Group housing and nest building only slightly ameliorate the cold stress of typical housing in female C57BL/6J mice

Abstract

Huddling and nest building are two methods of behavioral thermoregulation used by mice under cold stress. In the laboratory, mice are typically housed at an ambient temperature (T_a) of 20°C, well below the lower end of their thermoneutral zone. We tested the hypothesis that the thermoregulatory benefits of huddling and nest building at a T_a of 20°C would ameliorate this cold stress compared with being singly housed at 20°C as assessed by heart rate (HR), blood pressure (BP), triiodothyronine (T3), brown adipose (BAT) expression of Elovl3 mRNA, and BAT lipid content. A series of experiments using C57BL/6J female mice exposed to 20°C in the presence or absence of nesting material and/or cage mates was used to test this hypothesis. Mice showed large differences in HR, BP, shivering, and core body temperature (T_b) when comparing singly housed mice at 20°C and 30°C, but only a modest reduction in HR with the inclusion of cage mates or bedding. However, group housing and/or nesting at 20°C decreased T3 levels compared with singly housed mice at 20°C. Singly housed mice at 20°C had a 22-fold higher level of BAT Elovl3 mRNA expression and a significantly lower triacylglycerol (TAG) content of BAT compared with singly housed mice at 30°C. Group housing at 20°C led to blunted changes in both Elovl3 mRNA and TAG levels. These findings suggest that huddling and nest building have a limited effect to ameliorate the cold stress associated with housing at 20°C.

the mouse (mus musculus) is an important model system for mammalian studies. Its genetic tractability with a short reproductive time, coupled with advances in the miniaturization of physiological tools, create an increased ease for the merging of physiological studies and “omic” studies. The ambient temperature (T_a) of animal housing facilities is within a standard range that, while comfortable for human caretakers, falls well below the mouse thermoneutral zone (TNZ), the range of T_as in which endotherms do not elevate their metabolic rate to maintain core body temperature (T_b) (19, 23). The T_a at which metabolic rate begins to rise to offset heat loss (i.e., the lower critical temperature) is ∼29–30°C for mice (18). Housed at temperatures below this zone, mice are under cold stress (8). To elevate metabolic rate and minimize heat loss when exposed to cooler temperatures, endotherms engage one of many behavioral changes and/or several physiological adaptations, which vary across species and which are not mutually exclusive (15, 30, 38). Behavioral changes to a cool T_a typically reduce heat loss to the environment by 1) moving to a warmer environment (thermotaxis), 2) minimizing exposed surface area (through changes in body position or social huddling), and 3) extensive nest building, which reduces the thermal gradient for heat loss by trapping heat locally outside of the body (13, 15). Physiological mechanisms include actions to reduce heat loss to the environment, which include piloerection and a reduction in blood flow to the periphery, and actions that generate heat, which include shivering and nonshivering thermogenesis.

Brown adipose tissue (BAT) is a thermogenic organ that uses uncoupling protein 1 (UCP1) to dissipate the proton gradient within mitochondria and is a major heat-generating organ for small mammals like mice (9, 29). In response to an acute bout of cold exposure in mice, sympathetic nervous system activation of BAT results in increased activity of the UCP1 protein. Chronic exposure to cold results in growth of the organ, including mitochondrial biogenesis and increased expression of the UCP1 mRNA and protein (9). Interestingly, the most inducible gene in BAT of mice in response to a lowered T_a may not be involved in heat generation directly. That gene is Elovl3 (elongation of very long chain fatty acids-like 3) that generates a 30-kDa glycoprotein product that localizes to the ER and is reported to be responsible for the elongation of fatty acids in the C20–C24 range (24, 43, 45). The Elovl3 mRNA is chronically elevated 20–100-fold in response to a lowered T_a (43). The induction of Elovl3 mRNA in response to cold is likely a product of activation of BAT by the sympathetic nervous system in that Elovl3 mRNA is also induced in mice given norepinephrine while housed in their TNZ (43). While the Elovl3 protein product may not be involved with heat generation or metabolic rate directly, the highly cold-inducible expression pattern of the Elovl3 mRNA in BAT can serve as a potential marker for measuring the cold stress and/or BAT activation in mice. In addition to the production of heat from brown adipose tissue as an important adaptation during cold exposure, it has recently been suggested that skeletal muscle can also participate in adaptive thermogenesis in mice (2), as has been observed in mammals and birds that lack brown fat (37).

