Type 1 dopamine receptor (D1R)-independent circadian food anticipatory activity in mice

Circadian rhythms are entrained by light and influenced by non-photic stimuli, such as feeding. The activity preceding scheduled mealtimes, food anticipatory activity (FAA), is elicited in rodents fed a limited amount at scheduled times. FAA is thought to be the output of an unidentified food entrained oscillator. Previous studies, using gene deletion and receptor pharmacology, implicated dopamine type receptor 1 (D1R) signaling in the dorsal striatum as necessary for FAA in mice. To further understand the role of D1R in promoting FAA, we utilized the Cre-lox system to create cell type-specific deletions of D1R. We were unsuccessful in obtaining conditional deletion of D1R when using transgenically driven D1R-Cre. We then created a conditional deletion of D1R in GABA neurons using Vgat-ires-Cre line, which had attenuated FAA, but the amount was higher than expected based on prior results using a constitutive knockout of D1R, D1R KODrago. This result prompted us to re-test the original D1R KODrago line, which expressed less FAA than controls, but only moderately so. To determine if genetic drift had diminished the effect of D1R deletion on FAA, we re-established the D1R KODrago knockout line from cryopreserved samples. The reestablished D1R KODrago-cryo had a clear impairment of FAA compared to controls, but still developed increased activity preceding mealtime across the 4 weeks of timed feeding. Finally, we tested a different deletion allele of D1R created by the Knockout Mouse Project. This line of D1R KOKOMP mice had a significant impairment in the acquisition of FAA, but eventually reached similar levels of premeal activity compared to controls after 4 weeks of timed feeding. Taken together, our results suggest that D1R signaling promotes FAA, but other dopamine receptors likely contribute to FAA given that mice lacking the D1 receptor still retain some FAA.


INTRODUCTION
Circadian rhythms link molecular processes within cells to large scale phenomena within organisms such as metabolism, hormonal signaling, and behavior [1][2][3]. The circadian timekeeping mechanism for photoentrainment has been linked to core transcriptional-translational feedback loops in many types of cells [4]. In mammals, the suprachiasmatic nucleus (SCN) serves as the main time keeper, orchestrating the 24 hour rhythms in many other brain regions and peripheral organs [5]. Through photoreceptors within the retina, the SCN is synchronized by daily light exposure [6,7].
Aside from photic input, the availability of food is an external cue that synchronizes circadian clocks in the brain and peripheral tissues independently of the SCN [8]. This is demonstrated in rodents with time-restricted food access during their rest phase, which causes a shift of circadian activity cycles while retaining nocturnal activity driven by the SCN. This shift is associated with a bout of locomotor activity that anticipates the daily scheduled meal by about two hours [9]. Remarkably, this food anticipatory activity (FAA) is robustly maintained in rodents with SCN lesions, indicating the presence of a food entrained oscillator separate from the SCN [10]. It is not clear if the molecular components of the food entrained oscillator are fully, partially, or non-overlapping with the light entrained oscillator present within the SCN [11][12][13]. The location of the neurons responsible for the anticipation of food and other related stimuli have been elusive [9,14,15] [ 16,17]. Recent studies have implicated everything from liver-derived ketones (requiring intact period gene in the liver) [18] to neuronal expression of Reverb-ɑ (Nr1d1) [19] to dopamine receptors 1 and 2 as important for mediating FAA [20][21][22].
Within the brain, dopamine signaling is essential for orchestrating movement and plays a major role in reward and motivation [23][24][25]. The dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) innervate several parts of the brain, with SN neurons projecting primarily to the dorsal striatum which regulates goal-directed locomotion, while VTA neurons project to the nucleus accumbens to drive motivation and learning [25].. Studies have shown functional overlap and interactions between the SNdorsal striatum and VTAnucleus accumbens dopamine circuits [26,27]. Dopamine neurons function by activating two distinct types of G-protein coupled receptors, D1-like and D2-like, which are expressed in abundant, intermingled populations in the striatum and nucleus accumbens, among other regions [28,29]. Activation of D1-like receptors increases intracellular cAMP production to increase neuronal activity and excitability while D2-like receptors function to lower the intracellular production of cAMP, thereby depressing activity in D2-expressing neurons [29]. Dopamine signaling is modulated by circadian machinery and, conversely, dopamine signaling influences circadian rhythms [20][21][22][30][31][32][33][34][35]. For example, Reverb-α, a circadian nuclear receptor, is a negative regulator of tyrosine hydroxylase (TH) mRNA levels; TH levels were found to be highest at night when REV-ERBα levels were lowest [34]. In addition, mice with REV-ERBα gene deletion had higher dopamine production and firing rates than did controls [34].
Another study reports that extracellular levels of dopamine are controlled by the dopamine transporter protein (DAT or Slc6a3) with levels of dopamine correlating the behavioral activity cycle [32]. Furthermore, Hood and colleagues demonstrated that period 2 (Per2) protein expression in the striatum follows a circadian pattern and is dependent on dopamine signaling, as rats with unilateral lesions to dopamine neurons lost their Per2 rhythm in the lesioned side but not contralateral side [31]. Therefore, further understanding of the timing and release of dopamine is necessary for the treatment of many disorders, including drug addiction [36].
Daily access to food leads to anticipatory behavioral activity that may also be dependent on dopamine signaling [30,37,38]. Previous studies of mice with a constitutive knockout (KO) of D1R showed attenuated FAA, whereas D2R KO mice showed normal FAA [21,39]. The D1R KO mice had a weakened PER2 RNA expression in their striatum as well. In mice lacking TH, viral reintroduction of dopamine production in the dorsal striatum was sufficient for FAA, suggesting that dopamine signaling to D1R neurons in the dorsal striatum permits the expression of FAA [21]. In addition, timed daily D1R agonist injection caused anticipatory activity even in mice fed ad libitum (AL), suggesting that D1R neurons can control behavioral rhythms [21]. Taken together, these results suggested that the SN dopamine-striatal D1R neurons may constitute a critical component of the circadian response to scheduled feeding. Thus, resolving the D1R-dopamine circuitry required for FAA would advance the field toward a circuit-level understanding of food as a zeitgeber. To that end we examined conditional deletion mutants of D1R and also re-examined constitutive D1R mutants. Both males and females were used for all studies.

