Response of NPY immunoreactivity in the tadpole brain exposed to energy rich and energy depleted states

Accepted Manuscript Response of NPY immunoreactivity in the tadpole brain exposed to energy rich and energy depleted states

Swapnil Shewale, Ishfaq Ali, Kavita Hadawale, Shobha Bhargava PII: DOI: Reference:

S0143-4179(18)30014-3 doi:10.1016/j.npep.2018.06.001 YNPEP 1877

To appear in:

Neuropeptides

Received date: Revised date: Accepted date:

14 January 2018 12 June 2018 12 June 2018

Please cite this article as: Swapnil Shewale, Ishfaq Ali, Kavita Hadawale, Shobha Bhargava , Response of NPY immunoreactivity in the tadpole brain exposed to energy rich and energy depleted states. Ynpep (2017), doi:10.1016/j.npep.2018.06.001

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.

ACCEPTED MANUSCRIPT Response of NPY immunoreactivity in the tadpole brain exposed to energy rich and energy depleted states. Swapnil Shewalea,b,1 Ishfaq Alia,1, Kavita Hadawalea, Shobha Bhargavaa* a

Department of Zoology, Savitribai Phule Pune University, Ganeshkhind, Pune 411 007,

India Department of Zoology, Bhavan’s Hazarimal Somani College, Chowpatty, Mumbai 400

PT

b

Address for Correspondence: Dr. Shobha Bhargava

SC

*

RI

007, India

Department of Zoology,

NU

Savitribai Phule Pune University, Ganeshkhind Road,

India.

MA

Pune- 411 007,

[email protected]

Fax:

+91-020-25690617

Tel. No.:

+91-020-25601263

PT E

D

E-mail address:

Authors with equal contribution

*

Corresponding Author

AC

CE

1

ACCEPTED MANUSCRIPT Abstract. The central control of feeding in animals depends upon the alternating actions of orexigenic and anorectic peptides. Studies at understanding the food intake mechanisms have emphasised the role of Neuropeptide Y as a potent orexigenic peptide in the brain. The aim of this study is to investigate the response of NPY system to positive and negative energy states and elucidate a holistic response of NPY expression throughout the brain of a tadpole model.

PT

The pre-metamorphic tadpoles of Euphlyctis cyanophlyctis were subjected to fasting, or intracranially injected with glucose or 2-deoxy-D-Glucose (2DG)-a metabolic antagonist of glucose and the response of the NPY system in the entire brain was studied using

RI

immunohistochemistry. Glucose injections reduced the basal expression of NPY-

SC

immunoreactive perikarya (upto 20%) in the olfactory bulb, nucleus pre-opticus, infundibulum, raphe nucleus and the distal lobe of pituitary. These regions responded to the

NU

intracranial injections of 2DG by increasing the expression of NPY up to 30%. Animals deprived of food also possessed the same response except that the increase was much intense in the 2DG injected tadpoles. Our observations lead us to the conclusion that NPY containing

MA

neurons in the discrete brain areas may be involved in the maintenance of glucose homeostasis in amphibians and, since these regions also contain the glucose sensing neurons,

D

we further suggest that the release of NPY might be regulated by the glucose sensing neurons of the brain.

PT E

Keywords:

Neuropeptide Y, Food and feeding circuitry, Energy homeostasis, Tadpole. Abbreviations:

CE

AOB - Accessory olfactory bulb; AON –Anterior olfactory nucleus; AL – Amgdala pars lateralis; AVM – Area venteromedialis thalami; AVL- Area ventero lateralis thalami; BM

AC

Bed nucleus of striamedularis; DP –Dorsal pallium; EA – Anterior amygdala; HD – Habenula dorsalis; HV – Habenula ventralis; LFB - Lateral forebrain bundle; LP- Lateral pallium; ME - Median eminence; MGD – Magnocellular preoptic dorsal nucleus; MP Medial pallium; NAD - Nucleus antero dorsalis tegmenti mesencephali; NAS - Nucleus accumbens septi; NAV - Nucleus antero ventralis tegmenti mesencephali; NCER - Nucleus cerebella; NDB - Nucleus diagonal band of Broca; NLS – Nucleus lateralis septi; NMS – Nucleus medialis septi; NMNT- Nucleus of the oculomotor nerve; NPC - Nucleus posterocentralis thalami; NPL - Nucleus posterolateralis thalami; NPO - Nucleus preopticus; NPV - Nucleus posteroventralis tegmenti mesencephali; NR - Nucleus reticularis. Nucleus medialis septi; NRIS - Nucleus reticularis isthmi; OT - Optic tectum; PR - preoptic recess;

ACCEPTED MANUSCRIPT SCN - Suprachiasmatic nucleus; SGC - Striatum griseum central tecti; SGP - Striatum griseum periventricularis; SGS - Striatum griseum superficial tecti; TEO – Optic tectum; TS

AC

CE

PT E

D

MA

NU

SC

RI

PT

- Torus semicircularis; VM - Ventromedial thalamic nucleus.

ACCEPTED MANUSCRIPT Introduction Neuropeptide Y (NPY) has emerged as an important orexigenic agent in the vertebrate brain (Levine et al., 2004; Morton et al, 2006; Singru et al., 2008). While its role in food control is well established in mammals (Stanely, 1986; Schwartz et al., 2000, Zarjevski, 1993, Crespi, 2014, Kalra and Kalra., 2004), there are also increasing evidences of its role in the food and feeding regulation of sub-mammalian vertebrates. NPY has been shown to

PT

stimulate appetite and food consumption in various fish species including goldfish, zebra fish, orange spotted grouper, red tilapia, rainbow trout and salmon, (Aldegunde and Mancebo, 2006; Matsuda et al., 2012; Yokobori, et al., 2012; Wu et al., 2012; Volkoff et.al., 1999).