The cold stress on mice from a T_a that is comfortable for humans can have significant effects on the murine cardiovascular system, food balance, the immune system, skeletal muscle, and autonomic outflow (19, 41, 47). The comprehensive and drastic physiological and biochemical changes that occur in mice as a result of the housing conditions may influence translational success for human clinical trials and may place limitations on modeling of human disease (25). Because humans can effectively manipulate their environment, most humans in Western society live within their TNZ (12, 28), although this concept is not universally accepted (40). Housing mice at 30°C has been described as “humanizing” them (12), as moving them from a typical vivarium temperature up to 30°C removes the cold stress. We have shown that moving mice from a T_a of 20°C to 30°C changes the dynamic of autonomic outflow from primarily sympathetic outflow to almost entirely parasympathetic outflow (41), an autonomic balance that better reflects resting humans.

Given the known physiological effects of cold stress in mice caused by low T_a and the alleviation of this cold stress upon housing in a thermoneutral environment, it was hypothesized that the provision of nesting materials or group mates, either alone or in combination, would allow mice housed below their TNZ at 20°C to physiologically and biochemically resemble unstressed thermoneutral mice housed at 30°C. Mice build extensive nests at 20°C (13), with the effect of minimizing heat loss to the environment. Housing mice in groups allows for a collective reduction in the surface area-to-volume ratio of the group, allowing for lower metabolic rate at lower T_as (7, 19). This study found, however, that behavioral thermoregulation through nest building and huddling was insufficient to ameliorate the cold stress in young female C57BL/6J mice created by ambient temperature.

MATERIALS AND METHODS

Animals

C57BL/6J female mice were generated by breeding mice locally from mice purchased originally from Jackson Laboratories, being two to four generations from the original purchased breeders. Prior to the experiment, mice were housed in static, plastic cages (7.25″ × 11.5″ × 5″: Allentown) with a metal wire top that held food pellets (Teklad no. 7012 LM-485) and a water bottle, with a layer of sawdust bedding. Mice had ad libitum access to food and water throughout all of the experiments. Before the experiments began, the mice were housed with two to three other mice with shredded paper at a T_a of 30°C with a 12:12-h light-dark cycle. Mice used in experiments 1A and 1B were 6 mo old. Mice used for experiment 2 were 4 wk old. Mice used in experiment 3 were 8 wk old. All animal procedures were approved by the Williams College Institutional Animal Care and Use Committee.

Radiotelemeter Implantation

Each mouse in experiments 1A and 1B was implanted with a telemeter capable of either measuring blood pressure (BP), body temperature (T_b), heart rate (HR), and locomotor activity (HD-X11; Data Sciences International), or measuring BP, HR, and locomotor activity (PA-C10). For implantation, mice were anesthetized using 5% isoflurane in oxygen gas and maintained with 2.5–3% isoflurane for the duration of the implantation procedure. For the HD-X11 implant, the telemeter was implanted in the abdominal cavity, and ECG leads were placed subcutaneously, approximating a Lead II configuration and held in place by sutures used to close the body wall. The catheter for blood pressure measurement was tunneled subcutaneously from the original incision site to a second incision in the thoracic region. The left carotid artery was located and isolated. The artery was temporarily occluded and punctured to allow for the insertion of the blood pressure catheter. Once properly positioned, the catheter was secured in place with nonabsorbable 5-0 suture. Wound clips (7-mm size, Reflex Clips; Fine Science Tools) were used to close the abdominal incision, and the thoracic incision was closed with nonabsorbable 5-0 suture. In all cases, aseptic technique was used throughout the surgery. All mice received meloxicam (5 mg/kg sc) immediately at the end of the surgery, and a second dose 24 h later. During the postoperative recovery period, mice were housed individually at 30°C in cages placed half atop a heating pad. The mice were allowed to recover for 10 days before any experimentation began. Aside from the placement of ECG electrodes, the steps of the surgical implantation of PA-C10 telemeters are the same as described for HD-X11 implantation.

Experiments

Experiment 1A.

Physiological data were recorded from eight 6-mo-old female mice implanted with HD-X11 telemeters. After the postoperative recovery period, each mouse was randomly assigned to one of eight housing conditions: singly housed with no nesting material, singly housed with 10 g of nesting material (Eco Nest & Forage, FiberCore), group housed with two female siblings that were not implanted with telemetry devices, or group housed with two siblings and nesting material, at a T_a of either 20°C or 30°C. The amount of nesting was chosen from previous work that recommended 6–10 g of nesting material (13). Ambient temperature was maintained at these temperatures (±0.25°C) using a circulating heating and cooling system, moving the coolant through a copper tubing coiling system affixed to the inner wall of a well-insulated box with interior dimensions of 23″ × 13″ × 24″. Over 8 days, each mouse spent 24 h in each experimental condition in a repeated-measures randomized crossover design. Telemeter data were sampled from receivers (RPC-1, Data Sciences International) for 10 s each minute for 23 h, beginning at the onset of the dark phase until 1 h before the onset of the dark phase on the next day. The last hour of the light phase was reserved for animal care and changing the housing conditions. Data for each of the physiological parameters (BP, HR, and T_b) were averaged over the entire dark phase and over the first 11 h of the light phase using analysis software (Data Sciences International). For shivering analysis, raw data files of ECG recordings from the HD-X11 telemeters for ∼40 contiguous minutes (each 10 s) were imported into Ponemah Physiology Platform software (Data Sciences International). Noise detection was enabled, and waveforms were analyzed.

