INTRODUCTION:

Alcohol-related disorders have major social and economic implications creating an enormous impact around the globe (Moorman, 2018; Lim et al., 2012). Despite promising results from drugs like naltrexone, acamprosate, baclofen, there is no magic bullet for treating alcohol use disorders. (Palpacuer et al., 2017) An insight into the neurochemicals involved in addiction might facilitate developing novel curative strategies (Walker and Lawrence, 2016). The neuropeptide Orexin B has regulatory roles in various behavioural and physiological responses (Thorpe and Kotz, 2005.

Derrick L. Choi and Benoit, 2012, (Walker and Lawrence, 2016) including neuroendocrine regulation, anxiety (Jaz et al., 2017), feeding behaviour, (Derrick L. Choi and Benoit, 2012) and reward-seeking. (Shoblock et al., 2010; Brown et al., 2013)In our laboratory, we studied the effect of Orexins on consummatory behaviour and reported that they play an important role (Rashmi et al., 2015; Rashmi et al., 2016; Mayannavar et al., 2014). Direct association between reward function and stimulation of Orexinergic neurons was reported by Harris et al. (Harris et al., 2005).It was also reported that a higher preference of alcohol showed stronger activation of Orexin neurons (Harris et al., 2007). Recent studies have reported attenuation of alcohol preference reward processing by OX₂ receptor block. (Shoblock et al., 2010; Brown et al., 2013)

The lateral hypothalamus (LH) and ventral tegmental area (VTA) are intricately connected. Orexinergic fibres emerging from LH end in VTA Dopaminergic fibres (Fadel and Deutch, 2002). Nucleus Accumbens also reportedly receive Orexinergic connections (Fadel and Deutch, 2002; Peyron et al., 1998). Nucleus Accumbens plays a prime role in the reward and addictive effects of drugs abuse and of alcohol. ((Henderson et al., 2010). Orexin’s actions in Nucleus Accumbens has been mainly attributed to OX₂R binding. (Martin et al., 2002, Marcus, 2001). Primarily, the contribution of NAc in reward and motivation has been linked to the role of Dopamine in NAc. (Berridge and Robinson, 1998). However, other studies in nucleus accumbens have suggested that Dopamine was not the only neurotransmitter that mediates addiction (Stefanie Rassnicka, 1993). Gremel et al. reported that alcohol induced conditioned effect relied upon NMDA receptors in NAc. (Gremel and Cunningham, 2009). The glutamate receptors namely, NMDA, AMPA and Kianate has been widely involved in mesolimbic brain regions mediating consummatory and reward-related behaviours. (Chen et al., 2013) Reports on involvement of glutamate in the regulation of ethanol consumption have been scant.

Further, role of Orexin B in nucleus accumbens mediating ethanol consumption and preference is rarely documented. We found infusion of Orexin into the NAcc had influence on food intake behaviour. In continuation, the present study was designed to evaluate the role of Orexin B receptors in NAc on voluntary ethanol consumption and preference, thereby its influence on addictive behaviour. In the first series of experiments, we tested, whether infusion of Orexin B and OX2 antagonist TCS-OX2-29 into NAc modulate voluntary ethanol consumption and preference in male Wistar albino rats. Subsequently, we tested whether changes in ethanol consumption is mediated by various neurotransmitters in nucleus Accumbens.

MATERIALS AND METHODS:

Animals:

Adult Wistar male albino rats (n=48), weighing (200-275 gms) were used for this study. The experimental animals were obtained and maintained in institutional Central animal house. (Reg.No.213/CPCSEA) Animals were housed individually in polypropylene cages (29cm x 22 cm x 14cm) with paddy husk bedding. Throughout the research period, all the animals were maintained under standard laboratory conditions. Rats were provided with ad libtum access to potable water and rat feed pellets (Pranav Agro Industries Ltd, Maharashtra, India) except in the groups as mentioned in the experimental requirement. Animals were segregated as Group 1(voluntary ethanol consumption group-single-bottle) and Group 2 (ethanol preference group – two-bottle) with 24 animals. These were subdivided into control group (n=6; saline infused), 3nm/µl Orexin B treated group (n=6), 30 nm/µl of Orexin B treated group (n=6) and Orexin B antagonist (10µg/µl; n=6) treated group. Before the commencement of the study, Institutional Animal Ethics Committee (I.A.E.C) approval was obtained. All experimental procedures approved by Institutional Animal Ethical (IAEC), (No: IAEC/KMC/57/2009-2010) and investigations were performed in accordance with the guideline of Committee for the Purpose of Control and Supervision of Experiments on Animals.

Ethanol intake:

Rats were acclimatized to 10% ethanol by presenting an ethanol drinking bottle for one week in their home cage. Based on our pilot study reports, the concentration of ethanol was standardised (Mayannavar et al., 2014). Ethanol (10% Solution) was prepared from absolute ethyl alcohol by adding 10 ml of ethanol and 90 ml of distilled water (Durga laboratories, Mangalore, India). Rats were exposed to ethanol ad libitum for five days and for the following 2 days they received only water (Schneider et al., 2007). Following this acclimatization, rats were fasted for 24 hours and then presented 10% ethanol drinking bottle. Measurement of parameters were done meticulously at the end of 1h, 2 hrs, 4 hrs, 12 hrs and 24 hrs after the infusion during the days of testing.

Two-bottle preference/ free choice test:

In two-bottle free choice test, rats were provided two drinking bottles for one week in their home cage. They were presented with two drinking bottles (Ganaraja and Jeganathan, 1999; Blizard et al., 2008), one containing plain drinking water and the other 10% ethanol. During the experimental period, rats had free access to either drink 10% ethanol or plain water. Positions of bottles were switched daily to minimize any confound produced by side bias during the experimental period. For drug infusion experiments, measurements were done at the end of 1 h, 2 hrs, 4 hrs, 12 hrs and 24 hrs after infusion during the days of testing from each bottle and intake was noted.

Surgical procedure:

Rats were anesthetized by a cocktail of Ketamine and Xylazine, (50mg/ml of ketamine and 20mg/ml of xylazine). Calculated volume of anesthetic agents were drawn from the vials to make a solution containing equivalent of 70mg/kg bodyweight of Ketamine and 10 mg/kg bodyweight of Xylazine and injected by intraperitoneal route (ip). Anesthetized rats were fixed in the stereotaxic apparatus. Using stereotaxic technique, stainless steel needle (22 gauge) as guide cannula, was bilaterally implanted. For Nucleus Accumbens, AP= 1.6 mm in front of Bregma, L= 0.8mm from the midline, V=7.2mm from the surface of the skull. (George Paxinos, 1998). The implanted guide cannula was placed with stylet post-surgery to prevent blockage. One week was allowed for them to recover from surgery and also to secure the cannula in place (Santhosh Mayannavar et al., 2013).

Micro-infusion procedure:

The infusion of Orexin B and the antagonist was done in the un-anesthetised free moving rat. The infusion cannula was connected to the Harvard Pico Plus pump fitted with Hamilton syringe and inserted into NAcc through a prefixed guide cannula. The slow infusion was done over 90 seconds at a flow rate of 0.6µl/min. (Mayannavar et al., 2014a).

The dose of Orexin B, Catalogue no. 06262, Sigma Aldrich, St Louis, USA, used in the present study was according to the earlier study by Edwards CMA et al. (Edwards, 1999). Two doses of Orexin B were administered bilaterally into specific nuclei as per the study protocol., TCS-OX2-29, (Orexin B antagonist - Catalog.No.3370; Tocris Bioscience, UK) was dissolved in 0.9% saline and infused at a dilution of 10 micrograms/µl (Ebrahimian et al., 2015). Orexin B was mixed with 0.9% saline and refrigerated. Controls received 0.9% saline (vehicle). Orexin B was administered at 3 nm/µl and 30 nm/µl for 90 seconds in respective groups. Normal saline was infused to control group rats.