RI

Intra cerebro-ventricular injections of NPY stimulate food intake in goldfish Carassius

SC

auratusand Channel fish Ictalaurus punctatusand NPY-antagonists have been seen to block this effect with no effect on the basal food intake (Silverstein JT et al., 2000; Narnaware YK,

NU

2001). Recently, Li et al, reported involvement of NPY in increasing appetite and growth hormone expression in Olive flounder (Li et al., 2016).

In amphibians, NPY induced food intake and its role in the modulation of feeding

MA

behaviour is well known. Crespi and Denver in 2004 reported fluctuations in the levels of NPY and its mRNA in the hypothalamus of X. laevis during different feeding conditions.

D

Intracerebroventricular (icv) administration of NPY has been observed to stimulate food intake which could be blocked by the treatment of NPY Y1 receptor antagonist (Lopez-

PT E

Patino et al., 1999; Shimizu et al., 2013). Along with its stimulatory role, NPY is also known to inhibit feeding in larval stage of western spade foot toad, while it continues to stimulate food intake in juveniles (Crespi and Denver., 2012). This opposite action has been associated

CE

with the differential expression of NPY receptors (Crespi and Denver, 2012). For the central regulation of feeding, NPY in mammals and lower vertebrates (fishes)

AC

is known to engage various neuroanatomical regions of the brain including the olfactory nucleus, nucleus preoptics (NPO), articulate nucleus (AVN), paraventricular nucleus (PVN), optic tectum and ventral telencephalic regions (Daisukhe et al., 2012, Halford et al., 2004, Kalra et al., 1999, Yoo S et al., 2011). Increased levels of NPY mRNA in the forebrain in response to prolonged fasting are also reported (Silverstein JT, 1998; Qi I et al., 2016). Given that NPY is widely distributed in the brain of amphibians (D’Aniello et al., 1996; Tuinhof R, et al., 1994, Ali et.al.; 2016), most of the reports in amphibians only discuss the physiological role of NPY in food and feeding and there is only one noteworthy study describing the neuroanatomical substrates exerting these physiological effects. Calle et al (2006) reported an increase in the NPY expression in the supra chiasmatic nuclei in Xenopus laevis following

ACCEPTED MANUSCRIPT a prolonged starvation. However, they did not find any significant difference in the rest of the food and feeding associated areas of the brain where NPY producing cells are present in abundance (D’Aniello et al., 1996). NPY neurons in the hypothalamus of mammals are known to reflect the prevailing energy state in mammals and also serve a glucosensory function (Mountjoy P et al., 2007; Polakof S et al., 2011; Sheng B et al., 2012; Kotagale N., 2014). The NPY system in fish is

PT

also known to respond to the manipulations of glucose levels in the brain (Polakof S et al., 2011; Polako S., 2012; RileyJr L et al., 2009). In the current study, we investigate the changes in the NPY system in the entire brain of tadpole exposed to energy rich as well as

RI

energy depleted states and to check whether the role of NPY in amphibians is conserved or

NU

SC

not.

2. Materials and methods:

MA

2.1 Animals collection and procedure for intracranial treatments: The egg clutches of Euphlyctis cyanophlyctis were collected from the pond in Savitribai Phule Pune University campus and brought to the laboratory where these were kept

D

in glass aquarium (70x40x20cm³)containing aged declorinated tap water. The eggs hatched

PT E

after 2-3 days. Tadpoles were provided with ad libitum boiled spinach that served as a food. Tadpoles of stage 38 (Gosner, 1960; Shewale et al., 2015) were used for all the experiments. Stage 38 is characterised by the increase in the length of individual toes and appearance of

CE

metatarsal tubercles (Gosner, 1960). At this stage the larvae feeds voraciously and all the physiological processes are stabilized (Gosner, 1960; Ali et al., 2016). All the experimental

AC

procedures were performed in accordance with the ethical guidelines established for animal usage by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and Savitribai Phule Pune University, Pune, India (Institutional committee for animal ethics, No. 538/CPCSEA). A group of tadpoles (n=5) were anesthetized with 2-phenoxyethanol (Sigma, St.Louis, Mo; 1:2000) and brains were dissected and fixed in Bouins fixative for 24 hrs. Additional tadpoles were divided into groups (n=5 in each) and used as follows. The animals in group 2 were used as saline treated control (placebo controls), and groups 3 and 4 were used for 2 Deoxyglucose (16 µg/gbody weight; SRL, India), glucose (16µg/g body weight; Sigma, St. Louis; Mo) treatments respectively. An additional group (n=5) were food deprived

ACCEPTED MANUSCRIPT for 5 days which acted as the fasted group. All the above treatments were given dosage by intracranial route. The needle (#28) was glued into a 21G needle in a way that only 2mm of the needle tip remained exposed. The other end was connected to a 10µL syringe. Conscious tadpole were immobilized in a wet cloth, the backwardly directed needle tip was positioned against the entry point and gently pushed through the skin and the solution was delivered into the cerebrospinal fluid (CSF)-filled space in the periphery of the brain. 1 µL of 0.9% saline

PT

was used as a vehicle in placebo control. The injection procedures were completed within 3035 seconds and the tadpoles were returned to the tank. The stress due to injection procedure seems to be negligible since the tadpoles readily resumed their normal activity like swimming

RI

to the surface at regular intervals to gulp air. After an interval of 2 hours post injection each

SC

animal was anesthetized with 2-phenoxyethanol (Sigma, Germany), brains dissected and fixed in Bouins fixative for 24 hours at 4°C, cryoprotected in 10% (2 hours), 20% (2 hours)

NU

and 30% (overnight at 4°C) sucrose solution in Phosphate Buffer Saline (PBS, 0.0.1M, pH 7.4). Tissues were then embedded in Shandon cryomatrix and serially cut on a cryostat (Leica CM1510) at 20µm thickness in transverse plane. Sections were mounted on slides charged

MA

with poly-L-lysine (Sigma, USA) and stored in -20°C until further use for

2.2 Immunohistochemistry:-

D

immunohistochemical staining.

