Dopamine D1 receptor signalling in the lateral shell of the nucleus accumbens controls dietary fat intake in male rats

Central


Introduction
The modern food environment exposes us to an abundance of palatable food choices that can be readily consumed throughout a 24 h cycle.This has promoted the consumption of saturated fat and sugars beyond our metabolic needs and drives the current global obesity epidemic (Popkin, Adair, & Ng, 2012;Yau & Potenza, 2013).Both sugar and fat are highly palatable and perceived as rewarding (DiFeliceantonio et al., 2018;F.;Johnson & Wardle, 2014).The mesolimbic dopamine system plays a key role in reward-related feeding behaviour, and the nucleus accumbens (NAc) is a key dopaminergic target in which enhanced dopamine signalling is associated with reward (Berke, 2018;Cox & Witten, 2019;Volkow, Wise, & Baler, 2017;Wise, 2004;Wise & Robble, 2020).Various dopamine receptors, of which the dopamine receptor 1 (D1R) and dopamine receptor 2 (D2R) have received most attention, have been implicated in the control of palatable food consumption and thus in the development of obesity (de Weijer et al., 2011;Hryhorczuk et al., 2016;van de Giessen et al., 2013;Wang et al., 2001).Conflicting data exist on the role of D2R on palatable feeding; for example, lentivirus-mediated knockdown of D2R in the dorsal lateral Abbreviations: AP, anteroposterior; CD, control diet; D1R, dopamine receptor 1; D2R, dopamine receptor 2; DA, dopamine; DV, dorsoventral; fcHFHS, free-choice high-fat high-sugar; h, hours; LH, lateral hypothalamus; ML, mediolateral; MSN, medium spiny neuron; NAc, nucleus accumbens; RM ANOVA, repeated measures analysis of variance.
Many of the studies that investigated the effects of dopamine infusions on palatable feeding were not specific regarding the location of the infusion sites.For example, several studies mention the NAc as target site, yet the NAc consists of several subdivisions, including the core and lateral and medial shell, that all receive input from anatomically and functionally distinct ventral tegmental area (VTA) neuron populations (Lammel et al., 2012).We recently showed that depletion of dopamine terminals in the lateral shell of the NAc, but not the medial shell of the NAc or the dorsolateral striatum (DLS), promotes the intake of saturated fat in rats that were consuming a multi-component free-choice high-fat high-sugar diet (fcHFHS) (Joshi, Faivre, la Fleur, & Barrot, 2021).However, it is currently unclear which specific dopamine receptor controls consumption of fat.
Because dopamine depletion in the lateral shell of the NAc preferentially promotes fat intake in rats consuming a fcHFHS diet (Joshi et al., 2021), we investigated if targeted infusion of the D1R antagonist SCH2 3390, the D2R antagonist raclopride, or a combination of SCH2 3390 and raclopride, enhanced fat intake in rats consuming a fcHFHS diet.As these experiments revealed an injection site-specific sensitivity, we next investigated the effects of the D1R inhibition on fat intake along the rostro-caudal axis of the lateral shell of the NAc.

Animals
Male Sprague Dawley rats (Janvier labs, France) weighing 250-300 g were group-housed (4 per cage) in a temperature-(21 • C-23 • C) and light-controlled room (standard 12:12 light conditions, lights on at 07:00-19:00) with ad libitum access to standard control diet (CD; Teklad global diet 2918, 18.6% protein, 44.2% carbohydrate, and 6.2% fat, 3.1 kcal/g, Envigo, Horst, the Netherlands), a bottle of tap water, and cage enrichment (gnawing stick and PVC shelter) during seven-day acclimatization to the animal facility of the Netherlands Institute for Neuroscience (Amsterdam, the Netherlands).All procedures were approved by the Animal Ethics Committee of the Royal Dutch Academy of Arts and Sciences (KNAW, Amsterdam, the Netherlands) and in accordance with the guidelines on animal experimentation of the Netherlands Institute for Neuroscience.