Measurements: For 24 hours prior to drug infusion, rats were deprived of food and water. Food and fluids were provided following infusion. Measurement of food consumption was done at 1 h, 2 hrs, 4 hrs, 12 hrs, and 24 hrs following drug infusion. The animals were given a measured quantity of food and water and alcohol in respective groups, and the intake was noted at the time intervals mentioned above. The four trials of Orexin B/antagonist infusion were carried out and an average was taken as the measure of food and ethanol intake in single-bottle test for ethanol, food, water and ethanol intake in two-bottle free choice test. The value of ethanol consumption were expressed in milliliter of ethanol consumed per 100 g of bodyweight. A gap of 72 hours were maintained between successive treatment days.

Analysis of neurotransmitters in the Nucleus Accumbens by ELISA:

All the brain sample collections were done soon after the measurement of experimental parameters after four trials. At the close of fourth trial, rats were sacrificed by decapitation, and the brains were removed quickly. Nucleus accumbens was quickly dissected out on a chilled petri dish placed on crushed ice. The freshly prepared brain sample covered in thin aluminium foil, was stored in the refrigerator immediately at –70°C until subsequent estimation of neurotransmitter. On the day of homogenization, the wet weight of the sample was estimated and transferred it into ice cooled solution containing 0.1 N HCl and 1 mmol EDTA (1ml/50mg wet weight brain tissue) and homogenisation was carried out. The sample was centrifuged at 15,000g for 15 min at 4° C and the supernatant were used for ELISA analysis. Dopamine, Noradrenaline, and Serotonin were quantified using commercially available Tricat ELISA kit (IBL, Germany catalogue No, RE-59395). Glutamate was quantified using a commercially available ENZY^TMCHROM Glutamate Assay Kit (Bioassay systems, USA; catalogue number: EGLT-100) according to the manufacturer’s protocol. The analysis was done using Bio Tek ELx-800 automated ELISA micro plate reader.

Statistical analysis:

RESULTS:

Effect of bilateral micro-infusion of Orexin B, TCS-OX2-29 into NAc on voluntary ethanol consumption.

Alcohol intake was significantly different among all the groups studied in single-bottle test for ethanol [(F=70.992), p<0.0001]. Among these groups, at dose of 3nm/µl of Orexin B infusion, there was no net/total increase in alcohol intake before and after the infusion, except a significant increase in alcohol intake at the end of 1 hr and 4 hrs when compared to their controls respectively (Table 1; p<0.001). Infusion of Orexin B at 30 nm/µl into NAc increased alcohol intake at the end of 1 h (p<0.001), 2 hrs (p<0.001), 4 hrs (p<0.001), 12 hrs (p<0.05) and 24 hrs (p<0.001) significantly compared to their respective controls (Table 1). In this experiment, the infusion of TCS OX2-29 (Orexin B antagonist) decreased alcohol intake significantly at the end of 2 hrs (p<0.001), 4 hrs (p<0.01) and 24 hrs (p<0.05) with respect to their respective controls (Table 1).

Food intake was also significantly different among all groups studied in single-bottle test for ethanol [(F=59.948), p<0.0001]. At 3nm/µl of Orexin B infusion, there was no significant change in food intake except increase at the end of 1 h and 4 hrs when compared to their respective controls (Table 1; p<0.001). At 30nm/µl of Orexin B infusion, significantly increased food intake was observed at one hr, 4 hrs, and 24 hrs compared to their controls (Table 1; p<0.001). With Orexin B antagonist infusion, there was a significant decrease in food intake at the end of 12 hrs and 24 hrs when compared to their respective controls (Table 1; p<0.01).