Experiment 1B: 10-day physiological recording.

Physiological data were recorded from 7-mo-old female mice, ∼1 mo after completion of experiment 1A, implanted with HD-X11 (the same eight mice used in experiment 1A) or PA-C10 telemeters (an additional four implanted female mice of the same age). Each mouse was singly housed at 20°C with no nesting material (n = 12) for 10 days, and then randomly assigned to one of two housing conditions: singly housed at 20°C with nesting material (n = 6), or group housed with two female nonimplanted siblings at 20°C (n = 6). The mice were left undisturbed in these housing conditions for 10 days aside from 1) one cage change after 7 days and 2) daily food measurement, which occurred in the last hour of the light phase. All mice were subsequently housed at 30°C for 7 days.

Experiment 2: endocrinological comparison of housing conditions.

Thirty-five female 4-wk-old mice were randomly assigned to one of three housing conditions, each at two temperatures 20°C and 30°C: singly housed with no nesting material, singly housed with 10 g of nesting material, or group housed with two female siblings (n = 6 for each group, except for singly housed mice at 30°C with nesting, where n = 5). Mice were left undisturbed with no changes to housing conditions for 4 wk, except for twice weekly cage changes. At the end of 4 wk, mice were euthanized ∼2 h after the start of the light phase, and blood was drawn directly from the heart using EDTA-coated syringes. Samples were centrifuged for 2 min in an Eppendorf 5415C, and the plasma layer was isolated, immediately frozen in liquid nitrogen, and stored at −80°C. Plasma samples were analyzed for levels of triiodothyronine using a rat/mouse total triiodothyronine (T3) ELISA kit (MyBioSource) and thyroxine using a Total thyroxine (T4) EIA (MP Biomedicals). All assays were run in duplicate.

Experiment 3: brown fat gene expression and fat content.

Seventeen 2-mo-old female mice were randomly assigned to one of three groups: 1) singly housed 20°C (n = 6), 2) group housed with 5 mice to a cage at 20°C (n = 5), or 3) singly housed (n = 6) at 30°C. Following 21 days of these housing conditions, mice were euthanized with carbon dioxide inhalation for tissue collection. Interscapular brown fat pads were collected and quick frozen in liquid nitrogen until further processing. RNA was extracted from one of the two brown fat pads. The samples were homogenized in 4 M guanidinium thiocyanate, 0.2 M sodium acetate, and 0.1 M β-mercaptoethanol in DEPC (diethylpyrocarbonate)-treated water. Following homogenization, the samples were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was then transferred to a new tube containing an equal volume of 70% ethanol. The mixture was then transferred onto a Qiagen RNeasy column, and RNA was isolated following the manufacturer's protocol. First-strand cDNA synthesis from 100 ng of RNA was performed using the One-Taq RT-PCR kit (New England BioLabs) with a random hexamer primer. The subsequent quantitative RT-PCR reactions were performed with SYBR Green real-time PCR Master Mixes (Bio-Rad). The PCR amplification of Elovl3 mRNA was carried out using a forward primer (5′-GTCGTCTATCTGTTGCTCATCG-3′) and reverse primer (5′-ACTTCCACATCCTCAGAGTACC-3′). Gapdh primers were purchased from Qiagen (catalog no. 4352339E) and were used as a housekeeping gene for data normalization. qRT-PCR was performed in triplicate. Each sample also included a no-reverse transcriptase control, and a no-template control. The relative changes in gene expression were calculated using the 2^−ΔΔCt method, normalized to Gapdh for each sample.

Extraction of lipids.

For all samples, the lipids from the second brown fat pad were extracted using a modification of the Bligh and Dyer protocol (6). Each fat pad was macerated in 3 ml of chloroform/methanol (1:2; vol/vol). Following the addition of another milliliter of chloroform, the samples were held for 7 h to allow for complete extraction of the lipids. To effect a phase separation, 1.5 ml of 1% NaCl was added to each sample, and they were centrifuged to obtain clear phases. The lower chloroform phase of each was transferred to a new tube and stored at 4°C.