Surgery
After seven days of acclimatization, rats were handled several times and rats underwent intracranial surgery during which two 26-gauge stainless steel guide cannula (C315G-SPC, cut 9 mm below pedestal, Plastics One, Bilaney Consultants GmbH, Düsseldorf, Germany), aimed bilaterally at the lateral shell of the NAc, were implanted at anteroposterior (AP): +1.8 mm, mediolateral (ML): ±2.9 mm, dorsoventral (DV): 6.0 mm (coordinates from bregma and using an angle of 2 • in the frontal plane, verticality was taken from dura).To investigate injection site-specific sensitivity, an additional cohort of rats was implanted with cannulas more caudally in the lateral shell of the NAc, at AP: +1.5 mm, ML ±2.9 mm, DV: 6.0 mm (with similar angle as described above).All rats were kept under adequate anesthesia during the surgery, with an intraperitoneal injection of a mixture of ketamine, xylazine, and atropine before the onset of surgery, and ketamine during the surgery.This mixture was prepared by combining 0.8 mL ketamine (100 mg/mL), 0.4 mL xylazine (20 mg/mL) and 0.2 mL atropine (0.05 mg/mL) and was injected at 1.4 mL/kg body weight.The animals were fixed in a stereotactic frame and guide cannulas were secured to the skull using dental cement and four screws.A 26-gauge stainless steel dummy cannula (C315DC without protection, Plastics One) was kept in the guide cannula.Immediately after surgery, rats received carprofen (0.5 mg/ 100 g body weight, subcutaneous) as an analgesic, and animals were housed individually.On post-surgery day 1, rats received a second carprofen injection.

Experimental design and infusion
One week after surgery, rats were provided with the fcHFHS diet, enabling them to choose between the following components: a container with nuggets of a nutritionally complete standard diet (Teklad global diet 2918, 18.6% protein, 44.2% carbohydrate, and 6.25 fat, 3.1 kcal/g, Envigo), a container with saturated fat (beef tallow Ossewit/Blanc de Boeuf, Vandemoortele, Belgium; 9 kcal/g), a bottle with 30% sucrose water (commercial-grade table sugar dissolved in tap water; 1.2 kcal/g), and a bottle with tap water.Consumption of diet components, as well as body weight, was measured daily.Consumption of the diet components was assessed by measuring pre-weighed food hoppers (CD diet), preweighed metal cups (fat), and pre-weighed bottles (water and 30% sucrose water).All rats gained weight constantly during the experiment.To familiarize the rats and to ensure that the cannulas did not stick or block, dummies caps were unscrewed twice per week.

Cohort 1
Following two weeks of fcHFHS diet consumption, 0.6 μg/0.5 μL SCH2 3390, 1 μg/0.5 μL raclopride, a combination of SCH2 3390 (0.6 μg/0.5 μL) and raclopride (1 μg/0.5 μL), or 0.9% saline was administered in a balanced crossover design.After each injection, 3 days of washout was allowed.Before infusions, all food components were removed from the cage at 09:00.Injector cannula (C315I/SPC, Plastics One, Bilaney Consultants GmbH, Düsseldorf, Germany), projecting 1 mm below the guide cannula, were inserted into the guide cannula at around 15:00, and animals received bilateral infusions of 0.5 μL fluid per site at a rate of 0.3 μL/min via a syringe infusion pump in a volume mode A. Joshi et al. (Harvard PHD 22/2000 Syringe Pump).Injections were confirmed by observing fluid movement in the tubing (0.46 mm diameter) with the help of a small air bubble.After completion of the injection, the injector was left in place for 1 min to allow for diffusion.Upon completion of the infusion, all food components were returned to the cage and individual food components were measured 2, 5, and 24 h following infusion.

Perfusion
Rats were anesthetized with an overdose of pentobarbital and transcardially perfused with ice-cold 0.9% saline followed by 4% paraformaldehyde in 0.9% saline, and collected brains were postfixed overnight.Brains were washed in phosphate buffered saline, cryoprotected in 30% sucrose at 4 • C overnight, and subsequently frozen on dry ice and stored at − 80 • C. Cryostat sections were cut at 35 μm and mounted on Superfrost Plus slides (Fisher, Gerhard Menzel GmbH, Germany) and stained for Nissl staining with thionine.Stained sections were examined under the microscope to determine the placement of the cannula.

Data analysis and statistics
Data are presented as mean ± SEM and were analysed using GraphPad Prism 8.04 (GraphPad Software, La Jolla California USA).Two group comparisons were performed by two-tailed Student's t-test (total kcal intake).Repeated measure analysis of variance (RM-ANOVA) was performed to compare drug treatment, individual timepoint, food component and interactions between these factors.Post hoc analysis was performed using Bonferroni's multiple comparison paired t-tests using saline treatment as control.For all cases, a p value < 0.05 was considered significant.See Results section for statistical details of individual experiments, including statistical tests used, t, p, F-values, and number of subjects or samples tested.