Mouse husbandry and strains
To genotype mice, DNA was obtained from tail clippings from 2-week old mice, which were then digested with proteinase K and DNA was purified by isopropanol precipitation. Genomic PCR was performed to amplify D1R and Cre alleles. The D1R KO KOMP mice were genotyped through a third party using qPCR (Transnetyx). Genomic DNA of the D1R KO Drago and D1R KO KOMP lines was assessed using single polymorphic nucleotides (SNPs) for differences between genetic background comparable to C57BL/6J and C57BL/6N WT mice (Table II) by the Jackson Laboratory.

Food intake, behavioral measurements and calorie restriction conditions
Food intake was measured at 9-10 weeks of age. To calculate 60% CR food allotment per cohort, daily food intakes were measured per mouse over 48 hours.
These values were then averaged per group and 60% of the daily average was determined as the daily CR value. Food intakes were adjusted by 0.1 g per day to prevent excessive weight loss for some individual mice (experimenters were blind to genotypes during the experiment).
On video recording days, home cages only contained a minimum of sanichip bedding (100ml) and no cotton nestlet. During video recording, dim red lighting was provided with High Power 42 SMT RED LED PAR38 from LEDwholesalers.com. Home cage behavior measurements were obtained by video recording mice from a perpendicular angle to their home cages and analyzing these videos using an automated-behavior recognition software, HomeCageScan 3.0 [41], which annotates for the following behaviors: remain low, pause, twitch, awaken, distance traveled, turn, sniff, groom, food bin entry, chew, drink, stretch, hanging, jumping, rearing, walking, and unassigned behaviors.
These data were binned in 24 one-hour bins to quantify the temporal structure of activity. The behaviors hanging, jumping, rearing, and walking are designated as high activity. Total high activity was then determined by summation of high activity bins, while FAA ratios were calculated by dividing the final 3h of high activity over the total high activity of each mouse.
All mice entering CR were at least 10 weeks of age. Mice were individually housed on AL for 5-7 days prior to beginning CR with food intake studies conducted at 2-4 days preceding CR. Mice were then fed 60% of their daily caloric intake every day for 28 days at ZT 6 beginning on Day 0, or the start of CR. Body weights were measured every 7th day prior to scheduled mealtime, beginning from Day -7 and ending at Day 28.