Table 1. Effect of Orexin B, TCS-OX2-29 on voluntary ethanol (10%) and food intake at following treatment (ml/100g Bodyweight)

Alcohol-(ml/100gm bwt)

Food (g)

12 h

24h

12 h

24h

Control

3.1±

0.48

2.9±

0.18

3.7±

0.34

4.54±

1.19

16.62±

1.20

2.3±

0.36

0.69±

0.20

0.34±

0.05

6.9±

0.71

10.26±

1.05

Orexin

(3nmol/μl)

6.8±

0.70***

3.78±

0.38

4.8±

0.35 *

7.16±

1.36

20.85±

1.53

5.39±

0.50 ***

0.83±

0.27

1.31±

0.08***

5.47±

1.01

13.01±

1.44

Orexin

(30nmol/μl)

10.1±

0.64***

5.28±

0.35 ***

6.64±

0.42 ***

8.76±

0.91*

26±

0.85***

6.2±

0.43***

0.94±

0.14

2.18±

0.09 ***

8.62±

0.60

17.96±

0.99 ***

TCS-OX2-29

1.93±

0.53

0.56±

0.24***

2±

0.31**

3.14±

0.64

5.90±

1.03 *

1.83±

0.31

0.29±

0.08

0.24±

0.02

3.17±

0.57**

5.54±

0.71**

p˂0.05, **p<0.01, ***p<0.001 Experimental group Vs. Sham operated control (0.9% saline)]. (n=6 rats /group). [Values are mean± SEM. Repeated measures ANOVA (Student-Newman-Keuls)

Effect of Orexin B and its antagonist on noradrenaline levels (ng/ml) in a single-bottle test for ethanol.

Noradrenaline (0.56±0.04) was significantly increased with Orexin B infusion (Fig 1a; p<0.01) and significantly decreased (0.32±0.02) with Orexin B antagonist infusion compared to controls (0.42±0.03) (Fig 1a; p<0.05)

Effect of Orexin B and its antagonist on serotonin (5-HT) (ng/ml) levels in single-bottle test for ethanol.

Serotonin levels of NAc (1.53±0.02) were non-significantly decreased with Orexin B infusion into NAc and Orexin B antagonist increased it (1.62±0.03) compared to controls (1.55±0.04; Fig 1a)

Effect of Orexin B and its antagonist on Dopamine levels (ng/ml) in single-bottle test for ethanol.

A significant increase in the NAc Dopamine level (81.33±5.42) was observed with Orexin B infusion into nucleus accumbens when compared to controls (55.03±4.21) (Fig 1b; p<0.01). A significant fall in the NAc Dopamine level (36.33±3.97) was seen with Orexin antagonist infusion into nucleus accumbens with respect to controls (55.03±4.21) (Fig 1; p< 0.05).

Fig 1a and 1b. Effect of Orexin B/antagonist on nucleus accumbens neurotransmitter levels (ng/ml of brain tissue homogenate) Noradrenaline (NA) and Serotonin (Fig 1a); Dopamine, (Fig 1b); post infusion into nucleus accumbens. **p<0.01 control vs. Orexin B; $<0.05 control vs.TCS-OX2-29.

Effect of Orexin B and its antagonist on glutamate levels in single-bottle test for ethanol.

Neurotransmitter glutamate was found to be decreased significantly in NAc (55.49± 0.48) with the infusion of Orexin B when compared to controls (72.5±0.45). (Fig 2: p<0.001). A significant increase in the level of nucleus accumbens glutamate (88±1.6) was seen with Orexin antagonist infusion (72.5±0.45) (Fig 2: p<0.001)

Fig 2. Effect of Orexin B and Orexin antagonist on NAc glutamate levels (mg/dl of tissue) post infusion into NAc. ***p <0.001, control vs. Orexin B; $$$p<0.001, control vs. TCS-OX2-29.

Effect of bilateral micro-infusion of Orexin B and TCS-OX2-29 into NAc on preference for fluid (ethanol vs. water) in two-bottle free choice test.