PT E

Sections were used for the immunohistochemical localization of NPY following the standard protocol (Shewale et al., 2015). Briefly, the sections were washed with PBS thrice and treated with 0.3% hydrogen peroxide in methanol for 1h. Sections were then

CE

washed in PBS and incubated with blocking agent containing 0.5% Bovine Serum Albumin and 0.5% gelatin in PBS for 1h. After washing thrice, sections were incubated for 1 h in

AC

normal goat serum (1:40 dilution, Vectastain). After incubation, excess goat serum was blotted and then sections were incubated with Rabbit monoclonal antibody against NPY (1:7000, Sigma) containing 0.5% BSA and gelatin overnight at 4°C. Sections were then washed in PBS thrice and incubated with biotinylated goat anti-rabbit IgG antibody at room temperature (1:200, Vectastain). Sections were washed and incubated with ABC reagent for 1 hour at room temperature (Vectastain, ABC Kit, 1:100). After washing, sections were incubated with 3,3diamino-benzidine tetra-hydrochloride (DAB) in Tris buffer (0.05 M, pH 7.2) containing 0.02% H2O2 for 8-10 minutes. Slides were washed in distilled water, dehydrated, cleared in xylene, mounted in distyrene plasticizer xylene (DPX, Merck, India) and photographed.

ACCEPTED MANUSCRIPT

2.3 Morphometry:Digital images of NPY immunoreactivity were obtained on a Zeiss imager A2 microscope equipped with a ProgRes C3 digital camera run on ProgRes C3 software. For analysis, care was taken to match sections through the same region of the brain and at the same level using anatomic landmarks with the aid of frog stereotaxic atlas (Neary and

PT

Northcutt, 1983; Wada et al., 1980). For cell counting, all visible cell bodies stained within the defined brain region were counted manually keeping the same counting area for all the treated groups. Data from each brain region in an animal was calculated by taking the average

RI

counts from five brain slices. Data from each slice was calculated by taking the average

SC

counts from the left and right sides of brain region of interest. Alternate sections exhibited robust staining in the same neuroanatomical regions. The size of the areas analyzed was kept

NU

the same for all experimental groups.

2.4 Statistical analysis:-

MA

For each treatment in different areas of the brain, cell counts were plotted as box plots. The hypothesis that there was no significant difference in the cell counts of all the

D

treated groups and was tested using Kruskal-Wallis test. A post hoc analysis using MannWhitney U test with Bonferroni correction was performed to check the difference between a

PT E

pair treatment at a time. All statistical analysis was done in freeware PAST version 2.14 (Hammer et al., 2001). Total count of the number of immunoreactive cells is compared with

3. Results

AC

CE

the respective treatment group and is expressed in terms of percentage in the result section.

Serial sections of the tadpole brain were observed for differential immunostaining of Neuropeptide Y. Antisera against NPY was checked for its specificity before any immunoreactions. The NPY immunoreactivity seems to be specific as omission of primary or secondary antibody from reaction mixture resulted in absolute loss of immunoreactivity (Fig. 1A). Furthermore, pre-adsorption of primary antibody with pure NPY peptide completely abolished all immunostaining (Fig. 1B). Also positive control section shows NPY cells (arrows) of nucleus infundibularis ventralis (NIV) region in bright field (Fig.1C) and

ACCEPTED MANUSCRIPT immunofluorescent (Fig. 1D) preparations. Specificity of this antibody in the frog has been shown earlier in our laboratory (Ali et al., 2016; Heigrujam et al., 2017).

NPY is one of the most conserved peptide in the vertebrates with a striking similarity from cartilaginous fishes to mammals (90%). NPY in Torpedo and mice differ by only three amino acids (Blomqvist et al, 1992). Neuroanatomical distribution and relative expression of

PT

NPY peptide in the tadpole brain of the frog Euphlyctis cyanophlyctis was studied in the response to the food deprivation and intracranial administration of 2-deoxyglucose (2DG) and glucose. NPY immunoreactive cell and fibers were seen throughout the brain and the

RI

pituitary gland. Brain areas were characterized by using stereotaxic atlas for frog brain

SC

provided by Wada et al., (1980) and “nuclear organization of bullfrog diencephalon” by Neary and Northcutt, (1983). The distribution of NPY peptide in the brain and pituitary gland

NU

of tadpoles in response to different treatments are described below. The basal distribution of NPY immunoreactivity remained the same as reported earlier in Euphlyctis cyanophlyctis (Ali et al., 2016), Rana esculenta (D’Aniello et al. 1996),

MA

Rana catesbeiana (Calliez et al.1987), Xenopus laevis (Tuinhof et al. 1994). NPY-ir cells was found in the pallium, septum and accessory olfactory bulb of telencephalon, Nucleus

D

preopticus, superchiasmatic nucleus, epithalamus, thalamus and hypothalamus of diencephalon, and tegumentum region, raphe nucleus of mesencephalon. Given below is the

stimulation.

CE

3.1 Telencephalon

PT E

detailed description of the brain areas which displayed a significant response to feeding

In the telencephalon, although, the reaction was present in the pallium and septum

AC

regions, NPY immunoreactive cells and fibers differed markedly in the accessory olfactory bulb (AOB) and anterior olfactory nucleus (AON) region in different groups (Fig. 3A-F). There seems to be an increased number of NPY immunoreactive cells and fibers in the fasted animals (18%) than the normal fed groups. In addition, a differential increase in the number of immunoreactive cells of animals injected with 16ppm of 2DG (17%) as compared with the saline treated and glucose treated groups (P < 0.05). Immunoreactive cells with fibers were scattered and denser in the AON region of the fasted group. The immunoreactive cells were small having round to oval nuclei. Profound immunoreactivity was seen in the 2DG injected animals (Fig. 3C) as compared with the glucose injected group.