Inhibition of D1R in the lateral shell of the NAc promotes fat intake during consumption of a fcHFHS diet
To determine which receptor mediates the effect of dopamine on fat intake when animals are consuming a fcHFHS diet, we infused the D1R antagonist SCH2 3390 and the D2R antagonist raclopride in the lateral shell of the NAc (Fig. 1A and B).From the 24 animals that underwent cannula placement surgery, 12 animals showed a correct placement (depicted in Fig. 1C as blue dots).Cumulative diet component intake was measured over the first 2 h, over 5 h, and over 24 h following infusion (Fig. 1D-G).Two-way RM ANOVA on the 2 h data revealed a significant food type * drug effect (F (6, 66) = 2.660, p = 0.0226), and post hoc RM analysis on mean kcal intake for different diet components revealed a significant drug effect for fat consumption at 2 h (F (2.5, 27.0) = 5.4, p = 0.0075), but not for consumption of chow, sucrose, or total caloric consumption over 2 h following infusion (Fig. 1D).Bonferroni's multiple comparisons paired t-tests revealed that infusion of SCH2 3390 (p = 0.0023) and the co-infusion of SCH2 3390 and raclopride (p = 0.0478) promoted fat intake 2 h following infusion (Fig. 1D).Fig. 1E depicts the individual changes per animal between the saline and SCH2 3390 infusion.No significant difference for any diet component or total caloric consumption was observed for consumption over the 5 h or 24 h following infusion (Fig. 1F and G).

Inhibition of D1R in the rostral, but not caudal, region promotes fat intake during consumption of a fcHFHS diet
As mentioned above, we observed that inhibition of D1R in the lateral shell of the NAc promotes fat intake during consumption of a fcHFHS diet.Furthermore, detailed inspection of infusion sites along the rostro-caudal axis of the lateral shell of the NAc revealed that two out of three rats with cannulas that were placed more caudally (bregma <1.8 mm) had normal fat consumption in response to D1R inhibition.Thus, to investigate this infusion site-specificity along the rostro-caudal axis of the lateral shell of the NAc, we performed a follow-up experiment with additional infusions sites along this axis.From the 23 rats that underwent cannula placement surgery, eight rats had correct placements in the rostral lateral shell of the NAc and seven rats showed correct placements in the caudal lateral shell of the NAc (see Fig. 2A and B and 3A,B, respectively).Within the rostral extent of the lateral shell of the NAc, infusion of SCH2 3390 promoted fat consumption at 2 h following infusion (Fig. 2C; t 7 = 3.3, p = 0.013).Fig. 2D depicts the individual changes per animal between the saline and D1-antagonist infusion (Fig. 2D).No effects were observed at other time points or for other diet components (Fig. 2C,E,F).Within the caudal extent of the lateral shell of the NAc, only a small but significant effect of SCH2 3390 on sugar intake was observed at 24 h (Fig. 3F; t 6 = 2.5, p = 0.045).No additional drug effect on mean kcal intake for other diet components was observed over 2 h (Fig. 3C) or over 5 h (Fig. 3E) following infusion.Fig. 3D depicts the individual changes for fat intake per animal between the saline and SCH2 3390 infusion at 2 h.In addition, we also combined the data of Figs. 2 and 3 in a separate analysis to test for interaction effect between drug and canula placement.The RM repeated ANOVA detected an effect of drug (F (1.13) = 10.325,p = 0.0007), but no interaction effect.Taken together, we show that inhibition of D1R in the lateral shell of the NAc promotes fat intake during consumption of a fcHFHS diet and that this effect is stronger in the rostral part of the lateral shell of the NAc than in the caudal part.