Tissue histology and antibody labeling
For immunolabeling, brains were perfusion-fixed using 4% paraformaldehyde (Sigma) and post-fixed for 24h before being sectioned at room temperature using a vibrating blade microtome (VT-1000S, Leica Instruments).
Sections were imaged by confocal microscopy using a Nikon Eclipse Ti-E inverted microscope system linked to a computer system with NIS-Elements Imaging Software and FIJI was used for image processing [42].

Statistical analysis
Behavioral data were exported from HomeCageScan 3.0 as excel files, which were analyzed using MATLAB programs to sum, average, and visualize data. Statistical tests and graphs for all data described were performed using GraphPad Prism 9. Sample sizes for each experiment are indicated in the figure legends. Food intake data, which fell on a normal distribution, were tested for significance using an Unpaired T Test. Body weights were analyzed for differences using mixed-effects analysis with Tukey's post-test to examine differences between groups at each time point. For home-cage behavioral data, we also used mixed-effects analysis to examine the effects of genotype, time, and the interaction between time and genotye. We used Sidak's multiple comparisons test to compare behavior at each time point. To analyze a contingency table for mouse genotypes, a Chi-Square Test was performed to determine the dependency of genotype on viable offspring.

Conditional Deletion of D1R using D1R-Cre May Be Embryonically Lethal
To follow up on our previous work, which utilized a constitutive deletion mutant of D1R (denoted as D1R KO Drago ), we attempted to conditionally delete D1R by crossing D1R flox/flox mice [43] to D1-Cre transgenic mice [44] (Table I).
Surprisingly, backcrossing F 1 progeny (D1-Cre +/-; D1R wt/flox ,) to the parental D1R flox/flox line did not produce cKO progeny (D1-Cre +/-,D1R flox/flox ): out of 47 progeny examined, all were negative for this genotype (Table III; P<0.0001, Chi-Square). These results suggest that conditionally deleting D1R in all D1R-Cre expressing cells is embryonically lethal. However, since the location of the D1R-Cre transgene was not determined, the lack of cKO progeny can also be attributable to a genetic linkage of the D1R-Cre transgene with the D1R locus.
Nevertheless, the focus of our investigation does not concern the cause of embryonic lethality and therefore we turned to a different Cre driver line to achieve deletion of D1R.

