Chemogenetic activation of arcuate nucleus NPY and NPY/AgRP neurons increases feeding behaviour in mice

Neuropeptide Y (NPY) plays a crucial role in controlling energy homeostasis and feeding behaviour. The role of NPY neurons located in the arcuate nucleus of the hypothalamus (Arc) in responding to homeostatic signals has been the focus of much investigation, but most studies have used AgRP promoter-driven models, which do not fully encompass Arc NPY neurons. To directly investigate NPY-expressing versus AgRP-expressing Arc neurons function, we utilised chemogenetic techniques in NPY-Cre and AgRP-Cre animals to activate Arc NPY or AgRP neurons in the presence of food and food-related stimuli. Our findings suggest that chemogenetic activation of the broader population of Arc NPY neurons, including AgRP-positive and AgRP-negative NPY neurons, has equivalent effects on feeding behaviour as activation of Arc AgRP neurons. Our results demonstrate that these Arc NPY neurons respond specifically to caloric signals and do not respond to non-caloric signals, in line with what has been observed in AgRP neurons. Activating Arc NPY neurons significantly increases food consumption and influences macronutrient selection to prefer fat intake.


Introduction
Energy balance is modulated in the brain by the activity of different populations of neurons, which can act to increase or decrease food intake.The arcuate nucleus of the hypothalamus (Arc) regulates food intake by evaluating energy needs and modifying behavioural outputs accordingly (Ellacott and Cone, 2006;Ellacott and Cone, 2004).This critical process is modulated by two distinct populations of neurons in the Arc, which respond to peripheral hormones such as leptin, insulin, and ghrelin in order to produce opposing effects on food intake (Timper and Brüning, 2017;Hewson et al., 2002).Pro-opiomelanocortin (POMC) neurons can suppress food intake in response to homeostatic signals that indicate positive energy balance (Sasaki, 2017;Cone, 1999;Sohn, 2015).By contrast, Agouti-related peptide (AgRP) neurons increase food consumption in response to negative energy states (Zhang et al., 2019;Krashes et al., 2011a;Krashes et al., 2016).In humans and rodents, AgRP neurons are restricted to Arc; they release and co-express AgRP, gamma-aminobutyric acid (GABA) and neuropeptide Y (NPY) (Dailey and Bartness, 2009).
NPY, a 36-amino acid peptide, has critical functions in regulating feeding behaviours (Zimanyi et al., 1998;Cansell and Luquet, 2012;Cone et al., 2001).Stimulation of NPY-expressing neurons or administration of NPY into different regions of the hypothalamus, including the paraventricular, dorsomedial, ventromedial and Arc subregions, significantly increases food consumption in rodents (Krashes et al., 2011a;Bunner et al., 2020a;Dailey and Bartness, 2009;Minor et al., 2009).Intracerebroventricular (ICV) infusion of NPY increases consumption of palatable solutions, sucrose and saccharin, regardless of caloric value (Lynch et al., 1993), and induces a preferential intake of carbohydrates compared with fat (Stanley et al., 1989;Stanley et al., 1985), which extends to preferences between highly palatable high-fat or highcarbohydrate diets (Welch et al., 1994).Additonaly, both centraland hypothalamic paraventicular blockage of NPY signalling suppresses food consumption during the dark cycle when animals consume most of their daily energy intake (Dube et al., 1994;Hulsey et al., 1995;Shibasaki et al., 1993).Finally, expression of the NPY gene increases during anticipation of food in food-deprived animals (Minor et al., 2009), an effect which is reversed during re-feeding (Perboni et al., 2013).Targeted knockdown of the NPY gene causes reduced food intake during the first 9 days of a palatable meal-feeding schedule (Sindelar et al., 2005).
