for neuroendocrine stress responses (Yao et al., 2004; Ziegler and Herman, 2002). Similarly, Yao et al. (2004) observed an increased expression of anti-stress peptides in the NPO of juvenile frogs, and suggested its role in stress modulation of amphibians. Presence of NPY-ir in the regions associated with stress regulation and its salinity induced increase in our study establishes the role of NPY in modulating the consequences of osmotic stress in amphibians. Furthermore, Epithalamus (NHV, NHD), Thalamus (NPC, NPL) and hypothalamus (NIV, NID) are also involved in the behavioural responses to the stress, pain and anxiety in mammals. (Wirtshafter et al., 1994; Heldt et al., 2006; Dedovic et al., 2009). Therefore, increase in NPY immunoreactivity in these areas also supports its role as an anti-stress neurotransmitter in anuran brain. Salinity-induced increase in the NPY-ir in magnocellular neurons of hypothalamic supraoptic (SON) and periventicular (PVN) nuclei of rats have been reported by Larsen, et al. in 1992. Increase in the NPY-ir in the supra-optic neurons of magnocellular hypothalamus in vasopressin deficient homozygous Brattleboro (di/di) rats, against the control long evans rats has also been reported (Bundzikova et al., 2008, 2010). Hooi et al. (1989) demonstrated a close anatomical relationship between nerve terminals containing NPY and vasopressin (ADH) in the hypothalamic paraventricular (PVN) and supraoptic nuclei of mammalian brain. The overall distribution of NPY-ir in our study also correlates with the distribution of vasopressin-ergic cell bodies in the amphibian brain (see review Moore and Lowry, 1998) suggesting its involvement in the regulation of endogenous salt adaptation. Our results also display an increase of NPY-ir in the infundibular (NIV, NID) and pre-optic nuclei which are analogous to the magnocellular neurons in mammals. Furthermore, our observations draw a strong support from the fact that injections of NPY into the SON increased plasma concentrations of arginine vasopressin (ADH)-an endogenous hormone which maintains body osmolality-in the rat (Hooi et al., 1989). Thus, based on our findings we suggest that NPY may contribute to the pathways known to mediate the autonomic and endocrine responses to osmotic changes in the environment. Acknowledgments Authors acknowledged the financial Grant from Department of Science and Technology (DST), Government of India, New Delhi, India. Authors are grateful to Dr. N.K. Subhedar (IISER Pune, India) for his valuable suggestions. Ishfaq Ali thanks DST for providing junior research fellowship. We thank Amul sakharkar for proofreading the manuscript and Swapnil Shewale for statistical analysis. References Ali, I., Bhargava, S., 2016. Neuropeptide Y in the brain of Euphlyctis cyanophlyctis tadpoles responds to hypoxic stress. Gen. Comp. Endocrinol. Alldredge, B., 2010. Pathogenic involvement of neuropeptides in anxiety and depression. Neuropeptides 44, 215–224. Beck, B., Pourié, G., 2014. Ghrelin, neuropeptide Y, and other feeding-regulatory peptides active in the hippocampus: role in learning and memory. Nutr. Rev. 71 (8), 541–561. http://dx.doi.org/10.1111/nure.12045. Bentley, P.J., 2002. The amphibia; endocrines and osmoregulation, a comparative account in vertebrates second ed. of vol. 1. (Chapter 6). In: Bradshaw, S.D.,
7