Food Anticipatory Activity
A second attempt at a broad conditional deletion of D1R was made using a well-characterized Cre driver for inhibitory neurons, Vgat-Cre [45]. The only known D1R-expressing neurons in the dorsolateral striatum are GABAergic mediumsized spiny neurons [46]; thus, we expected that D1R should be deleted throughout the striatum in Vgat-Cre; D1R flox/flox mice. We verified deletion of D1R protein using antibody labeling. Both D1R and TH staining can be seen in control striatum tissue (Fig. 1A,C,E), whereas D1R antibody labeling is completely absent in Vgat-Cre; D1R flox/flox forebrain tissue (Fig 1B,D,F).
We measured daily food intake levels for control and Vgat-Cre; D1R flox/flox mice, and found no difference in mean food intakes: control animals ate 4.2g (+/-0.6g SD) and Vgat-Cre; D1R flox/flox 4.5 (+/-0.8 SD) for cKO (P=0.385, unpaired t test; Fig 1G). When food intake is normalized to body weight, the cKO mice are hyperphagic compared to controls (unpaired t test, P=0.0009, Fig 1H). Notably, cKO body weights are much lower than controls: prior to starting CR, the average  Fig 1I). However, when body weight is normalized by dividing each value by the day 0 body weight, it is clear that the response of body weight to timed 60% CR feeding is similar between Vgat-Cre; D1R flox/flox and control mice, which both lost similar fractions of weight across the experiment (mixed-effects analysis, fixed effects: time P<0.0001, genotype P = 0.26, time x genotype P = 0.77; P>0.05 for all pairwise comparisons; Fig 1J).
Both groups displayed similar waveforms of normalized high activity behaviors (defined as the sum of walking, cuddling, hanging vertically, hanging upside down, and jumping) on day 0, the first day of timed CR feeding (Fig 1K).
On day 14 of CR, the duration of nighttime activity appeared prolonged in the Vgat-Cre; D1R flox/flox mice while the premeal activity was lower amplitude and had a later oneset (Fig 1L). On day 28 of timed CR feeding, both groups showed premeal activity but the control group had an increased amplitude of FAA in comparison to Vgat-Cre; D1R flox/flox mice (Fig 1M). We plotted total activity levels (in seconds), observing that the Vgat-Cre; D1R flox/flox mice were considerably more active overall than controls: on days 0, 7, 12, and 21 activity was significantly increased (mixedeffects analysis, fixed effects: time P = 0.023, genotype P < 0.0001, time x genotype P = 0.187; for pairwise comparisons P = 0.038 day 0, P < 0.0001 day 7, P = 0.0154 for day 14, P = 0.0196 for day 21). For pre-meal (ZT 4-6) high activity behaviors in seconds, there was a strong effect of time (P<0.0001) but no interaction of genotype (P = 0.565), and a significant effect of time x genotype (P = 0.0269, mixed-effects analysis; Fig 1O). However, pairwise comparison of these data yielded no significant differences at any time point (P>0.05). To account for differences in total activity levels between Vgat-Cre; D1R flox/flox and control groups, we divided the ZT 4-6 high activity by the 24-hour total high activity, expressing activity as a fraction of total (Fig 1P). This normalization revealed strong fixed effects of time (P < 0.0001), genotype (P = 0.0029) and time x genotype (P < 0.0001). Pairwise comparisons demonstrated that Vgat-Cre; D1R flox/flox had significantly less FAA as compared to controls on days 14, 21, and 28 of CR (Sidak's multiple comparison, P < 0.0001 day 14, P=0.0076 day 21, P = 0.024 for day 28). We also noted that by day 28 of timed CR, all control mice showed at least 20% of their activity during ZT 4-6 whereas only a small fraction of the Vgat-Cre; D1R flox/flox mice exceed 20% and many showed very low activity during this preprandial time window.

Food Anticipatory Activity
Given that Vgat-Cre; D1R flox/flox mice demonstrated more FAA than we had predicted (we expected a near complete loss of FAA), we returned to the constitutive deletion strain D1R KO Drago that we previously demonstrated had a large impairment in FAA [21]. We verified D1R deletion using antibody staining of adult mice; D1R KO Drago  Prior to CR, D1R KO Drago mice were individually housed and measured for daily food intakes while on an AL diet. Mean food intake for control animals was 3.6g (+/-0.8 SD) while D1R KO Drago ate only 3.0g (+/-0.8 SD), which was significantly reduced (Fig 2G, P=0.020, unpaired t test). However, when food intake is normalized to body weight, the D1R KO Drago mice are hyperphagic compared to controls (P=0.035, unpaired t test, Fig 2H). Notably, the D1R KO Drago body weights are much lower than controls: prior to starting CR, the average weight of controls was 25.5g (+/-3.8 SD) while D1R KO Drago mice weighed only 18.3g (+/-2.8 SD) (Fig 2I). At time points prior to and during timed CR, the body weights of D1R KO Drago mice were less than that of controls (mixed-effects analysis: fixed effects of time P<0.0001, genotype P<0.0001, and time x genotype P<0.001; for pairwise comparisons using Sidak's multiple comparison, P<0.0001 for days -7, 0, 7, 14, 21, and 28; Fig 2I). However, when body weight is normalized by dividing each value by the day 0 body weight, it is clear that the response of body weight to timed 60% CR feeding is similar between D1R KO Drago  Both groups displayed similar waveforms of normalized high activity behaviors on day 0 (Fig 2K). On day 14 of CR, the amplitude of nighttime activity was higher in the D1R KO Drago mice, while their premeal activity was lower than controls ( Fig 2L). On day 28 of timed CR feeding, both groups showed premeal activity but the control group had an earlier and stronger increase in the amplitude of FAA in comparison to D1R KO Drago mice (Fig 2M). We plotted the total activity levels (in seconds), observing that the D1R KO Drago