Discussion
We have recently demonstrated that lesioning of dopaminergic inputs to the lateral part, but not to the median part, of the NAc shell promotes consumption of the dietary fat component in rats that are consuming a fcHFHS diet (Joshi et al., 2021).In this study, we demonstrate that inhibition of D1R, but not inhibition of D2R, in the lateral shell of the NAc promotes fat consumption in rats consuming fcHFHS diet.This highlights the role of D1R in the lateral shell of the NAc to mediate the effects of dopamine on fat preference in the fcHFHS paradigm.Moreover, this study also reveals that the effects of D1R inhibition are most pronounced in the rostral part of the lateral shell of the NAc.
We demonstrate that inhibition of D1R, but not inhibition of D2R, in the lateral shell of the NAc promotes fat consumption in rats consuming the fcHFHS diet.Although dopamine effects within the NAc on fat intake have been studied extensively (Baldo, Sadeghian, Basso, & Kelley, 2002;Durst, Konczol, Balazsa, Eyre, & Toth, 2019;Lardeux, Kim, & Nicola, 2015;Ragnauth, Znamensky, Moroz, & Bodnar, 2000;Will, Pratt, & Kelley, 2006), these studies often focused on the medial part of the NAc shell.For example, a D1 agonist injected in the median shell of rats suppressed consumption of fat food (Durst et al., 2019).However, several studies also failed to observe an effect of dopamine signaling manipulation in the NAc on feeding behavior in general, or on fat consumption in particular (Baldo et al., 2002;Lardeux et al., 2015;Will et al., 2006).These observations are in line with our own previous observation that local lesions of dopamine neuron terminals in the medial part of the shell of the NAc did not alter fat consumption in rats consuming the fcHFHS diet (Joshi et al., 2021).One study has targeted the lateral part of the ventral striatum (Inoue et al., 1995), which included the lateral shell of the NAc; this study demonstrates that D2R antagonism and agonism in the ventral lateral striatum reduces and promotes consumption of a standard control diet, respectively, whereas D1R receptor activation or inhibition did not alter feeding behavior in female rats (Inoue et al., 1995).Consumption of fat intake was unfortunately not investigated in this study (Inoue et al., 1995).
Given the proposed role for dopamine in enhancing reward value (Berke, 2018;Cox & Witten, 2019;Volkow et al., 2017;Wise, 2004;Wise & Robble, 2020), it might seem surprising that we observed an increase in fat intake following D1R inhibition in the lateral shell of the NAc.A potential explanation could be that the downstream areas of the lateral NAc are distinct from the medial part and involve inhibiting projections to neuronal populations that are involved in the promotion of fat consumption.Thus, when removing this inhibitory dopamine tone, this may promote fat consumption.Exploring the downstream anatomical targets is a logical next step in understanding the role of the lateral shell of the NAc in feeding behavior.
Against our expectation, we did not observe effect of D2R inhibition on the consumption of any fcHFHS diet component.Systemic administration of a relatively low and high dose of raclopride, increases and decreases, respectively, the consumption of a high fat diet (Baker, Osman, & Bodnar, 2001).Raclopride increases dopamine release, whereas quinpirole decreases dopamine release, in the striatum of freely moving rats (See, Sorg, Chapman, & Kalivas, 1991).A potential explanation is that D2-MSNs in the lateral shell of the NAc are not involved in feeding-related behavior under the conditions tested in our study.
Observations in our first experimental cohort revealed a larger effect of D1R inhibition on fat consumption when the cannula was placed more rostrally in the lateral shell of the NAc.We therefore inhibited D1R in either the more rostral part or the caudal part of the lateral shell of the NAc in a second experimental cohort.Interestingly, D1R inhibition in the rostral part of the lateral shell of the NAc promoted fat consumption in all animals tested, but D1R inhibition in the caudal part of the lateral shell of the NAc failed to reach significance.However, we do have to be cautious with our conclusion that there is a clear difference between the results observed following rostral or caudal placement, as we did not observe an interaction effect.Furthermore, some animals with caudal placements did increase their fat intake following infusion.Earlier studies have revealed contrasting control over appetitive and aversive states along the rostro-caudal axis of the NAc shell.Microinjections of opioids in the rostral shell exert positive hedonic orofacial reactions to sucrose taste, whereas in the caudal shell opioids exert an aversive response, and such a difference along the rostro-caudal axis has been shown for the ventral pallidum as well (e.g.(Castro & Berridge, 2014;Castro, Terry, & Berridge, 2016;Ho & Berridge, 2014;Mahler, Smith, & Berridge, 2007;Mitchell, Berridge, & Mahler, 2018;Smith & Berridge, 2007).In addition, blocking glutamate signaling in the rostral NAc shell promotes eating, whereas in the caudal shell it elicited defensive behavior to a predator; and these effects were dependent on NAc dopamine (Reynolds & Berridge, 2002;Richard & Berridge, 2011).Therefore, it is possible that the lateral shell of the NAc might have a similar hedonic hotspot organization along its rostro-caudal axis in response to dopamine receptor modulation.However, it seems that this would be opposite to the organization as described for opioid and glutamate modulation as our data point to a suppressing effect of dopamine on palatable fat consumption.In addition, a heterogenous function over the rostro-caudal axis may be due to different innervation and neurotransmitter and modulator systems along this axis (Delfs, Zhu, Druhan, & Aston-Jones, 1998;Park, Aragona, Kile, Carelli, & Wightman, 2010;Vaccarino & Rankin, 1989) or different downstream targets, clearly warranting further future investigation in relation to consumptive behavior of (palatable) food.Our experiments were performed at the end of the light period (ZT8), when the drive to consume calories increases in order to prepare for the active (dark) period.As often relative little information is given regarding specific timing of experiments (Nelson, Bumgarner, Walker, & DeVries, 2021), it is difficult to compare our data with previous studies.Dopamine levels in the striatum increase during the time window in which we assessed caloric intake (Castaneda, de Prado, Prieto, & Mora, 2004), thus it would be of interest to study whether the effect of D1R inhibition on fat intake is specific to this time window of increasing dopamine levels or whether the effect would be similar during the active (dark) period.
In this study we focused on effects of dopamine signaling in the NAc on fat intake in male rats, as our previous work on dopamine depletion and fat intake was also performed in males (Joshi et al., 2021).However, it is currently unclear how female rats respond to the fcHFHS diet.Therefore, future experiments should also address this critical point to include studies in female rats, in order to characterize their response to the diet, and to determine how dopamine is involved in feeding behavior in females.
In summary, our observations highlight a role for D1R in the lateral shell of the NAc in control of dietary fat intake.Moreover, our data emphasizes the need for further research on the specific neural mechanisms and anatomical circuits that mediate the effects of dopamine signaling on (palatable) food consumption.