Burggren, W., Heller, H.C., Ishii, S., Langer, H., Neuweiler, G., Randall, D.J. (Eds.), Zoophysiology, 39. Springer-Verlag, Berlin, Heidelberg, pp. 155–186. Brady, L.D., Griffiths, R.A., 2000. Developmental responses to pond desiccation in tadpoles of the British anuran amphibians (Bufo bufo, B. calamita and Rana temporaria). J. Zool. 252 (1), 61–69. Bundzikova, J., Pirnik, Z., Zelena, D., Mikkelsen, J.D., Kiss, A., 2008. Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats. Cell. Mol. Neurobiol. 28 (8). Bundzikova, J., Pirnik, Z., Zelena, D., Mikkelsen, J.D., Kiss, A., 2010. The alpha2adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons in the long evans and brattleboro rats. J. Physiol. Pharmacol. 61 (4). Colmers, W., Wahlestedt, C., 1993. The biology of neuropeptide Y and related peptides. Crespi, E.J., Vaudry, H., Denver, R.J., 2004. Roles of corticotropin releasing factor, neuropeptide Y and corticosterone in the regulation of food intake In Xenopus laevis. J. Neuroendocr. 16 (3), 279–288. Cullinan, W.E., Herman, J.P., Battaglia, D.F., Akil, H., Watson, S.J., 1995. Pattern and time course of immediate early gene expression in rat brain following acute stress. Neuroscience 64, 477–505. D’Aniello, B., Vallarino, M., Pinelli, C., Fiorentino, M., Rastogi, R., 1996. Neuropeptide Y: localisation in the brain and pituitary of the developing frog (Rana esculenta). Cell Tissue Res. 285, 253–259. Dedovic, K., Duchesne, A., Andrews, J., Engert, V., Pruessner, J.C., 2009. The brain and the stress axis: the neural correlates of cortisol regulation in response to stress. Neuroimage 47, 864–871. Ebersole, T.J., Conlon, J.M., Goetz, F.W., Boyd, S.K., 2001. Characterization and distribution of neuropeptide Y in the brain of a caecilian amphibian. Peptides 22 (3), 325–334. Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16 (3), 183–190. Griffiths, R.A., 1997. Temporary ponds as amphibian habitats. Aquat. Conserv. Mar. Freshwater Ecosyst. 7 (2), 119–126. Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electronica 4 (1), 9. Heilig, M., 2004. NPY in stress, anxiety and depression. Neuropeptides 38, 213–224. Heldt, S.A., Ressler, K.J., 2006. Lesions of the habenula produce stress-and dopamine-dependent alterations in prepulse inhibition and locomotion. Brain Res. 1073, 229–239. Hoar, W.S., 1988. The physiology of smelting salmonoids. In: Hoar, W.S., Randall, D. J. (Eds.), Fish Physiology. Academic Press, San Diego, pp. 275–343. Hooi, S.C., Richardson, G.S., McDonald, J.K., Allen, J.M., Martin, J.B., Koenig, J.I., 1989. Neuropeptide Y (NPY) and vasopressin (AVP) in the hypothalamoneurohypophysial axis of salt-loaded or Brattleboro rats. Brain Res. 486, 21– 220. Hopkins, G.R., Brodie Jr, E.D., Neuman-Lee, L.A., Mohammadi, S., Brusch IV, G.A., Hopkins, Z.M., French, S.S., 2016. Physiological responses to salinity vary with proximity to the ocean in a coastal amphibian. Physiol. Biochem. Zool. 89 (4). Hopkins, G.R., Brodie Jr, E.D., 2015. Occurrence of amphibians in saline habitats: a review and evolutionary perspective. Herpetological Monogr. 29 (1), 1–27. Khan, M.S., 1997. A new subspecies of common skittering frog Euphlyctis. Pak. J. Zool. 29 (2), 107–112. Khan, M.S., 2014. The filter feeding mechanism in tadpoles. Bull. Chicago Herpetological Soc. 49 (8), 116–119. Kormos, V., Gaszner, B., 2013. Role of neuropeptides in anxiety, stress, and depression: from animals to humans. Neuropeptides 47, 401–419. Kubo, T., Okatani, H., Nishigori, Y., Hagiwara, Y., Fukumori, R., Goshima, Y., 2004. Involvement of the medial amygdaloid nucleus in restraint stress-induced pressor responses in rats. Neurosci. Lett. 354 (1), 84–86. Larhammar, D., Söderberg, C., Blomquist, A.G., 1993. Evolution of the neuropeptide Y family of peptides. In the Biology of Neuropeptide Y and Related Peptides. Humana Press, pp. 1–41. Larsen, P.J., Sheikh, S.P., Mikkelsen, J.D., 1992. Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway. Brain Res. 573, 181–189. Larsson, T.A., Olsson, F., Sundstrom, G., Lundin, L.G., Brenner, S., Venkatesh, B., Larhammar, D., 2008. Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions. BMC Evol. Biol. 8 (1), 1. Lopez, J.M., Gonzalez, A., 2002. Ontogeny of NADPH diaphorase/nitric oxide synthase reactivity in the brain of Xenopus laevis. J. Comp. Neurol. 445, 59–77. Luo, Y., Kaur, C., Ling, E., 2000. Hypobaric hypoxia induces Fos and neuronal nitric oxide synthase expression in the paraventricular and supraoptic nucleus in rats. Neurosci. Lett. 296 (2–3), 145–148. Mathieu, M., Yon, L., Charifou, I., Trabucchi, M., Vallarino, M., Pinelli, C., Fournier, A., Rastogi, R.K., Vaudry, H., 2001. Ontogeny of pituitary adenylate cyclaseactivating polypeptide (PACAP) in the frog (Rana ridibunda) tadpole brain: immunohistochemical localization and biochemical characterization. J. Comp. Neurol. 431 (1), 11–27. McCormick, S.D., Bradshaw, D., 2006. Hormonal control of salt and water balance in vertebrates. Gen. Comp. Endocrinol. 147 (1), 3–8. Moore, F.L., Lowry, C.A., 1998. Comparative neuroanatomy of vasotocin and vasopressin in amphibians and other vertebrates. Comp. Biochem. Physiol. C: Pharmacol. Toxicol. Endocrinol. 119 (3), 251–260. Morales-Medina, J.C., Dumont, Y., Quirion, R., 2011. A possible role of neuropeptide Y in depression and stress. Brain Res. 1314, 194–205.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015
8
E. Heigrujam et al. / General and Comparative Endocrinology xxx (2017) xxx–xxx
Mousley, A., Polese, G., Marks, N.J., Eisthen, H.L., 2006. Terminal nerve-derived neuropeptide y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum). J. Neurosci. 26 (29), 7707–7717. Neary, T.J., Northcutt, R.G., 1983. Nuclear organization of the bullfrog diencephalon. J. Comp. Neurol. 213, 262–278. Saha, S., Batten, T.F.C., Henderson, Z., 2000. A GABA-ergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study. Neuroscience 99, 613–626. Shewale, S.A., Gaupale, T.C., Bhargava, S., 2014. Temperature dependent changes in cocaine- and amphetamine regulated transcript (CART) peptide in the brain of tadpole, Sylvirana temporalis. Gen. Comp. Endocrinol. 220, 61–69. Shoemaker, V.H., Nagy, K.A., 1977. Osmoregulation in amphibians and reptiles. Annu. Rev. Physiol. 39, 449–471. Tuinhof, R., Gonzalez, A., Smeets, W.J.A.J., Roubos, E.W., 1994. Neuropeptide Y in the developing and adult brain of the South African clawed toad Xenopus laevis. J. Chem. Neuroanat. 7, 271–283. Uchiyama, M., Maejima, S., Wong, M.K.S., et al., 2014. Changes in plasma angiotensin II, aldosterone, arginine vasotocin, corticosterone, and electrolyte
concentrations during acclimation to dry condition and seawater in the crabeating frog. Gen. Comp. Endocrinol. 195, 40–46. Uchiyama, M., Konno, N., 2006. Review: hormonal regulation of ion and water transport in anuran amphibians. Gen. Comp. Endocrinol. 147, 54–61. Wada, M., Urano, A., Gorbman, A., 1980. Stereotaxic atlas for diencephalon nuclei of the frog Rana pipiens. Arch. Histol. Jpn. 43, 157–173. Wirtshafter, D., Asin, K.E., Pitzer, M.R., 1994. Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula. Brain Res. 633, 21–26. Wu, G., Feder, A., Wegener, G., Bailey, C., Saxena, S., Charney, D., Mathé, A.A., 2011. Central functions of neuropeptide Y in mood and anxiety disorders. Expert Opin. Ther. Targets 15, 1317–1331. Yao, M., Westphal, N.J., Denver, R.J., 2004. Distribution and acute stressorinduced activation of corticotrophin-releasing hormone neurones in the central nervous system of Xenopus laevis. J. Neuroendocrinol. 16, 880– 893. Ziegler, D.R., Herman, J.P., 2002. Neurocircuitry of stress integration: anatomical pathways regulating the hypothalamo–pituitary–adrenocortical axis of the rat. Integr. Comp. Biol. 42, 541–551.
Please cite this article in press as: Heigrujam, E., et al. NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. Gen. Comp. Endocrinol. (2017), http://dx.doi.org/10.1016/j.ygcen.2017.01.015