In addition to their role in consummatory behaviours, NPY neurons also affect appetitive responding, including searching for and obtaining food.In mice, ICV administration of NPY increases lever pressing for milk in a progressive ratio schedule of reinforcement (Flood and Morley, 1991).This suggests that NPY plays a role in driving motivation to eat, which may help account for the role of NPY neurons in increasing food consumption.Chemogenetic activation of Arc NPY neurons also promotes food-seeking by increasing physical activity during the light cycle and reducing sleep duration in favour of foraging (Goldstein et al., 2018).Importantly, this increase in locomotor activity is only evident in the absence of food (Krashes et al., 2011a), indicating the specific role of NPY neurons in searching for and obtaining food.Together, these findings indicate that NPY neurons play an important role in driving motivation for, and intake of, palatable food.
To date, most studies investigating NPY in the Arc have used AgRPpromoter driven models, as the majority of NPY neurons (~85%) in the Arc are co-localized with AgRP neurons (Luquet et al., 2005;Dietrich et al., 2012).However, there is a subpopulation of Arc NPY neurons (~15%) which do not co-express AgRP, and there is growing evidence that this population has some distinct features compared with AgRP neurons (Luquet et al., 2005;Dietrich et al., 2012).The result of a recent study by Qi and collegues demonstrated the orexigenic effect of AgRPnegative NPY neurons and showed that chemogenetic activation of this subpopulation of Arc NPY neurons increased feeding during ad libitum access to chow.Their results further demonstrated that in contrast with AgRP-positive NPY neurons, which only respond to energy deficit, AgRP-negative NPY neurons enhance feeding during prolonged positive energy balance in addition to fasting and negative energy states (Qi et al., 2023).
The current study aimed to investigate the differential function of broader NPY neurons, encompassing both AgRP-positive and negative NPY-expressing neurons, compared to NPY/AgRP-expressing neurons within the Arc, focusing on their impact on feeding behaviour.In addition to the AgRP-Cre mouse line widely used in previous studies (Bunner et al., 2020b;Krashes et al., 2013;Krashes et al., 2011b), we used and NPY-Cre mouse line which has the advantage of expressing Cre in both AgRP-positive and AgRP-negative NPY neuron populations.This allowed us to elucidate the shared and specific roles of broader of NPY neurons in the Arc on regulating energy and food consumption.Using AgRP Cre/+ and NPY Cre/+ animals, this study examined the effects of chemogenetic activation of Arc NPY or AgRP neurons on food consumption, preferences between, and operant responding for a) two macronutrients (fat and carbohydrates) and b) caloric and non-caloric sweeteners (sucrose and saccharin).