Fatty-acid esterification and gas chromatography.

An aliquot of each lipid extract was used for fatty acid methyl esterification using boron trifluoride/methanol (31). After heating at 100°C for 12 min, the fatty acid methyl esters were extracted using hexane. The prepared fatty-acid methyl esters were analyzed with the Hewlett-Packard 5890 bench top chromatograph with a flame ionization detector on a 15 m × 0.25 mm DB-23-fused silica capillary column. A standard mixture of fatty acids was injected into the GC to identify the major fatty acids based on retention times.

Thin-layer chromatography.

Aliquots of the lipid extracts were spotted on silica gel H-coated TLC plates (EMD Sciences). The initial solvent system consisted of chloroform/methanol/acetic acid/water (85:15:15:3 vol/vol) and was used to separate polar lipids along the bottom half of the plate. The second solvent system consisted of petroleum ether/ethyl ether/acetic acid (80:20:1 vol/vol) and was used to separate nonpolar lipids along the top half of the plate. The separated lipid classes were stained with iodine vapor. The plates were digitally photographed, and each lane was analyzed using the gel densitometry function in ImageJ.

Data Analysis

All results are reported as means ± SE. Statistical analyses were performed in SPSS 15.0 (IBM). In experiments 1A and 1B, a repeated-measures ANOVA with ambient temperature, nesting, and cage mates as within-subjects factors, followed by Bonferroni post hoc testing, was performed. For experiment 2, a 1 × 6 ANOVA with ambient temperature, nesting, and cage mates as between-subjects factors, followed by Bonferroni post hoc testing, was performed. For experiment 3, a 1 × 3 ANOVA, followed by Bonferroni post hoc testing, was performed. P values less than 0.05 were considered statistically significant.

RESULTS

Experiment 1A

To assess the potential cardiovascular and thermoregulatory benefits of group housing and nest building, mice were randomly cycled through eight housing conditions over 8 days, spending 24 h in each of the conditions. Figure 1 shows a 23-h plot of multiple physiological variables (HR, BP, and T_b) from the same mouse while housed singly without nesting at 20°C and a second 23-h plot, while housed at 30°C. Each recorded physiological variable was averaged for all mice over the dark phase (Table 1) and over the light phase (Fig. 2). The average HR recorded at 20°C was significantly higher than that recorded at 30°C (P < 0.05), regardless of housing condition, for both light and dark phases (Table 1 and Fig. 2A). Within each T_a grouping, there was no significant difference in the average HR in each housing condition except during the light phase, where group housing + nesting significantly lowered HR from other groups housed at 20°C (Fig. 2). Similarly, there was a main effect due to T_a in the mean blood pressure (BP) of the mice (P < 0.05), but no significant differences due to nesting or group housing (Table 1 and Fig. 2B). The T_a of each housing condition had a significant effect on core T_b as well. The average T_b of all mice at 20°C was significantly lower than that of mice housed at 30°C, but not different among the housing conditions at any one given T_a (Table 1 and Fig. 2C). General cage activity of the mice, as measured by the telemeters, did not differ among any of the groups (data not shown).

Fig. 1.

Typical physiological tracings of a mouse housed at 20°C and 30°C. C57BL/6J mice were implanted with HD-X11 telemeters and housed in one of several conditions. Typical tracings from this telemeter are shown from a single mouse in two different conditions: singly housed without nesting at 20°C (black trace) and singly housed without nesting at 30°C (gray trace). These telemeters report heart rate (A), mean blood pressure (B), and T_b (C). The dark phase is the first 12 h, as indicated by the dark bar.

Table 1.

Experimental conditions and dark phase physiological measurements for experiment 1

Condition Name	Ta	Grouping	Nesting	Heart Rate, bpm	Mean BP, mmHg	Tb, °C
CSN	20°C	Single	Nest	625 ± 17	117 ± 6	36.5 ± 0.2
CSB	20°C	Single	No nest	626 ± 12	118 ± 4	36.3 ± 0.2
CGN	20°C	Group	Nest	611 ± 12	121 ± 7	36.8 ± 0.1
CGB	20°C	Group	No nest	624 ± 18	120 ± 9	36.8 ± 0.1
WSN	30°C	Single	Nest	496 ± 10a	98 ± 4a	37.1 ± 0.1a
WSB	30°C	Single	No nest	497 ± 12a	103 ± 2a	37.1 ± 0.1a
WGN	30°C	Group	Nest	491 ± 8a	110 ± 2a	37.3 ± 0.1a
WGB	30°C	Group	No nest	494 ± 10a	105 ± 2a	37.2 ± 0.1a

^aP < 0.05 denotes significance between T_as with the same housing condition (group, nest, or group + nest).