In two-bottle free choice experiments, saline infused control animals were consuming more of water as a choice over 10% ethanol during the course of observation of 24 hrs. After the Orexin B treatment (30nmol/µl), rats started consuming ethanol preferentially in comparison with water. Ethanol consumption was significantly increased with 30 nm/µl of Orexin B when compared to their respective controls (Table 2; p<0.001). Ethanol consumption was not significantly increased with 3 nm/µl of Orexin B infusion (Table 2; p >0.05). The total volume of fluid intake was increased in Orexin B treated rats (3nm/μl and 30nm/μl) when compared to saline infused control group. It was significantly evident during 12 hrs (6.80±0.13) of 3 nm/µl of Orexin B infusion (Table 2; p<0.001) and 12 hrs (9.40±0.36) and 24 hrs (18.51±0.56) of 30 nm/µl of Orexin B infusion with respect to their controls (3.96±0.79 and 10.48±.24) (Table 2; p<0.001). Alcohol preference was found to be significantly increased with the infusion of 30nmol/µl of Orexin B at the end of 1, 2,4,12 and 24 hrs (p<0.001)

In Orexin B antagonist infused group, there was significant decrease in total fluid intake at the end of 24 hrs (7.06±0.67) post treatment compared to control group (10.48±1.24) (Table 2; p<0.001). Alcohol intake was decreased at the end of 24 hrs (1.85±0.32) compared to control (2.17±0.40) (Table 2; p<0.01). 3 nm/µl of Orexin B increased food intake at the end of 12 hrs significantly (5.64±0.48) compared to control (3.86±0.25) (Table 2; p<0.001). Significant increase in food intake with the infusion of 30 nm/µl of Orexin B infusion was observed at the end of 1 h (5.38±0.33) and 24 hrs(12.39±0.41) with respect to controls (2.97±0.30 and 8.07±.62) (Table 2; p<0.01). Similarly food intake was decreased with the antagonist infusion at the end of 12 hrs (2.44±0.17) and 24 hrs (5.99±0.18) (Table 2; p<0.001, p<0.01).

Table 2. Food intake, alcohol intake and water intake (gm %), in two-bottle preference test group, alcohol preference ratio following Orexin B and its antagonist infusion into NAc.

		Nucleus Accumbens
Parameters	Groups	Duration (h)
Control		1 hr	2 hr	4 hr	12 hr	24 hr
	Alcohol	0.66±0.23	0.48±0.07	0.38±0.24	0.64±0.11	2.17±0.40
	water	2.45±0.50	1.01±0.30	0.82±0.33	3.47±0.83	8.20±0.83
	Total	3.40±0.37	1.74±0.32	1.37±0.32	3.96±0.79	10.48±1.24
	Alcohol Preference ratio	0.19±0.009	0.28±0.01	0.28±0.009	0.16±0.006	0.21±0.005
Orexin B (3 nm/µl)	Alcohol	0.62±0.22	0.36±0.17	0.61±0.11	1.61±0.11	3.21±0.17
	water	1.52±0.24	0.79±0.16	0.79±0.14	5.25±0.14	8.36±0.28
	Total	2.28±0.43	1.04±0.14	1.36±0.26	6.80±0.13***	11.49±0.48
	Alcohol Preference ratio	0.27±0.004	0.35±0.004***	0.45±0.004	0.24±0.008	0.28±0.007***
Orexin B (30 nm/µl)	Alcohol	2.92±0.30***	1.94±0.19	1.07±0.18	4.01±0.27***	9.95±0.31***
	water	1.62±0.19	0.95±0.19	0.74±0.16	5.42±0.22	8.73±0.31
	Total	4.53±0.31	2.82±0.30	1.76±0.30	9.40±0.36***	18.51±0.56***
	Alcohol Preference ratio	0.64±0.003***	0.69±0.008***	0.61±0.003***	0.43±0.002***	0.54±0.006***
Treated (TCS-OX2-29)	Alcohol	0.39±0.10	0.44±0.05	0.38±0.24	0.63±0.09	1.85±0.32**
	water	1.2±0.21	0.85±0.21	0.79±0.21	2.20±0.24**	5.05±0.45***
	Total	1.73±0.24	1.33±0.27	1.23±0.35	2.76±0.26	7.06±0.67***
	Alcohol Preference ratio	0.23±0.003	0.33±0.004	0.31±0.006	0.23±0.003	0.26±0.006
Food	Control	2.97±0.30	0.69±0.21	0.54±0.22	3.86±0.25	8.07±0.62
	Orexin B (3 nm/µl)	3.0±0.21	1.02±0.17	0.51±0.17	5.64±0.48$$$	10.66±0.41
	Orexin B (30 nm/µl)	5.38±0.33***	1.53±0.33	0.93±0.12	2.94±0.21	12.39±0.41***
	TCS-OX2-29	2.00±0.16	1.02±0.17	1.02±0.17	2.44±0.17##	5.99±0.18###