Animals
All experimental and animal care procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales (UNSW Sydney, Australia).Male and female NPY Cre/+ and AgRP Cre/+ mice on a C57BL/6 background were bred at Australian BioResources and delivered to UNSW at 6-12 weeks of age.Mice were group-housed in standard cages maintained at a temperature of 22 ± 2 • C on a 12-h light/dark cycles.Animals had ad libitum access to water and standard chow (Specialty Feeds; 60% carbohydrate, 19% protein, and 4.6% fat, digestible energy 3.4 kcal/g), unless otherwise described.All animals were acclimated and handled for one week prior to the start of the experiments.Equal numbers of males and females were used in the study where possible, and since no effects of sex were observed on consumption of food, supplementary data fig.1,the data have been combined.

Diets
All animals had ad libitum access to standard chow unless otherwise described.
Sucrose solution: 10% sucrose solution dissolved in tap water with a total caloric value of 0.4 kcal/ ml.

Measuring food and liquid intake in the BioDAQ system
Three weeks following stereotaxic surgery, mice were transferred and individually housed in BioDAQ cages supplied with standard rodent chow.The BioDAQ food and liquid monitoring system (BioDAQ, Research Diets, NJ, USA) was used to continuously monitor food and water intake.BioDAQ was used to monitor each animal's continuous feeding activity, including bout numbers, bout size, feeding bout, bout seconds (bout duration), and post-bout interval automatically.The system provides two hoppers in which liquid bottles or different pellets can be placed to allow researchers to investigate animal food preferences.A computer records the animal's undisturbed, native intake behaviour for each meal.A bout is defined using the parameters interbout interval (IBI) and minimum (min) food intake.The IBI is the time that must pass between feeding events to distinguish bouts (300 s), and the min value dictates the minimum amount of food an animal must consume to qualify as a bout (0.02 g).Therefore, food intake is considered a new bout only when bouts are separated by ≥5 min, and food consumption is ≥0.02 g.Animals were given one week to acclimate to the BioDAQ cages before experimental testing.Testing occurred in well-fed animals during the early light cycle.Clozapine N-oxide (CNO, Sigma-Aldrich, St. Louis, MO; 1 mg/kg) or sterile saline (5 ml/kg) was administered intraperitoneally (i.p.) immediately prior to experimental recording.A counterbalanced design was used with a two-day rest period between CNO and saline injections to guarantee a complete CNO washout.Prior to testing for macronutrient preference (fat vs carbohydrate), animals were exposed ad libitum to 4% intralipid and 10% sucrose in the BioDAQ for 3 consecutive days (6 h/day).Animals were similarly pre-exposed ad libitum to 10% sucrose and 0.1% saccharin before testing for preference of caloric and non-caloric sweeteners.Wellfed animals were subjected to macronutrient preference and caloric/ non-caloric tests using the BioDAQ system.They were provided access to intralipid and sucrose, as well as sucrose and saccharin simultaneously.
The feeding behaviour data, including food and liquid intake, bout numbers, and bout duration, for all the experiments, were collected by BioDAQ and assessed cumulatively at 1-, 2-, 4-, and 6-h following drug administration using BioDAQ Data Viewer software (Research Diets, New Brunswick, NJ).

Self-administration of sucrose and intralipid solutions
Behavioural training and testing procedures were conducted in operant chambers (Med Associates Inc. St Albans, VT, US).Each chamber contained a grid floor with standard bedding in a tray placed below, a house light on the left wall, and two retractable levers on the right wall with a recessed magazine placed between levers.The magazine was attached to the switchable dipper that delivered 0.01 cc liquid.The chambers were equipped with sound-attenuating and ventilation fans which produce a constant low-level white noise to prevent the distraction of the animals.All procedures were monitored and recorded by MedPC software (Med Associates Inc. St Albans, VT, US).
Animals were trained to lever press for intralipid and sucrose solutions, on alternative days, in 60-min sessions for 14 days.In the first two training sessions, to foster autoshaping, animals earned 4% intralipid or 10% sucrose solutions for each lever press.To make animals familiar with the reinforcer and its delivery location, animals also received a reward every 5 min.Animals were then trained on increasing fixed ratio (FR) schedules FR1, FR3, and FR5 in which 1, 3, and 5 levers press response/s delivered one drop (0.02 cc) of intralipid or sucrose solutions, respectively, over 2 weeks.Following FR programs, animals were exposed to 5-7 days progressive ratio (PR) schedule of reinforcement which increased based on the following schedules: 1, 2, 4, 6,9,12,15,20,25,32,40,50,62,77,95,118,145,178,219,268,328,402,492,693,737,901.The PR program ended if animals failed to press the lever for 20 min.Prior to the tests, mice received CNO or saline in a counterbalanced design (Tracy et al., 2008).An inactive lever was presented during FR3, FR5 and PR.Responding on the inactive lever was recorded but had no scheduled consequence.

Perfusion
Following the completion of behavioural experiments, animals were injected with CNO (1 mg/kg) and after two hours were euthanized via lethal injection of sodium pentobarbital (120 mg/kg, i.p) and transcardially perfused with 1× phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) solution.Fixed brains were extracted and postfixed in 4% PFA solution for 2 h and then stored in 30% sucrose in PBS until sectioning.