Fig. 2.

Implanted C57BL/6J female mice were housed in one of eight conditions in a randomized cross-over design, such that each mouse was housed in each condition for 24 h (CSN = 20°C, singly housed, nesting: CSB = 20°C, singly housed, no nesting: CGN = 20°C, group housed, nesting: CGB = 20°C, group housed, no nesting: WSN = 30°C, singly housed, nesting: WSB = 30°C, singly housed, no nesting: WGN = 30°C, group housed, nesting: CGB = 30°C, group housed, no nesting). Mice housed at an ambient temperature (T_a) of 30°C displayed a significantly reduced HR (A) and BP (B) relative to those measurements in the same mice housed at an T_a of 20°C. Neither group housing nor the provision of nesting material significantly influenced BP at either T_a. HR was significantly lowered by ∼8% when mice were housed in a group (with or without nesting) compared with singly housed (with or without nesting). C: T_b was significantly lower in mice housed at 30°C vs. 20°C, independent of cage mates or nesting material. Data are shown as means ± SE. ^aP < 0.05 denotes significant difference between T_as with same housing condition (group, nest, or group + nest). ^bP < 0.05 denotes significant difference between group vs. singly housed mice (with or without nesting) at the same T_a.

As a proxy for shivering, the ECG tracings (Fig. 3) were analyzed for noise using the Ponemah software program. The noise in the baseline of the ECG tracings was quantified from every mouse in all eight housing conditions. Recordings of 40-min duration were selected from the light phase during periods of relative inactivity, based on activity counts from telemetry data. There was significantly more noise in the ECG tracings of singly housed mice at 20°C than at 30°C (6.17 ± 0.15 vs. 3.87 ± 0.07 arbitrary units; P < 0.05). However, the addition of cage mates and nesting had no impact on the noise quantification from the ECG tracing, suggesting that mice housed at 20°C shivered significantly more than mice housed at 30°C, regardless of the availability of nesting or group mates. It should be noted that these mice were raised at 30°C and first experienced 20°C (either singly, with nesting, or with cage mates) at the initiation of this experiment. As these mice were not acclimated to the cooler temperature, the elevated noise on the ECG tracing is likely only acute in nature as shivering diminishes over time with acclimation to 20°C housing (3, 8).

Fig. 3.

Shivering is significantly elevated in mice acutely housed at 20°C vs. 30°C. Noise from ECG tracings was quantified as a proxy for shivering. A: ECG tracing from a mouse singly housed at 20°C shows significant baseline noise. B: ECG tracing from the same mouse at 30°C shows significantly less noise in addition to a slower HR than was recorded at 20°C. C: noise of the tracings was quantified using Ponemah software and was expressed in arbitrary units. Please see the Fig. 2 caption or Table 1 for the description of each group. A repeated-measures ANOVA followed by a post hoc Bonferroni test found a main effect of T_a but no interaction between group housing or nesting on shivering. Mice housed at 20°C had significantly more noise than those housed in the warm temperature, independent of the presence of other mice or nesting material. Data are shown as means ± SE. ^aP < 0.05 denotes significance between T_as with the same housing condition (group, nest, or group + nest).

Experiment 1B

Because no significant interactions due to housing conditions were seen using 24-h exposures (except for HR in the light phase), we became concerned that the stress from handling the mice daily overwhelmed any changes that might have been observed by housing conditions. Therefore, we designed a longer-term study of the effects of housing condition to test whether group housing and/or nest building provides thermoregulatory benefits after multiple days of acclimation. Mice were initially singly housed without nesting at 20°C for 10 days, then randomly assigned to one of two groups: singly housed with nesting at 20°C or group-housed at 20°C for 10 days. All mice were then singly housed at 30°C for 1 wk. HR and BP were averaged for the final three 24-h periods for each condition. During the light phase, the HR of mice housed at 20°C was significantly higher than the HR of mice singly housed at 20°C with nesting or group housed at 20°C (Fig. 4A). Group housing also lowered the dark-phase HR, although dark-phase HR was not significantly lower with the use of nesting at 20°C. In all cases, HR was significantly lower at 30°C compared with any of the housing conditions at 20°C. Similarly, BP was lower in the mice when housed at 30°C compared with any of the housing conditions at 20°C (Fig. 4B). However, unlike HR, there was no effect of the different housing conditions at 20°C on blood pressure (Fig. 4B). Daily caloric intake in mice either singly housed at 20°C or housed with nesting at 20°C was significantly greater, and at least 50% more, than mice singly housed at 30°C (Fig. 4C).

Fig. 4.