Declaration of competing interest
None.

Fig. 1 .
Fig. 1.D1R and D2R inhibition in the lateral shell of the NAc and fcHFHS diet consumption.Experimental timeline for the infusion study (A).An example of cannula placement, with injection site indicated by an asterisk symbol (B).Reconstruction of correct cannula placement (indicated by blue dots), based on the atlas of Paxinos and Watson (2014); the distance in mm from bregma is given on the right side of each neuroanatomical reconstruction image (C).Consumption of fcHFHS diet components following infusion with saline, SCH2 3390 (D1-anta), raclopride (D2-anta), or both (infusions were done in randomized crossover design) over 2 h following infusion (D), with enlarged figure for fat consumption following SCH2 3390 infusion (E), and consumption over 5 h (F) and over 24 h (G) following infusion.*, p < 0.05; **, p < 0.01.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 .
Fig. 2. D1R inhibition in the rostral part of the lateral shell of the NAc and fcHFHS diet consumption.An example of cannula placement, with injection site indicated by an asterisk symbol (A).Reconstruction of correct cannula placement (indicated by blue dots), based on the atlas of Paxinos and Watson (2014); the distance in mm from bregma is given on the right side of each neuroanatomical reconstruction image (B).Consumption of fcHFHS diet components following infusion with saline or D1-antagonist SCH2 3390 (infusions were done in randomized crossover design) over 2 h following infusion (C), with enlarged figure for fat consumption following D1-antagonist SCH2 3390 infusion (D), and consumption over 5 h (E) and over 24 h (F) following infusion.*, p < 0.05.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3 .
Fig. 3. D1R inhibition in the caudal part of the lateral shell of the NAc and fcHFHS diet consumption.An example of cannula placement, with injection site indicated by an asterisk symbol (A).Reconstruction of correct cannula placement (indicated by blue dots), based on the atlas of Paxinos and Watson (2014); the distance in mm from bregma is given on the right side of each neuroanatomical reconstruction image (B).Consumption of fcHFHS diet components following infusion with saline or SCH2 3390 (infusions were done in randomized crossover design) over 2 h following infusion (C), with enlarged figure for 2 h fat consumption following D1-antagonist SCH2 3390 infusion (D), and consumption over 5 h (E) and over 24 h (F) following infusion.*, p < 0.05.(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)