All results represent mean± SEM. Repeated measures ANOVA followed by Student Newman Keuls multiple comparison tests were used to determine the significance of differences among groups. [F value significance; Alcohol-p<0.0001(F=106.45); Water-p<0.0001 (F=59.12); Total fluid intake-p<0.0001 (F=94.01); Alcohol preference ratio - p<0.001 (F=751.72); Food- P<0.0001 (F=51.49)]; **p<0.01, ***p<0.001 Orexin B ((30 nm/µl) vs sham control (saline infused); ##p<0.01, ###p<0.001 TCS-OX2-29 vs control; $$$p<0.0001 Orexin B (3 nm/µl vs control)]

DISCUSSION:

Addictive behaviour is a chronic, relapsing disorder that consists of a compulsive pattern of seeking and taking the substance at the expense of other activities. To elucidate the mechanism by which the transition from use to addiction, research is directed toward identifying and characterizing brain systems that mediate the rewarding effects of addictive agents. Harmful alcohol consumption is responsible for 2.5 million deaths annually, causing illness and. Alcohol acts on neuronal pathways and neurotransmitter systems (Moorman, 2018, Lim et al., 2012). A literature survey suggested the active involvement of the nucleus accumbens in addictive behaviour (Henderson et al., 2010). More recently, attention has been given to the role of neuropeptides in modulating the mesocorticolimbic system, including the neuropeptide Orexin.

In the present study, we investigated the possibility of the orexinergic system in the causation of changes in the addictive behaviour towards alcohol. We tested this hypothesis using normal inbred rats by infusing the Orexin B into the nucleus accumbens in different dosages of 3 nm/µl and 30 nm/µl and testing the alcohol intake. We had already established that Orexinergic pathways, particularly Orexin B had influence on ingestive behaviour. (Rashmi et al., 2015) Further, Orexin B antagonist was also used in the present study as a confirmatory proof for the role of Orexin and the possible involvement of Dopaminergic, glutamatergic and nor-adrenergic neurotransmitter levels.

The preference for ethanol after bilateral infusion of Orexin B into NAc was evident in the two-bottle free choice test. When the preference for ethanol was tested after the infusion of Orexin B, rats chose to drink ethanol. Ethanol preference ratio was also found to be increased during post-infusion period. There was an increase in alcohol intake and a decrease in water intake in these animals during the post-infusion observation period.

A strong association between drug preference and stimulation of Orexin neurons in LH has been reported. (Harris et al., 2005, Harris et al., 2007) Orexin A and Orexin B injection-induced increase of ethanol consumption was observed in the anterior paraventricular nucleus of thalamus. (Barson et al., 2015a, Barson et al., 2015b) However, there were few studies reporting the association between Orexin infusion into NAc and ethanol consumption. (Schneider et al., 2007). In the present study, normal inbred Wistar rats reared in the institutional animal house were used rather than alcohol-preferring rats. These rats have been exposed to ethanol training during acclimatising process by giving access to ethanol. Our study signifies the role of Orexin B in preference for alcohol consumption with the active involvement of NAc.

