Article Figures & Data
Figures
- Figure 1.
Vitamin D3 levels modulate food intake (FI) and body weight (BW). A, HF and HF-D mice exhibited no difference in net body weight between groups over the first 50 d of diet exposure, but a significant increase for the HF-D mice was observed for days 50-100 (n = 10, 10). B, Binned change in body weight for HF and HF-D groups from day 50 to day 101 (n = 10, 10). C, HF and HF-D mice maintained significant differences in body weight throughout the transition from group-housed to single-housed environment from day 101 to day 116 (n = 5, 5). D, Cumulative food intake for HF and HF-D groups from day 101 to day 116 (n = 5, 5). E, Plasma levels of 25(OH)D3 from the HF and HF-D mice on day 165 of their diets (n = 5, 5) used in C and D. F, G, A separate cohort of HF and HF-D mice (n = 5, 5) was killed on day 50 of their diets (F), and serum analysis of 25(OH)D3 levels revealed a significant reduction, despite no differences in body weight (G). H, These mice exhibited a small yet significant reduction in serum calcium levels (n = 5,5). I, Single-housed mice chronically exposed to Ch or Ch-D diets had no significant difference in body weight between groups over days 0-50 or 50-101 (n = 5,5). J, These mice did, however, exhibit increased food intake over both time periods (n = 5, 5). K, At the end of this experiment on day 127, the Ch-D mice were found to have reduced plasma levels of 25(OH)D3. L, A separate cohort of group-housed Ch and Ch-D mice (n = 5, 5) was killed on day 50, and were found to have significantly reduced serum 25(OH)D3 levels. M, These mice displayed no differences in body weight (n = 5, 5). N, Additionally, no differences in serum calcium levels were detected (n = 5, 5).
- Figure 2.
A, After 26 d of chronic HF exposure, naive mice were treated with vehicle, calcitriol, or leptin, and displayed reduced net body weight in response only to calcitriol for the first day after single acute treatment (n = 8, 8, 8). B, Calcitriol treatment also reduced food intake for the first day after treatment in these mice (n = 8, 8, 8). C, An additional long-term HF exposure cohort was generated to assess the effects of acute calcitriol treatment on serum calcium levels. After 32 d of the HF diet, mice were treated in the short term and killed 24 h later. No changes in serum calcium levels were observed (n = 8, 8). D, CPA was performed on a separate cohort of naive mice to assess potential aversive effects of calcitriol that could contribute to altered feeding behavior. E, After 4 d of alternating conditioning treatments to specific chambers, there was no difference between groups in the change in the amount of time spent in the paired chamber, yet a significant reduction in net body weight was observed (n = 8, 8). All error bars indicate the SEM.
- Figure 3.
VDR is expressed in dopamine-producing, as well as in dopamine-receiving, neurons. A, Colocalization of TH (green) with VDR (purple) in subregions of the midbrain including the VTA and SN. White scale bars indicate a distance of 40 µm. B–E, Colocalization of Cre recombinase (green) with VDR (purple) using the D1R-Cre and D2R-Cre transgenic mouse lines in the nucleus accumbens (Acb; B, C), as well as in the dorsal striatum (Str; D, E).
- Figure 4.
Calcitriol treatment modulates gene expression differentially throughout dopamine circuitry. A–D, Mouse brain atlas diagrams (Paxinos and Franklin, 2004) showing typical dissections for qPCR analysis (red areas). E–H, Naive mice treated acutely with vehicle (−) or calcitriol (+; n = 5, 5) 6-7 h prior to being killed revealed differential effects of calcitriol in the midbrain, accumbens, and medial and lateral striatum (Str), as assessed by qPCR. All error bars indicate the SEM.
- Figure 5.
Calcitriol pretreatment enhanced dopamine release in mice and rats in response to amphetamine or dopamine transporter inhibition. All animals received acute pretreatment with calcitriol or vehicle 6-7 h prior to subsequent treatments. A, Microdialysis measuring tonic dopamine release in naive mice (n = 6, 6) revealed the effects of calcitriol on amphetamine-induced dopamine release (red arrow, amphetamine treatment). B, C, FSCV experiments in rats revealed the effects of calcitriol on evoked dopamine release upon treatment with amphetamine (calcitriol, n = 5; vehicle, n = 4) or dopamine transporter inhibition (GBR-12909 dihydrochloride; calcitriol, n = 4; vehicle n = 7). D, E, Representative traces for FSCV experiments. All error bars indicate the SEM.
- Figure 6.