Activity
Given that the Drd1 Drago KO strain showed more FAA than expected based on prior results (Gallardo et al., 2014), we speculated that mutations or selection for alleles present in this strain that promote FAA appeared in our line of D1R KO Drago mice. To test this hypothesis, we obtained the line again from the cryorepository at Jackson lab (Bar Harbor, ME, USA); we denote this line as D1R KO Drago-cryo to indicate that it is newly established in our colony (Table I). Prior to CR, mice were individually housed and measured for daily food intakes while on an AL diet. Mean food intake for control animals was 3.8g (+/-0.4 SD) while D1R KO Drago-cryo ate 4.4g (+/-0.6 SD) per day, which was significantly increased (Fig 3A,   P=0.012, unpaired t test). When food intake is normalized to body weight, D1R KO Drago mice are even more obviously hyperphagic compared to controls (P=0.0001, unpaired t test, Fig 3B). As with the other deletion strains, the D1R KO Drago-cryo body weights are much lower than controls: prior to starting CR, the average weight of controls was 25.7g (+/-5.8 SD) while D1R KO Drago-cryo mice weighed only 18.4g (+/-2.8 SD) (Fig 3C). At time points prior to and during timed CR, the body weights of D1R KO Drago- Fig 3C). However, when body weight is normalized by dividing each value by the day 0 body weight, it is clear that the response of body weight to timed 60% CR feeding is similar between D1R KO Drago-cryo and control mice, which both lost similar fractions of weight across the experiment (mixed-effects analysis, fixed effects: time P<0.0001, genotype P=0.597, time x genotype P=0.0656; P>0.05 for all pairwise comparisons; Fig 3D).
Both groups displayed similar waveforms of normalized high activity behaviors on day 0, with the D1R KO Drago-cryo KO showing a wider amplitude of nighttime activity (Fig 3E). On day 14 of CR, the amplitude of nighttime activity was higher in the D1R KO Drago-cryo mice, while their premeal activity was lower than controls ( Fig 3F). On day 28 of timed CR feeding, nocturnal activity continued to be pronounced in D1R KO Drago-cryo KO mice and they had much less noticeable premeal activity compared to the control group (Fig 3G). We plotted the total activity levels (in seconds), observing that the D1R KO Drago-cryo mice were more active than controls on days 0 and 7, but not at other measurements (mixed-effects analysis, fixed effects: time P<0.0001, genotype P=0.257, time x genotype P<0.0001; for pairwise comparisons P=0.807 day -7, P=0.0009 day 0, P=0.0430 for day 7, and P>0.05 days 14, 21, and 28; Fig 3H). of their daily activity during ZT 4-6, while the other half failed to show activity during this time window (Fig 3J).

A Novel Constitutive Deletion of D1R (Knockout Mouse Project), D1R KO KOMP , Has Delayed Acquisition of Food Anticipatory Activity
We next tested a novel constitutive KO of D1R, which we term D1R KO KOMP (Table I). This particular deletion strategy was attractive because it used a "knockout first" approach, creating a mutant allele that can later be reactivated by FLP recombinase [40]. In addition, the "flipped" allele can be deleted in a conditional manner by Cre recombinase [40]. As such, we first confirmed that the allele was disrupted by antibody labeling forebrain tissue using D1R antibody ( Fig   4A-F). While TH staining is apparent in both WT and D1R KO KOMP forebrain tissue, no D1R was present in the D1R KO KOMP forebrain tissue (Fig. 4A-F). In contrast, control samples had ample D1R staining in the striatum and other areas within the forebrain (Fig. 4C,E).
Daily average food intake was similar between controls, which at 3.2g (+/-0.7 SD), and D1R KO KOMP mice, which ate 3.0g (+/-0.7 SD) (P=0.43, unpaired t test; Fig 4G). When food intake is normalized to body weight, there was no difference between groups, unlike for all the other D1R mutants tested (P=0.62, unpaired t test, Fig 4H). In contrast to the other D1R deletion strains described above, the D1R KO KOMP mice had only a subtle decrease in body weight (Fig 4I).
In this experiment, all mice displayed similar waveforms of normalized high activity behaviors on day 0 ( Fig 4K). On day 14 of CR, the D1R KO KOMP mice clearly developed pre-meal activity, but it was of lower amplitude than controls (Fig 4L).
By day 28 of timed CR feeding, both groups showed strong pre-meal activity ( Fig   4M). We plotted the total activity levels (in seconds), observing that the D1R KO KOMP mice were similar to controls and that both groups tended to be less active overall as the experiment progressed (mixed-effects analysis, fixed effects: time P<0.0001, genotype P=0.550, time x genotype P=0.029; for pairwise comparisons P>0.05 for all measurements, Sidak's multiple comparison; Fig 4N). For pre-meal (ZT 4-6) high activity behaviors in seconds, there was a strong effect of time (P<0.0001), genotype (P<0.0001), and time x genotype (P<0.0001, mixed-effects analysis; Fig 4O). Pairwise comparison of these data showed significant differences on days 14, 21 and 28 of CR (P=0.0313 for day 14, P=0.0019 for day 21, and P=0.0057 for day 28; P>0.05 at all other measurements, Sidak's multiple comparisons test). To account for differences in total activity levels between D1R KO KOMP and control groups, we assessed activity as a fraction of total (Fig 4P)