ACCEPTED MANUSCRIPT 3.2 Diencephalon In the Diencephalon, there seems to be a robust increase in the NPY immunoreactive cells in the nucleus preopticus (NPO) of fasted groups (44%) as compared with normal fed control group (Fig. 4A-B). The perikaryon appears to be oval in shape with small process. The axonal processes of these neurons were projecting away from the ventricle. Isolated cells with beaded fiber system were present in the NPO of glucose and

PT

saline injected group. The group injected with 2DG exhibited a robust increase in the number of intensely stained immunoreactive cells (68%) than the glucose treated group (Fig. 4F; P < 0.05). The immunoreactive cells were present on the lateral sides of the pre-optic recess. The

RI

cells were oval in shape with their axons projecting away from the center. Strong and dense

SC

immunoreactive fibers were also observed in all the groups.

In the hypothalamus, the extensive distribution of NPY-ir neurons was seen in the

NU

Infundibulum. In nucleus infundibularis dorsalis (NID) intense immunoreactivity was seen in the fasted group as compared to the normal fed group (Fig. 5A-B). The neuronal axons were projecting away from the ventricle. A similar pattern of immunoreactivity with highly

MA

pronounced changes was observed in the nucleus infundibularis ventralis (NIV). The cells were round to oval in shape with their axons projecting towards the infundibular recess. Also

D

there seems to be a sort of communication and intermingling between the axonal fibers of the neurons (Fig. 5C). In the 2DG injected group profound immunoreactivity and increased

PT E

number of communicating cells (34%) were seen in comparable with the glucose injected group (Fig. 5C-D; P < 0.05). NPY immunoreactive fiber system was seen in all the treated

CE

groups with comparable intensity in 2DG treated group (Fig. 5C).

3.3 Rhombencephalon

AC

In the raphe nucleus (RA), differential immunoreactivity is seen in all the treatments. A robust increase in the immunoreactive cells is observed in the fasted group (36%) when compared with the normal fed group (Fig. 6A-F). The cells were round to oval in shape with axonal endings away from the ventricle. Pronounced number of NPY immunoreactive cells was observed in the 2DG injected group (43%) as compared with the glucose and saline controls (Fig. 6F; P < 0.05). Also a remarkable increase in the beaded fiber content was observed in the RA of 2DG injected and the fasted animals.

3.4 Pituitary gland

ACCEPTED MANUSCRIPT Remarkable changes were observed in the pituitary gland of all the groups. The Distal lobe (DL) of the pituitary gland of fasted animals showed increased number of NPY immunoreactive cells (60%) as compared to a smaller amount of immunoreactive cells in the control group (Fig. 7 A-F). The cells were round to oval in shape with intense staining. Drastic increase in immunoreactive cells was clearly seen in the 2DG injected group (55%) and the food deprived group as compared to very few cells in the glucose treated group.

PT

NPY-ir fiber system was also observed in the nucleus anteroventralis tegmenti mesencephali (NAV) region of all the groups.

RI

4. Discussion

SC

Neuropeptide Y possesses a widespread distribution in the amphibian brain. The peptide is produced in the dorsal, medial and lateral pallium; medial septum; medioventral

thalamic nuclei, suprachiasmatic

NU

telencephalon; anterior pre-optic area of telencephalon, ventromedial, central and posterior nuclei, infundibulum of diencephalon, anteroventral

mesencephalic tegumentum of mesencephalon and central gray; cerebellar area; vestibular

MA

nuclei of rhombencephalon of larval forms(D’Aniello et al., 1996; Ali et al 2016). However the distribution becomes much restricted in adults where the peptide could only be localised

D

in the medial pallium, basal forebrain, preoptic area, Ventral and dorsal nuclei of the infundibulum, tegmentum and trigeminal nerves(Ebersolea et al., 2001; Danger et al., 1985).

PT E

The distribution of NPY in our studies corresponds to the distribution pattern of NPY in the developing larvae of Rana esculenta and Euphlyctis cyanophlyctis (Heigrujam et al., 2017, Ali et al., 2016, D’Aniello et al., 1996). In our model, NPY producing cells were

CE

seen in pallium, septum medialis, pars lateralis and pars medialis regions of amygdala and anterior pre-optic area in telencephalon; epithalamus, thalamus and hypothalamic nuclei of tegmentum

AC

diencephalon,

area

of

mesencephalon

and

the

raphe

nucleus

of

rhombencephalon. By studying the expression of NPY in different nutritional conditions, herein we have tried to investigate the role of NPY-system in energy homeostasis in the brain of E. cyanophlyctus tadpoles. NPY modulated feeding behaviour is already known in mammals (Stanely, 1986; Schwartz et al., 2000, Zarjevski, 1993, Crespi, 2014, Kalra and Kalra., 2004) as well as nonmammalian vertebrates (Aldegunde and Mancebo, 2006; Matsuda et al., 2012; Yokobori, et al., 2012, Wu et al., 2012; Volkoff et al., 1999). In mammals ICV injection of NPY into cerebral ventricles stimulates food consumption and decreases energy expenditure (Stanley, B. G. 1986; Schwartz MW et al., 1999). Also, continuous or repeated central administration

ACCEPTED MANUSCRIPT of NPY leads readily to obesity (Stanley, B. G et al., 1986; Zarjevski, N. et al., 1993) and NPY secretion reduces leptin / insulin signalling to the brain acting as an anabolic signalling molecule (Wilding, J. P. H. et al. 1993). NPY has been shown to stimulate appetite and food consumption in various fish species including goldfish, zebra fish, orange spotted grouper, red tilapia, rainbow trout and salmon. Intra cerebro-ventricular injections of NPY stimulate food intake in goldfish (Carassius auratus) and Channel fish (Ictalaurus punctatus)and NPY-

PT

antagonists have been seen to block this effect with no effect on the basal food intake (Silverstein JT et al., 2000; Narnaware YK 2001). Much recently, Li et al, reported NPY to increase appetite and growth hormone expression in Olive flounder (Li et al., 2016).