Vitamin D3 levels alter amphetamine-related behaviors in mice. A, Binned locomotor activity of HF and HF-D mice during habituation, saline injection (black arrow), and acute amphetamine treatment (red arrow). B, HF and HF-D cumulative activity for 20 min after amphetamine administration (n = 5, 5). C, D, Binned locomotor activity of Ch and Ch-D mice during habituation, saline injection (black arrow), and acute amphetamine treatment (red arrow; C), and Ch and Ch-D cumulative activity for 2 h after amphetamine (n = 4, 4; D). E, Binned locomotor activity in naïve mice pretreated with vehicle or calcitriol (n = 8, 8) 6-7 h prior to acute amphetamine treatment (red arrow). F, Cumulative activity of the pretreated mice over 2 h after amphetamine administration.
- Figure 7.
A, Oral amphetamine licks for HF and HF-D groups shown in 2 h bins across the 18 h test period (n = 5, 5). B, HF and HF-D group amphetamine preferences shown in 2 h bins across the 18 h test period. C, HF and HF-D amphetamine preference scores grouped for 0-8 and 8-18 h. D, E, Cumulative amphetamine licks for the HF and HF-D mice over the 18 h test period, as well as the total number of licks (amphetamine plus water). F, Oral amphetamine licks for naive mice after single acute calcitriol or vehicle treatment (6-7 h prior to testing) shown in 2 h bins across the 18 h test period (n = 12, 12). G, Amphetamine preference for the calcitriol- and vehicle-treated mice shown in 2 h bins across the 18 h test period. H, Calcitriol and vehicle amphetamine preference scores grouped for 0-8 and 8-18 h. I, J, Cumulative amphetamine licks over the 18 h test period, as well as the total number of licks (amphetamine plus water). All error bars indicate the SEM.
Tables
- Table 1:
Caloric breakdown of the different diets used for these experiments, as well as dietary vitamin D3 modifications
Diet Fat
(% kcal)Protein
(% kcal)Carbohydrate
(% kcal)Combined
(kcal/g)Dietary D3
(cholecalciferol) (IU/g)HF 35 15 50 4.3 1.10 HF-D 35 15 50 4.3 Range: 0.092-0.12 Ch 14 26 60 3.2 2.40 Ch-D 14 26 60 3.2 0.16 The range shown for the HF-D diet represents the values observed over multiple batches.
- Table 2:
Animal usage breakdown by cohort, experiment, species, diets, and days on the respective diets
Cohort Experiment Species Diets Diet length (d) 1 Chronic deficiency, body weight, FI Mouse, n = 10,10 HF, HF-D 116 (1) Chronic deficiency, plasma Mouse, n = 5, 5 HF, HF-D 165 2 Chronic deficiency, body Weight, FI Mouse, n = 5, 5 Ch, Ch-D 127 3 Chronic deficiency, body weight, serum Mouse, n = 5, 5 HF, HF-D 50 4 Chronic deficiency, body weight, serum Mouse, n = 5, 5 Ch, Ch-D 50 5 DIO, acute calcitriol, leptin, body weight, FI Mouse, n = 8, 8 HF 30 6 Acute calcitriol, CPA Mouse, n = 8, 8 Std Chow ∼63 7 qPCR analysis Mouse, n = 5, 5 Std Chow ∼63 8 Microdialysis Mouse, n = 6, 6 Std Chow ∼63 9 FSCV Rat, n = 9, 11 Std Chow n/a 10 Chronic deficiency, amph locomotor Mouse, n = 5, 5 HF, HF-D 166 (10) Chronic deficiency, amph licking Mouse, n = 5, 5 HF, HF-D 200 11 Chronic deficiency, amph locomotor Mouse, n = 4, 4 Ch, Ch-D 163 12 Acute calcitriol, amph locomotor Mouse, n = 8, 8 Std Chow ∼63 13 Acute calcitriol, amph licking Mouse, n = 12, 12 Std Chow ∼63 amph, Amphetamine; FI, food intake; Std, standard.
Gene Primer Drd1 Forward 5'-GAGCGTGGTCTCCCAGAT-3' Drd1 Reverse 5'-TCACTTTTCGGGGATGCT-3' Slc6a3 Forward 5'-CTGGTGCTGGTCATTGTTCT-' Slc6a3 Reverse 5'-AGCAGGGCTGTGAGGACTAC-3' Oprm1 Forward 5'-CCATCATGGCCCTCTATTCT-3' Oprm1 Reverse 5'-TGTTGGTGGCAGTCTTCATT-3' Tbp Forward 5'-AAAGGGAGAATCATGGACCAGAACAA-3' Tbp Reverse 5'-TGGACTAAAGATGGGAATTCCAGGAG-3'
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