DISCUSSION
Previously, we provided evidence that deletion of D1R in mice nearly eliminated behavioral anticipation but not the body temperature increase associated with a daily meal; we proposed that the circuitry sufficient for FAA involved D1R mediated signaling from the SN into the dorsal striatum [21]. More recently, we demonstrated that mice with hypomorphic mutations in paired-like homeodomain transcription factor 3, a transcription factor necessary for survival of 95% of SN dopamine neurons, defines a minimal population of dopamine neurons sufficient for FAA and metabolic entertainment to timed feeding [47]. To further resolve the circuitry of D1R-expressing neurons and their role promoting FAA, we created a conditional knockout of D1R in Vgat-Cre expressing neurons. This deletion eliminated D1R expression in all brain regions examined and significantly attenuated FAA, but some FAA was still present in the conditional KO mice. This led us to retrace our original findings: we repeated testing of FAA in D1R KO Drago and observed a less prominent attenuation of FAA than we expected based on prior findings. Similarly, testing of other constitutive knockouts of D1R (D1R KO Drago-cryo and D1R KO KOMP ) showed an impairment in FAA but also that the behavior was still evident (i.e. D1R promotes FAA but is not necessary for it to occur). In sum, our current study suggests that D1R is a key component of FAA but not essential for it to occur.
We considered methodological issues that could have influenced the outcomes of our study. Firstly, the use of D1R-Cre line to delete D1R with its endogenous promoter appeared to affect mice embryonically, such that no experimental mice were produced (Table III). This was surprising because of the several existing constitutive mutants of this allele, which show only slight decreases in viability, but have clear growth defects [48,49]. We speculate that depending on the timing of the deletion of the Drd1a gene, compensatory changes occur in the brain to permit viability. Another methodological issue that may be important in comparing results of food entrainment studies relates to the severity of calorie restriction. For example, LeSateur and colleagues (2018) observed that transgenic over-expression of D2R reduced FAA but only on a very mild restriction, whereas the same mice had ample FAA when temporally restricted to a short feeding time window. In prior studies, we have noted that 80% CR induced much lower FAA as compared to 60% CR [50]. It is possible that if we had increased the food allotment in our studies, that the amount of FAA demonstrated by the various D1R knockout strains may have been lessened.
The dissection of the neural circuitry required for FAA has remained elusive.
One study demonstrated a strong role for liver-derived ketones in driving food entrainment, yet the brain region(s) mediating responses to ketones has yet to be identified [18]. Furthermore, it appears that there are several brain regions capable of controlling activity cycles in dopamine-dependent manners [51]. Future studies using conditional genetic approaches may be able to delineate the contributions of separable dopamine and/or D1R populations in mediating the behavioral expression of FAA and/or the underlying food-entrained oscillator. Furthermore, the recently appreciated connection between dopamine signaling in the SCN and out of phase eating resulting in diet-induced obesity demonstrates that this is a complex area of research that deserves additional study [52].