RI

Brain regions responding to the altered glucose levels included accessory olfactory

SC

bulb (BOA), anterior olfactory nucleus (NOA) of forebrain, Nucleus pre-optics (NPO) and hypothalamus (NIV, NID) of midbrain and raphe nucleus (RA) of hindbrain. Further, the

NU

distal lobe of pituitary (DL) and Nucleus anteroventralis tegmenti mesencephalic (NAV) also responded to a similar increase and decrease of NPY against positive and negative nutritional states respectively. Increased levels of NPY-ir in the fasted and 2-deoxy-glucose (2DG)

MA

injected tadpoles against the normally fed and glucose injected tadpoles suggests its involvement in feeding regulation as reported earlier for mammals and sub-mammalian

D

vertebrates (Muroya et al., 1999, Silverstein et al., 2000; Narnaware 2001). In our study, bulbus olfactorious accessorious (AOB) and nucleus olfactorious

PT E

anterior (AON) in the forebrain presented a much conspicuous increase in NPY immunoreactive cells and fibres in glucose deprived state against the normally fed tadpoles. There was a two-fold increase in the fasted and 2DG injected animals. To our knowledge,

CE

this is the first report on the presence of NPY-ir material in these regions which becomes significant during food deprivation. AOB and AON are known to receive sensory neurons

AC

from the olfactory nerve to process the olfactory information and communicate it to the higher centers of the brain (Mori et al, 1999; Morton et al., 2006). Fasting enhances olfactory sensing by the expression of appetite-stimulating peptides which increase the ability to locate food from an infinite array of environmental constituents (Tong et al., 2011). Increase in the NPY immunoreactive fibers during glucose deficient state may suggest its role as an appetizer and a possibility that NPY might be enhancing the sensory input of olfactory system to ensure efficient foraging. Calle et al in 2006, while studying feeding in Xenopus laevis adults, reported an increase of NPY-ir in supra chiasmatic nucleus during starvation. However, they did not find any significant change of NPY expression in any other brain regions like olfactory nucleus,

ACCEPTED MANUSCRIPT nucleus pre-optics, infundibular nuclei which is surprising because these regions contain huge clusters of NPY immunoreactive cells in amphibians (Ali et al., 2016) and are also reported to be involved in the NPY mediated orexigenic responses in fishes and mammals(Stanley, B. G. 1986; Schwartz MW et al., 1999; Zarjevski, N., 1993; Kalra 1991). Nucleus pre-optics, in our studies, also seems to respond to different nutritional status of the brain. This nucleus is already known to process feeding information through NPY secretion in mammals (Kalra et

PT

al., 1999). Furthermore, there was a significant difference in the NPY immunoreactivity of the distal lobe of pituitary between animals of different nutritional conditions. Earlier, Hanson and Dallman in 1995 have suggested an integrative role of NPY to process the

RI

feeding related information through the hypothalamo-pituitary-adrenal (HPA) axis. Strongly

SC

stained immunoreactive fibers in the distal lobe of pituitary during glucose deficient conditions points towards a similar functioning of neuropeptide Y in our model. Changes in

NU

the NPY immunoreactivity in the hypothalamic regions in our study are consistent with the findings of Calle et al and also draws strong support from the known involvement of NPY-ir cells in hypothalamic feeding-center model in mammals and fishes (Muroyya et al., 1999,

MA

Silverstein et al., 2000; Narnaware 2001; Schwartz et al., 1999; Zarjevski, 1993; Kalra 1999, Wang et al 2015).

D

Although the effect of NPY on food intake and feeding behaviour is well known (Muroyya et al., 1999, Silverstein et al., 2000; Narnaware 2001, Calle et al in 2006; Suyama

PT E

et al., 2017) but how the glucose levels in brain effect the expression of NPY is not understood. Burdakov et al., (2005) reported an inhibition in appetite promoting NPY neurons in the hypothalamic arcuate nucleus of mice brain. Arcuate nucleus in mammals is

CE

homologous to the infundibular regions of the frog brain. Glucose administration, in our study, decreased the expression of NPY in the nucleus infundibularis (NIV, NID). ICV

AC

administration of deoxy-glucose exhibited an upregulation of NPY in the parallel regions of tadpole brain. Increase in the expression levels of NPY during glucose deprivation hints towards a possible regulation of NPY expression through glucose sensing neurons in the hypothalamus.

5. Conclusion The expression of NPY in the olfactory bulb, nucleus preopticus, nucleus infundibularis ventralis, nucleus infundibularis dorsalis, raphe nuclei and pituitary was up regulated following energy depletion and reduced during energy enriched states. We conclude that the NPY system in these areas may be sensitive to glucose and play a role in

ACCEPTED MANUSCRIPT regulating the feeding behaviour and energy homeostasis. The current study on amphibians suggests that this function of the NPY system may be a feature highly conserved across the entire vertebrate lineage.

Declaration of Interests It is declared that there is no conflict of interest that could be perceived as prejudicing the

PT

impartiality of the research reported.

Funding

RI

Authors acknowledged the financial grant from University Grant Commission, New Delhi,

SC

India (UGC, New Delhi), Department of Science and Technology – Science and Engineering Research Board (DST- SERB, Government of India, New Delhi, India; DST F GOI-A-719),

NU

Department of Science and Technology – Promotion of University Research and Scientific Excellence (DST-PURSE, Government of India, New Delhi, India) and Department Research

MA

and Development Program (DRDP), Government of India, New Delhi, India.

D

Aknowledgement

Swapnil Shewale and Ishfaq Ali thanks UGC and DST for providing junior research

PT E

fellowship.