Long-term housing (10 days) with nesting or group mates only partially ameliorated the tachycardia but not elevated blood pressure associated with housing at 20°C. Implanted C57BL/6J mice were singly housed at 20°C for 10 days. The mice were then split into one of two groups: singly housed at 20°C with nesting, or group-housed at 20°C, for an additional 10 days. Then, the mice were housed at 30°C for 7 days. The HR, BP, and food consumption data shown are the average over the last 3 days in each of the conditions. A: HR was significantly reduced (∼10%) in group housing or with nesting compared with single housing without nesting and was significantly elevated relative to singly housed mice at 30°C. In the dark phase, only group housing led to a lower HR, while housed at 20°C. B: BP was unchanged by housing condition at 20°C and significantly elevated relative to housing at 30°C in both the dark and light phases. C: food consumption when mice were group-housed at 30°C was significantly lower relative to singly housed mice at 20°C with or without nesting but not significantly different to group-housed mice at 20°C. Food consumption from group-housed mice at 20°C was not different compared with any other condition. Data are shown as means ± SE. ^aP < 0.05 denotes significant difference vs. singly housed mice at 20°C. ^bP < 0.05 denotes significant difference vs. group housed mice at 20°C. ^cP < 0.05 denotes significance difference vs. singly housed + nesting mice at 20°C.

Experiment 2

In a group of young female mice, blood plasma was collected after a 4-wk acclimation period in one of six randomly assigned housing conditions. Plasma T3 levels were significantly higher in mice singly housed without nesting at 20°C than in mice housed with nesting material or in groups at 20°C or mice housed at 30°C (Fig. 5A). Plasma T4 levels in mice housed with nesting at 30°C were significantly higher than plasma T4 levels of mice in any condition at 20°C (Fig. 5B).

Fig. 5.

In a second experiment, C57BL/6J mice were assigned to one of six groups (singly housed at 20°C or 30°C, group-housed at 20°C or 30°C, or singly housed with nesting at 20°C or 30°C). The mice were maintained in those conditions for 28 days. The sample size for all experimental groups was 6, except the 30°C nesting group, in which n = 5. A: singly housed mice at 20°C had significantly higher levels of circulating T3 than the other five groups. B: circulating thyroxine (T4) was elevated in all groups housed at 30°C vs. 20°C, independent of cage mates or nesting material. Within a given T_a, plasma T4 levels were not significantly different between the groups. Data are shown as means ± SE. ^aP < 0.05 denotes significant difference vs. singly housed at 20°C. ^bP < 0.05 denotes significant difference with the same housing condition between 20°C and 30°C.

Experiment 3

In this experiment, female C57BL/6J mice were housed in one of three conditions: 1) 20°C singly, 2) 20°C in a group of 5, or 3) 30°C singly. The mice were maintained in these conditions for 21 days before brown fat was removed and analyzed for gene expression and fat content. Expression of the Elovl3 mRNA in brown fat was elevated 22-fold in the 20°C singly housed mice relative to the 30°C singly housed mice (Fig. 6). Group housing at 20°C resulted in expression of Elovl3 mRNA at intermediate levels relative to singly housed at either 30°C or 20°C (elevated nine-fold or diminished 60%, respectively). Brown fat was also analyzed for lipid content. Triacylglycerol (TAG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) contents of BAT were assessed by thin-layer chromatography. TAGs comprised ∼90% of the total lipid of BAT from mice housed at 30°C (Fig. 7A), which was significantly decreased to ∼80% in the BAT of mice housed at 20°C (Fig. 7A). Group housing at 20°C resulted in an intermediate composition of TAGs in BAT at 85%. PC and PE together make up much of the remainder of the lipid content of fat (∼6% in BAT of mice housed at 30°C and 10–14% in BAT of mice housed at 20°C). However, the ratio of PE:PC was increased by ∼40% in the BAT of mice housed at 20°C either singly housed or group housed compared with housing at 30°C (Fig. 7B). The fatty acid profiles for the brown fat samples were then obtained by gas chromatography. There were no significant differences in the proportion of 16:0 (palmitic acid), 18:1 (oleic acid), and 18:3 (α-linolenic acid) in any of the conditions (Fig. 7C). Compared with BAT from 30°C-housed mice, the BAT from mice singly housed at 20°C had significantly lower levels of 16:1 (palmitoleic acid)—levels not altered by group housing—and elevated levels of 18:0 (stearic acid)—levels mitigated somewhat by group housing. The proportion of 18:2 (linoleic acid) was elevated in both housing conditions at 20°C relative to 30°C. Additionally, the proportion of 20:4ω-6 (arachidonic acid) in BAT from mice singly housed at 20°C was three-fold higher than BAT from mice housed singly at 30°C (3.7 ± 0.4% vs. 1.1 ± 0.1%, P < 0.05, Fig. 7C). The elevation in the content of both 18:2 and 20:4ω-6 in brown fat in response to cold exposure has been repeatedly shown in mitochondria of brown fat (33–35), and we show here that the level of only one of these (20:4ω-6) was mitigated by group housing.