This finding was further confirmed by the use of Orexin B antagonist -TCS-OX₂-29 infusion experimentations. Treatment with OX₂ receptor antagonist reduced voluntary ethanol consumption in Wistar rats. The present reports are consistent with the study done by Brown et al. (Brown et al., 2013). The role of Orexins in rewarding effects of morphine has been demonstrated and this effect was associated with OX₂ receptors in morphine-dependent mice (Tabaeizadeh et al., 2013). Collectively, these findings implicate OX₂R in the NAc in mediating the reinforcing effects of ethanol. However, to the best of our knowledge, for the first time, the effect of Orexin B and its antagonist, TCS-OX2-29 in NAc on ethanol consumption and preference in Wistar albino rats, were explored.

Orexins can modulate Dopaminergic (Korotkova et al., 2003) and noradrenergic (Hagan et al., 1999) systems. Enhanced Dopaminergic activity in NAc and increased noradrenaline levels in alcohol consumed rats compared to controls. There are no other studies that investigated the effect of Orexin B on brain neurotransmitter levels in alcohol consumption. Further, it was reported that VTA has direct input of Orexinergic neurons and which was extended to prefrontal cortex and Nucleus Accumbens (Monti and Monti, 2007).Innervation of mesolimbic region by Orexin and Orexin receptors in these nuclei may be the potential circuitry involved in alcohol addiction.

Interaction between the glutamatergic system and the orexinergic system was implicated in drug abuse. The release of Glutamate in NAc in the addictive behaviour was documented by series of investigators (LaLumiere and Kalivas, 2008; Pierce et al.; 1996, McFarland et al., 2003; Kalivas and O'Brien, 2008). We observed decreased glutamate levels in NAc following Orexin B infusion. These results suggest an association between Orexin B mediated ethanol consumption and glutamatergic systems in NAc (Gass et al., 2011). Martin et al. (Martin et al., 2002) reported that an application of Orexin B decreased postsynaptic NMDA receptors, which support our findings. Acute alcohol exposure also showed a drop in the extracellular glutamate levels in NAcc. (Carboni et al., 1993) It was also reported that alterations in the functions of both NMDA receptors (Lovinger et al., 1989) and metabotropic glutamate subtype 5 receptors (Blednov and Harris, 2008) might affect glutamate action. Orexin was shown to regulate drug self-administration by modulating glutamatergic transmission in VTA (Borgland et al., 2006; Ungless et al., 2001). Notably, NAc receives far less Orexin projections than VTA. (Plaza-Zabala et al., 2013). These findings indicate that there may be differential roles played by Orexin B in ethanol reward and glutamatergic neurotransmission. The effect of Orexin B on ethanol reward and glutamatergic transmission might also depend on site-specificity. The rewarding effects of food and addictive drugs might mediate their actions through common molecular substrates in the brain. (Harris and Aston-Jones, 2006)

Collectively, these results indicate that Orexin B infusion facilitates and its blockade through Orexin 2 receptor blocker was effective in reducing the reinforcing effects of ethanol reward. Therefore, it can be inferred that Orexin B is a prospective neuropeptide mediating addictive behaviour and NAc is the major site of action of OX₂ receptors. There might be a presence of intricate Hypothalamic NAc circuits responsible for behavioural observations between natural rewards and drugs of abuse. Further, studies are necessary to target the neurobiology of the Orexin system focusing on the therapeutic potential towards the treatment of alcohol used disorders.

CONFLICT OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

FUNDING:

Department of Biotechnology, Ministry of Science and Technology, New Delhi, Government of India.

ACKNOWLEDGEMENT:

We acknowledge the support provided by the management of Kasturba Medical College., Mangalore, India by providing animal facility and laboratory.

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Received on 23.05.2020 Modified on 02.07.2020

Research J. Pharm. and Tech. 2020; 13(12):6224-6230.

DOI: 10.5958/0974-360X.2020.01085.9