References:

CE

Aldegunde, M., & Mancebo, M. (2006). Effects of neuropeptide Y on food intake and brain biogenic amines in the rainbow trout (Oncorhynchus mykiss). Peptides, 27(4), 719-727.

AC

Ali, I., & Bhargava, S. (2016). Neuropeptide Y in the brain of Euphlyctis cyanophlyctis tadpoles responds to hypoxic stress. General and Comparative Endocrinology 251, 3845.

Bi S, Kim Y J, Zheng F (2012). Dorsomedial hypothalamic NPY and energy balance control. Neuropeptides 46,309–314. Blomqvist A. G., Söderberg C., Lundell I., Milner R. J., Larhammar D. (1992). Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones. Proc. Natl. Acad. Sci. U.S.A. 89, 2350–2354.

ACCEPTED MANUSCRIPT Burdakov, D., Luckman, S. M., & Verkhratsky, A. (2005). Glucose-sensing neurons of the hypothalamus. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 360(1464), 2227-2235. Cailliez, D., Danger, J.M., Andersen, A.C., Polak, J.M., Pelletier, G., Kawamura, K., Kikuyama, S., Vaudry, H., (1987). Neuropeptide Y (NPY)-like immunoreactive neurons in the brain and pituitary of the amphibian Rana catesbeiana. Zool. Sci. 4, 123–134.

PT

Calle, T., Kozicz, M., Van der, L.E., Desfeux, A., Veening, J.G., Barendregt, H.P., Roubos, E.W., (2006). Effect of starvation on Fos and neuropeptide immunoreactivities in the brain and pituitary gland of Xenopus laevis. Gen. Comp. Endocrinol. 147, 237–246.

RI

Crespi, E. J., & Denver, R. J. (2012). Developmental reversal in neuropeptide Y action on

SC

feeding in an amphibian. General and comparative endocrinology, 177(3), 348-352. Crespi, E. J., & Unkefer, M. K. (2014). Development of food intake controls: neuroendocrine

NU

and environmental regulation of food intake during early life. Hormones and behavior, 66(1), 74-85.

Crespi, E.J., Vaudry, H., Denver, R.J., (2004). Role of corticotropin releasing factor,

MA

neuropeptide Y and corticosterone in the regulation of food intake in Xenopus laevis. J. Neuroendocr. 16 (3), 279–288.

D

Daisuke Kohno, Toshihiko Yada, (2012). Arcuate NPY neurons sense and integrate peripheral metabolic signals to control feeding. Neuropeptides, 46 (6), 315-319.

PT E

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.

CE

Danger, JM., Guy, J., Benyamina, M., Jegou, S., Leboulenger, F., Cote, J., Tonon, MC., Pelletier, G., Vaudry, H., (1985). Localization and identification of neuropeptide Y

AC

(NPY)-like immunoreactivity in the frog brain. Peptides 6, 1225–36. Ebersolea, T.J.J., Conlonb, M., Goetza, F.W., Boyda, S.K., (2001).Characterization and distribution of neuropeptide Y in the brain of a caecilian amphibian. Peptides 22, 325–334. Gosner, K.L., (1960). A simplified table for staging anuran embryos and larvae with notes on identification, Herpetologica 16 (3), 183–190. Halford, J. C., Cooper, G. D., & Dovey, T. M. (2004). The pharmacology of human appetite expression. Current Drug Targets, 5(3), 221-240. Hammer, Ø., Harper, D.A.T., Ryan, P.D., (2001). PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4 (1),9.

ACCEPTED MANUSCRIPT Hanson, E. S., & Dallman, M. F. (1995). Neuropeptide Y (NPY) may integrate responses of hypothalamic feeding systems and the hypothalamo‐pituitary‐adrenal axis. Journal of neuroendocrinology, 7(4), 273-279. Heigrujam, E., Ali, I., & Bhargava, S. (2017). NPY up-regulation in the tadpole brain of Euphlyctis cyanophlyctis during osmotic stress. General and Comparative Endocrinology 251, 46-53.

PT

Kalra, S. P., & Kalra, P. S. (2004). NPY and cohorts in regulating appetite, obesity and metabolic syndrome: beneficial effects of gene therapy. Neuropeptides, 38(4), 201-211.

RI

Kalra, S. P., Dube, M. G., Pu, S., Xu, B., Horvath, T. L., & Kalra, P. S. (1999). Interacting appetite-regulating pathways in the hypothalamic regulation of body weight 1. Endocrine

SC

reviews, 20(1), 68-100.

Kalra, S. P., Dube, M. G., Sahu, A., Phelps, C. P., & Kalra, P. S. (1991). Neuropeptide Y

NU

secretion increases in the paraventricular nucleus in association with increased appetite for food. Proceedings of the National Academy of Sciences, 88(23), 10931-10935. Kotagale N R, Upadhya M, Hadole PN, Kokare DM, Taksande BG (2014). Involvement of

MA

hypothalamic neuropeptide Y in pentazocine induced suppression of food intake in rats. Neuropeptides 48, 133–141.

with

neuropeptide

Y:

effects

on

ingestive

behaviors

and

energy

PT E

journey

D

Levine, A. S., Jewett, D. C., Cleary, J. P., Kotz, C. M., & Billington, C. J. (2004). Our

expenditure. Peptides, 25(3), 505-510. Li, M., Tan, X., Sui, Y., Jiao, S., Wu, Z., Wang, L., & You, F. (2016). The stimulatory effect of neuropeptide Y on growth hormone expression, food intake, and growth in olive

CE

flounder (Paralichthys olivaceus). Fish physiology and biochemistry, 1-8. Lopez-Patino, M.A., Guijarro, A.I., Isorna, E., Delgado, M.J., Alonso-Bedate, M, de Pedro,

AC

N., (1999). Neuropeptide Y has a stimulatory action on feeding behavior in goldfish (Carassius auratus). Eur. J. Pharmacol. 377 (2–3), 147–153. Matsuda, K., Sakashita, A., Yokobori, E., & Azuma, M. (2012). Neuroendocrine control of feeding

behavior

and

psychomotor

activity

by

neuropeptide

Y

in

fish. Neuropeptides, 46(6), 275-283. Mori, K., Nagao, H., & Yoshihara, Y. (1999). The olfactory bulb: coding and processing of odor molecule information. Science, 286(5440), 711-715. Morton, G. J., Cummings, D. E., Baskin, D. G., Barsh, G. S., & Schwartz, M. W. (2006). Central nervous system control of food intake and body weight. Nature, 443(7109), 289295.