Fig. 6.

Group housing lowers expression of the T_a-sensitive gene, ELOVL3. C57BL/6J mice were either singly housed at 30°C (WS), singly housed at 20°C (CS), or group-housed at 20°C (CG) for 21 days. Brown fat was removed and assessed for Elovl3 mRNA expression by quantitative PCR, expressed relative to Gapdh mRNA. Relative to WS, Elovl3 mRNA is significantly elevated in the CS group and CG group (^bP < 0.05 vs. WS). However, group housing lowered the steady-state levels of Elovl3 mRNA relative to singly housed (^aP < 0.05 vs. CS). Data are shown as means ± SE.

Fig. 7.

Group housing of mice at 20°C influenced brown adipose lipid content. Brown fat was extracted from one interscapular fat pad from mice that were singly housed at 20°C (CS), group housed at 20°C (CG), or singly housed at 30°C (WS). A: triacylglycerol content (TAG) was assessed by thin-layer chromatography (TLC). Densitometric analysis showed that housing at 20°C significantly lowered the TAG content of brown fat compared with housing at 30°C (^bP < 0.05 vs. WS) and that group housing (CG) was intermediate in TAG content (^aP < 0.05 vs. CS). B: from the same TLC analysis as A, the ratio of phosphatidylethanolamine (PE) relative to phosphatidylcholine (PC) was calculated to be significantly greater in either of the 20°C housing conditions relative to housing at 30°C (^bP < 0.05 vs. WS). C: fatty acid profiles within brown fat samples were analyzed by gas chromatography. Brown fat from CS and CG mice had significantly depressed levels of 16:1 and elevated levels of 18:2 relative to WS (^bP < 0.05), and neither of these fatty acids were altered with group housing. In contrast, the proportion of 18:0 and 20:4ω-6 in BAT was elevated in CS mice relative to WS mice (^bP < 0.05 vs. WS), and the elevation of both of these fatty acids in housing at 20°C was partially blunted in the CG mice (^aP < 0.05 vs. CS). Data are shown as means ± SE.

DISCUSSION

The results confirm that young female mice housed at 20°C exhibit physiological and biochemical indicators of cold stress. We tested the hypothesis that manipulating the environment of the mouse, without changing the T_a from 20°C, would alleviate the cold stress through behavioral thermoregulation (13, 16). When mice are housed in groups of two or more, mice typically huddle, decrease their collective surface area, and conserve heat (21). The density of a huddle increases with a corresponding decrease in T_a (21). Huddling confers a thermoregulatory advantage; group-housed infant mice (4 days postnatal) exhibit a delay in brown-fat-induced thermogenesis upon cold exposure but no significant long-term inhibition of BAT activity (11). The provision of nesting material in mouse cages also lends thermoregulatory benefits. Mice use shredded paper, intended to mimic grasses found in the wild, to build insulating dome-shaped nests (13, 22). The T_a within a nest has been shown to approach thermoneutrality, even at T_as associated with cold stress (20). In the current studies, we tested our hypothesis that inclusion of either nesting material or the addition of cage mates to promote behavioral thermoregulation would alleviate the physiological and biochemical changes that are associated with housing mice at 20°C.