Immunohistochemistry
Brains were sectioned in a cryostat (Leica Biosystems CM1900, North Ryde, NSW, Australia) at 40 μm thickness in a 1-in-4 series of hypothalamic sections.Slices between Bregma − 1.2 to − 1.6 (Paxinos and Franklin Mouse Brain Atlas) were used to asses viral expression and immunohistochemical detection of c-Fos protein.
Brain sections were washed with PBS three times and incubated for 2 h in a blocking solution containing 10% normal goat serum (NGS, Sigma-Aldrich, St. Louis, MO) and 0.5% Triton X-100 in PBS.Next, sections were incubated for 24-48 h in primary antibody (rabbit anti-c-Fos 1:500, Cell Signalling), 2% NGS, and 0.2% Triton X-100 in PBS.
Brain sections were washed with PBS three times and incubated for 2 h with a secondary antibody (goat anti-rabbit, 1:200 Alexa Fluor® 488, Abcam), 2% NGS, and 0.2% Triton-X 100 in PBS.Sections were washed a further three times in PBS before being mounted onto glass microscope slides, coverslipped, and imaged for c-Fos in the Arc using an Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan).

Statistical design
Statistical analyses were performed using GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA).Food consumption data was analysed using a two-way mixed analysis of variance (ANOVA), with genotype (AgRP Cre/+ vs NPY Cre/+ ) as a between-subjects factor and drug (CNO vs Saline) as a within-subjects factor.Paired t-tests were used to compare the number of bouts and time spent feeding between CNO and saline-treated days.

Viral-mediated DREADD expression in the arcuate nucleus of the hypothalamus
Expression of the Cre-dependent AAV-DIO-hM3Dq-mCherry viral vector in the Arc of NPY Cre/+ and AgRP Cre/+ mice was confirmed via confocal microscopy (Fig. 1 B, C).The animals showed mCherry expression only in the Arc of the hypothalamus.To assess the effectiveness of the DREADD activation strategy, animals were injected with CNO two hours prior to perfusion.Chemogenetic activation of NPY neurons resulted in robust expression of c-Fos protein in the Arc (Fig. 1  E), with a high degree of overlap with hM3Dq-transduced neurons, all c-Fos positive cells were also mCherry positive.(Fig. 1D, E and F).

Chemogenetic activation of Arc NPY neurons increases intake of fat compared with carbohydrates
To examine the effects of chemogenetic activation of NPY neurons on macronutrient preference, NPY Cre/+ and AgRP Cre/+ hM3Dq mice were given i.p. injections of CNO and saline across two test days and exposed to 4% intralipid (fat) and 10% sucrose (carbohydrate).Both macronutrients were prepared as solutions and were calorie-matched to prevent the influence of form (solid vs. liquid) and caloric value.CNO treatment significantly increased the intake of intralipid compared with saline treatment (Fig. 3A) in NPY Cre/+ hM3Dq (F(3, 96) = 4.298, p = 0.0009) and AgRP Cre/+ hM3Dq (F(3, 104) = 7.71, p = 0.0001) animals.However, no effect of CNO injection was observed on the consumption of sucrose solution in any of the expermintal groups, when compared to control (Fig. 3B).For control group data, see supplementary data fig.2.

Chemogenetic activation of Arc NPY neurons increases operant responding for fat but not carbohydrates
Our results demonstrated that activation of NPY or AgRP Arc neurons significantly increase intake of intralipid (fat) but not sucrose (carbohydrate) solutions.This finding raises the question of whether the activation of Arc NPY or AgRP neurons increases motivation to obtain fat over carbohydrates in mice.For control group data, see supplementary data fig.2. To investigate the effect of activation of Arc NPY and AgRP neurons on operant responding, animals were trained to lever press for 10% sucrose and 4% intralipid under fixed ratio 1 (FR1), FR3, FR5, and progressive ratio (PR) schedules (Fig. 4A-D).Animals were treated with either CNO or saline on FR5 and PR test days.Chemogenetic activation of NPY and AgRP NPY neurons via CNO administration significantly increased FR5 responding for intralipid compared with sucrose in both NPY Cre/+ (t (5) =2.75, p = 0.0204) and AgRP Cre/+ (t (5) = 2.18, p = 0.0538) hM3Dq animals (Fig. 4E).The NPY Cre/+ and AgRP Cre/+ hM3Dq animals also exhibited a notable rise in operant responding for fat over carbohydrates, as evidenced by significant increases (t (5) =4.65, p = 0.0009) and (t (5) =2.02, p = 0.0709) respectively, under the PR schedule.(Fig. 4F).For control group data, see supplementary data fig.3.