ACCEPTED MANUSCRIPT Mountjoy P. D., Bailey S. J., Rutter G. A., (2007). Inhibition by glucose or leptin of hypothalamic neurons expressing neuropeptide Y requires changes in AMP-activated protein kinase activity. Diabetologia, Volume 50(1); 168–177. Muroya Shinji; Yadaad Toshihiko; Shiodac Seiji; Takigawab Morikuni (1999). Glucosesensitive neurons in the rat arcuate nucleus contain neuropeptide Y. Neuroscience Letters, 264 (1-3), 113-116.

PT

Narnaware, Y. K., & Peter, R. E. (2001). Effects of food deprivation and refeeding on neuropeptide Y (NPY) mRNA levels in goldfish. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 129(2), 633-637.

RI

Neary, T.J., Northcutt, R.G., (1983). Nuclear organization of the bullfrog diencephalon.J.

SC

Comp. Neurol. 213, 262–278.

Polakof S, Panserat Stéphane, José L. Soengas, Thomas W. Moon, (2012). Glucose

NU

metabolism in fish: a review. Journal of Comparative Physiology, 182:8, 1015–1045. Polakof S, Thomas P., Mommsen JL., Soengas, (2011).

Glucosensing and glucose

homeostasis: From fish to mammal. Comparative Biochemistry and Physiology,

MA

160:4, 123-149.

Qi Y, Fu M, Herzog H (2016). Y2 receptor signalling in NPY neurons controls bone

D

formation and fasting induced feeding but not spontaneous feeding. Neuropeptides 55, 91–97.

PT E

RileyJr LG, .Walker AP, .Dorough CP, .Schwandt SE, Grau, Gordon, (2009). Glucose regulates ghrelin, neuropeptide Y, and the GH/IGF-I axis in the tilapia, Oreochromis mossambicus. Comparative Biochemistry and Physiology, 154(4), 541-546.

CE

Schwartz, M. W., Baskin, D. G., Kaiyala, K. J., & Woods, S. C. (1999).Model for the regulation of energy balance and adiposity by the central nervous system. The American

AC

journal of clinical nutrition, 69(4), 584-596. Schwartz, M. W., Woods, S. C., Porte, D., Seeley, R. J., & Baskin, D. G. (2000). Central nervous system control of food intake. Nature, 404(6778), 661-671. 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 Shimizu S., Azuma M., Morimoto N., Kikuyama S., Matsuda M. (2013). Effect of neuropeptide Y on food intake in bullfrog larvae. Peptides 46, 102–107. Silverstein, J. T., & Plisetskaya, E. M. (2000). The Effects of NPY and Insulin on Food Intake Regulation in Fish. American Zoologist, 40(2), 296-308.

ACCEPTED MANUSCRIPT Silverstein, J. T., Breininger, J., Baskin, D. G., & Plisetskaya, E. M. (1998). Neuropeptide Ylike gene expression in the salmon brain increases with fasting. General and comparative endocrinology, 110(2), 157-165. Singru, P. S., Mazumdar, M., Barsagade, V., Lechan, R. M., Thim, L., Clausen, J. T., & Subhedar, N. (2008). Association of cocaine-and amphetamine-regulated transcript and neuropeptide Y in the forebrain and pituitary of the catfish, Clarias batrachus: a double

PT

immunofluorescent labeling study. Journal of chemical neuroanatomy, 36(3), 239-250. Stanley, B. G., Kyrkouli, S. E., Lampert, S., &Leibowitz, S. F. (1986). Neuropeptide Y

hyperphagia and obesity. Peptides, 7(6), 1189-1192.

RI

chronically injected into the hypothalamus: a powerful neurochemical inducer of

SC

Suyama S, Lei W, Kubota N, Kadowak T, Yada T (2017). Adiponectin at physiological level glucose-independently enhances inhibitory postsynaptic current onto NPY neurons in the

NU

hypothalamic arcuate nucleus. Neuropeptides 65, 1–9.

Tong, J., Mannea, E., Aimé, P., Pfluger, P. T., Yi, C. X., Castaneda, T. R., & Julliard, K.

MA

(2011). Ghrelin enhances olfactory sensitivity and exploratory sniffing in rodents and humans. Journal of Neuroscience, 31(15), 5841-5846.

D

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. Journal

PT E

of chemical neuroanatomy, 7(4), 271-283. Volkoff H., Bjorklund J. M., Peter R. E. (1999). Stimulation of feeding behavior and food consumption in the goldfish, Carassius auratus, by orexin-A and orexin-B. Brain

CE

Res. 846, 204–209.

Wada, M., Urano, A., Gorbman, A., (1980). Stereotaxic atlas for diencephalon nuclei of the

AC

frog Rana pipiens. Arch. Histol. Jpn. 43, 157–173. Wang, B., Sheriff, S., Balasubramaniam, A., & Kennedy, M. A. (2015). NMR based metabolomics study of Y2 receptor activation by neuropeptide Y in the SK-N-BE2 human neuroblastoma cell line. Metabolomics, 11(5), 1243-1252. Wilding J.P.H.; Gilbey S.G.; Lambert P.D.; Ghatei M.A.; Bloom S.R., (1993) Increases in Neuropeptide Y Content and Gene Expression in the Hypothalamus of Rats Treated with Dexamethasone Are Prevented by Insulin. Neuroendocrinology (1993). 57:581–587. Wu, Q., Clark, M. S., & Palmiter, R. D. (2012).Deciphering a neuronal circuit that mediates appetite. Nature, 483(7391), 594-597.

ACCEPTED MANUSCRIPT Yokobori, E., Azuma, M., Nishiguchi, R., Kang, K. S., Kamijo, M., Uchiyama, M., & Matsuda, K. (2012). Neuropeptide Y stimulates food intake in the zebrafish, Danio rerio. Journal of neuroendocrinology, 24(5), 766-773. Yoo SB, Ryu V, Park EY, Kim B, Kang D, Lee JH,Jahng JW (2011). The arcuate NPY, POMC, and CART expressions responding to food deprivation are exaggerated in young female rats that experienced neonatal maternal separation. Neuropeptides 45, 343–349.

PT

Zarjevski, N., Cusin, I., Vettor, R., Rohner-Jeanrenaud, F., & Jeanrenaud, B. (1993). Chronic intracerebroventricular neuropeptide-Y administration to normal rats mimics hormonal

SC

RI

and metabolic changes of obesity. Endocrinology, 133(4), 1753-1758.

NU

Figure legends

Figure 1.

Transverse section through NPO of tadpole brain of E. cyanophlyctis shows no

MA

immunoreactions following omission of primary or secondary antibody from the reaction mixture (A). Transverse section through NPO shows loss of immunoreactivity following

D

application of antisera preadsorbed with pure NPY peptide (B). Positive control section showing NPY cells (arrows) of NIV region in brightfield (C) and immunoflorescent (D)

PT E

preparations. Scale bar = 100µm for A-C; 50µm for D.

Figure 2.

CE

Schematic frontal diagrams of rostrocaudal series of transverse sections (A-J) of the tadpole brain of E. cyanophlyctis subjected to varying nutritional states. The cytoarchitectonic areas

AC

are on the left with NPY neurons (circles) and fibres (dots) on the right side respectively. For details of cytoarchitectonic regions, please refer to the list of Abbreviations. Scale bar = 1mm.

Figure 3. Transverse section showing differential distribution of NPY peptide in the accessory olfactory bulb (AOB) in response to fasting, 2DG and glucose treatments (some of the dense NPY-ir cells are shown by arrows). In AOB increased NPY immunoreactivity was seen in the fasted and 2DG injected group are compared with glucose and saline treated groups (Fig. CE). Fig F shows the graphical distribution of all the treatments. Kruskal-Wallis value for

ACCEPTED MANUSCRIPT AOB is H=20.97 and P=0.0003212. The same superscripts over the bars represent the data which are not significantly different. Scale bar, A-E, 100 μm. For Abbreviation see list.

Figure 4. NPY immunoreactive cells in the Nucleus pre-opticus (NPO) of tadpole brain exposed to differential nutritional states (A-E). Increased NPY immunoreactive cells shown was seen in

PT

the fasted animals are compared to normal feeding group (Fig. A-B). Few strong positive cells are shown by arrows. Also there seems to be a profound increase in the density of NPY ir cells and fibers of 2DG injected group are compared other treatments (Fig. C-E). Fig F

RI

shows the comparative distribution of all the groups. Kruskal- Wallis value for NPO is

SC

H=21.61 and P=0.0002391. The same superscripts over the bars represent the data which are

NU

not significantly different. Scale bar, A-E, 100 μm. For Abbreviation see list.

Figure 5.

Transverse section showing profound NPY immunoreactivity in the infundibular region of

MA

frog E. cyanophlyctis in response to fasting, 2DG and glucose treatments (NPY-ir cells are shown by arrows). NPY immunoreactive cells and fibers were observed in Nucleus

D

infundibularis dorsalis (NID) and Nucleus infundibularis ventralis (NIV) (A-E). Arrows indicate a few immunoreactive cells. Increased immunoreactivity was observed in the 2DG

PT E

and the fasted group in comparison with other treated groups (A-E). Fig F shows the collective number of immunoreactive cells of all the treatments. Kruskal- Wallis value for NIV is H=22.46 and P=0.0001621. The same superscripts over the bars represent the data

AC

Figure 6.

CE

which are not significantly different. Scale bar, A-E, 100 μm. For Abbreviation see list.

Differential expression of NPY in the Rhombencephalon region of tadpole brain energy rich and depleted states (NPY-ir cells are shown by arrows). Increased NPY immunoreactivity was seen in the fasted animals are compared to normal feeding group (Fig. A-B). Also a comparable increase in the density of NPY ir cells (arrows) and fibers was seen in 2DG injected group as compared to other treatments (Fig. C-E). Fig F shows the comparative distribution of all the groups. Kruskal- Wallis value for RA is H=20.63 and P=0.0003741. The same superscripts over the bars represent the data which are not significantly different. Scale bar, A-E, 100 μm. For Abbreviation see list.

ACCEPTED MANUSCRIPT Figure 7. Differential expression of NPY in the pituitary gland (PIT) of larval E. cyanophlyctis. Increased immunoreactivity was observed in the fasted and 2DG animals compared to normal feeding group (Fig. A-C). Fig F shows the comparative distribution of all the groups. Kruskal- Wallis value for RA is H=19.81 and P=0.0005432. The same superscripts over the bars represent the data which are not significantly different. Scale bar, A-E, 100 μm. For

AC

CE

PT E

D

MA

NU

SC

RI

PT

Abbreviation see list.

ACCEPTED MANUSCRIPT Highlights:

CE

PT E

D

MA

NU

SC

RI

PT

NPY system is up regulated in energy depleted state Rich energy state down regulates NPY expression in the brain NPY neurons may play a role in energy sensing

AC

  

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7