Chemogenetic activation of Arc NPY neurons increases consumption of 10% sucrose, but not a non-caloric sweetener
To investigate whether chemogenetic activation of Arc NPY and AgRP neurons influences calorie-seeking or palatability-seeking behaviour, we exposed NPY Cre/+ and AgRP Cre/+ hM3Dq animals to a 10% sucrose and non-caloric but palatable 0.1% saccharin solution.Animals were treated with i.p. injections of CNO and saline across two test days.
For control group data, see suplimentary data Fig. 4.

Discussion
In the present study, we investigated the role of both AgRP and NPY neurons in the Arc on food consumption and food selection in response to caloric and non-caloric cues.We activated NPY or AgRP neurons in the Arc of NPY Cre/+ as well as a widely-used AgRP Cre/+ mouse line.While AgRP-Cre mice express Cre in ~85% of NPY neurons (which coexpress AgRP), NPY Cre/+ mice have the advantage of expressing Cre in all NPY-expressing neurons in the Arc.Therefore, the results of our study provides a more comprehensive understanding of how this population of neurons is involved in various aspects of feeding behaviour.Our findings indicate that chemogenetic activation of Arc NPY and AgRP neurons in  fed mice increases chow consumption.This is consistent with previous findings demonstrating that chemogenetic or optogenetic activation of Arc AgRP neurons increases food consumption in fed mice (Krashes et al., 2011b;Aponte et al., 2011).
The results of our second experiment indicate that chemogenetic activation of Arc NPY and AgRP neurons influences macronutrient preferences.We observed that activating either Arc NPY or AgRP neurons increases the consumption of intralipid (fat) solution in well-fed mice, without observable influence on the consumption of sucrose solution.This finding contrasts with several previous studies which suggest ICV administration of NPY induces preferential intake of carbohydrates over fat when animals can choose between both palatable diets (Stanley et al., 1989;Stanley et al., 1985;McConn et al., 2018).In our study, intralipid and sucrose solutions were calorie-matched.Therefore, our findings suggest that activating NPY neurons in the Arc induces a specific macronutrient preference for fat over carbohydrate that is unrelated to calorie-seeking.These data also suggest that increased intralipid consumption during chemogenetic activation of NPY neurons is also independent of the baseline palatability of fat, as we observed no preference for fat or carbohydrate in animals injected with control virus nor in saline treated NPY Cre/+ hM3Dq mice.
The findings from our study also demonstrate that activating Arc NPY and AgRP neurons can increase motivation to obtain palatable rewards.Chemogenetic activation of Arc NPY and AgRP neurons significantly increased operant responding to obtain a fat reinforcers, but not carbohydrates, under fixed and progressive ratio schedules.Consistent with our findings, Tracy and colleagues demonstrated that infusing AgRP into the Arc selectively increased operant responding for fat rather than carbohydrate (Tracy et al., 2008).Furthermore, other studies suggest that stimulating or administering NPY in different brain regions, such as the lateral hypothalamus, nucleus accumbens shell, or ventral tegmental area increases operant responding to palatable food (Brown et al., 1998;Pandit et al., 2014).To our knowledge, this study is the first to demonstrate that chemogenetic activation of the broader population of Arc NPY neurons increases operant responding for palatable rewards and induces a macronutrient preference for fat solutions over sucrose.Our results demonstrate the importance of Arc NPY neurons in operant responding to specific macronutrients.
We also examined whether increased responding to palatable cues was related to seeking calories or seeking palatable substances.Our findings indicate that Arc NPY and AgRP neurons are not sufficient to increase responding to non-caloric palatable solutions, as chemogenetic activation of Arc NPY and AgRP neurons resulted in increased sucrose solution consumption with no significant increase in the consumption of non-caloric sweetener (0.1% saccharin).In contrast to our findings, two previous studies demonstrated that ICV administration of NPY increased the consumption of both palatable sucrose and saccharine solutions (Lynch et al., 1993;Furudono et al., 2006).Furudono and colleagues demonstrated that ICV infusion of NPY increases the consumption of non-caloric sweeteners, indicating the effect of central NPY in regulating palatability-induced consumption (Furudono et al., 2006).Furthermore, another study demonstrated that ICV administration of NPY increased the consumption of both caloric (sucrose) and non-caloric (saccharin) palatable solutions (Lynch et al., 1993).These combined results emphasise that NPY may act differently across discrete areas of the brain to regulate distinct aspects of feeding behaviour.Notably, AgRP neurons are highly sensitive to caloric value and are inhibited during exposure to caloric nutrients, but not non-caloric palatable food (Beutler et al., 2017;Su et al., 2017).Su and colleagues also demonstrated that the replacement of non-caloric gel with the caloric diet decreased AgRP neuronal activity (Su et al., 2017).Therefore, co-expression of AgRP in Arc NPY neurons may account for the differing response of these neurons to noncaloric solutions compared with NPY neurons in other brain regions.
In summary, this study demonstrates that chemogenetic activation of the broader population of Arc NPY neurons has an equivalent effect on feeding behaviour as activation of Arc AgRP neurons.Considering Arc NPY neurons are responsive to cues related to hunger, it seems likely that activating these neurons induces calorie-seeking to regulate energy homeostasis and prevent the aversive sensation caused by activating these neurons.Our results also indicate that Arc NPY neurons mediate macronutrient selection by preferentially increasing fat intake.

Fig. 1 .
Fig. 1.Confirmation of expression of the Cre-dependent AAV-DIO-hM3Dq-mCherry viral vector in the Arc.(A and F) Stereotaxic microinjections were directed to the arcuate nucleus of the hypothalamus (Arc) in NPY-Cre and AgRP-Cre mice.(C and G) Photomicrograph of hM3Dq-mCherry transduction in the Arc.Scale bars B = 10 μm, G = 100 μm.(D, I) Activation of hM3Dq-mCherry neurons resulted in increased c-Fos protein activity in the Arc.(E, J) Merged image shows co-localization of the hM3Dq-mCherry and Fos-protein in the Arc.Scale bars C, D, E = 20 μm and H, I, J = 10 μm.

Fig. 2 .
Fig. 2. Chemogenetic activation of NPY and AgRP neurons in the ARC increases food consumption.(A) CNO-treatment of NPY Cre/+ and AgRP Cre/+ hM3Dq animals increased chow intake 2-6 h following injection, compared to saline injected animals.(B) CNO treatment did not affect chow consumption in animals transduced with a control virus.(C, D) This increase in consumption of food in NPY Cre/+ (n = 14) and AgRP Cre/+ (n = 14) hM3Dq animals following CNO treatment was the result of the increase in both (C) feeding bout numbers and (D) meal duration.Data are means ± SEM (* p < 0.05).

Fig. 3 .
Fig. 3. Chemogenetic activation of NPY and AgRP neurons in the Arc increases intake of an intralipid, but not sucrose solution in a two bottle choice test.(A) CNO treatment in NPY Cre/+ (n = 14) and AgRP Cre/+ (n = 14) hM3Dq animals significantly increased the consumption of intralipid.(B) CNO treatment in NPY Cre/+ and AgRP Cre/+ hM3Dq animals did not affect the consumption of sucrose.(*p < 0.05).Data are means ± SEM.