The principle findings of this study demonstrated that the provision of group mates and nesting material has a limited impact on the physiological and biochemical consequences of the cold stress seen in C57BL/6J female mice housed at the standard laboratory ambient temperature of 20°C. Anecdotally, we found that the state of the nesting material was quite different in cages held at 20°C vs. 30°C. As Gaskill et al. (13) have observed, we found nesting material scattered around the cage floor when the cage was at 30°C, but in a tight nest when the cage was at 20°C. Further, our qualitative observation for group-housed mice was that these mice tended to be resting in a huddle at both T_as of 20°C and 30°C, although our observations were limited to the last hour of the light phase. When comparing the physiological and biochemical measurements for mice housed within their thermoneutral zone of 30°C, we found that inclusion of nesting or cage mates influenced some measurements, while others were not impacted. If housing mice at 30°C is considered baseline, and housing at 20°C without nesting or cage mates constitutes a cold stress (8, 11), then inclusion of nesting or cage mates for mice housed at 20°C only slightly normalized heart rate (particularly for longer-term housing), food intake, circulating T3, Elovl3 mRNA expression in brown fat, triacylglycerol content in brown fat, the relative content of the fatty acid 20:4ω-6 of brown fat, and the ratio of PE relative to PC in brown fat. The extent to which nesting or cage mates ameliorated the measurement was quite small. For example, the drop in HR that accompanied group housing for 10 days was only about 8%, and to a level that was still well above the intrinsic HR of the mouse (41). This agrees with previous research of pair-housed male mice at 21°C (39). Our data suggest that group housing at 20°C still results in a cardiovascular system that is predominantly controlled sympathetically, unlike resting humans or mice housed within their thermoneutral zone (41). Further, we examined Elovl3 mRNA expression in brown fat because the steady-state level of this mRNA is extremely responsive to chronic BAT activation (36, 43, 45), although it is unclear whether the protein product of this gene is involved with heat generation or metabolic rate. Long-term group housing caused a twofold drop in BAT Elovl3 mRNA relative to singly housed mice at the same cool T_a, but this level of expression was still nine-fold higher than that seen in mice housed at 30°C. While it is unclear if the fatty acid 20:4ω-6 is a product of this fatty acid elongase, the pattern of fatty acid content of 20:4ω-6 between the three groups qualitatively resembles that of the mRNA for the elongase (Fig. 7). Additionally, we found measurements that were not affected by nesting or cage mates when the mice were housed at 20°C. These parameters include BP, T_b, shivering, circulating T4, and most of the fatty acid types within brown fat. Although the housing density in experiment 3 (brown fat analysis) was greater than the density used in experiments 1 and 2, the conclusions that we draw from these different experiments are qualitatively the same. As a whole, we can conclude that inclusion of either nesting or cage mates is limited in the amelioration of the cold stress of C57BL/6J female mice housed at a typical vivarium T_a.

It should be noted that the mice used in these experiments, as well as the breeders purchased from Jackson Laboratories, were housed at 30°C before the onset of the experiments, which may influence the extent at which the T_a influences the physiological/biochemical measurements made in these mice. Research examining rearing temperature to examine possible influences of T_a on developmental and epigenetic factors is warranted. Further, we only used one strain of the mouse in the current study, C57BL/6J. We chose this strain because of its widespread use in research. Other strains of mice, however, have varying levels of social behavior and undergo varying levels of behavioral thermoregulation in response to relatively cool T_as (1, 27), and hence, our observations may be strain-specific. In addition, female mice (from a heterogeneous strain of HS/Ibg) huddle at both warm and cool T_as, whereas male mice huddle much less when exposed to warm T_as (5). Because we used only female mice in the current study, one should not extrapolate our findings to the impact of group mates on male mice. Finally, the bedding type that we used in this experiment was not deep enough for burrowing, another way in which mice can lower thermal demands in a relatively cool environment (17).

Given the above caveats, the results of this study impress the overarching effect of T_a on laboratory mouse physiology. The mouse has evolved to adapt to cooler environments as a T_a of 20°C is within the natural history of the mouse. The mouse engages numerous behavioral and physiological strategies to adapt to a cool T_a. These strategies include activation of heat production, decreasing skin and tail blood flow, thermotaxis, nest building, and huddling, of which the latter two are often intertwined (13, 14, 19, 20, 42). One relevant question to ask is whether the cold stress of housing mice at 20°C imposes a significant limitation on modeling human disease, such as energy balance and immune function (8, 25). Metabolic studies show a large impact of T_a on energy balance. Just one example is the observation that mice deficient in UCP1 become obese when housed in their thermoneutral zone, but exhibit a normal weight when housed at 22°C, a temperature at which metabolic rate is significantly elevated for thermoregulation (12). Further, the immune system is significantly impacted by housing temperature (25). For example, housing mice at subthermal neutral zone temperatures activates IL4-mediated alternative activation of macrophages and activation of these macrophages helps generate heat in brown fat in addition to uncoupling (32). Also, it was recently shown that housing at 20°C suppresses the antitumor immune response compared with mice housed at 30°C (26). Although it remains to be determined whether the inclusion of cage mates and/or nesting material will influence the outcome of mouse studies involving clinically relevant topics like the immune system, our data on the cardiovascular and BAT biochemical status lead to the prediction that these two environmental enhancers may not have the desired effect for mimicking the human state.

Perspectives and Significance

Our findings demonstrate that inclusion of either nesting or cage mates with mice housed in typical vivarium temperatures has a small, but measureable, ameliorative effect on cold stress associated with housing mice at 20°C. We had anticipated that nesting and inclusion of cage mates would considerably lessen cold stress. However, despite the inclusion of nesting or cage mates, the sympathetic nervous system appears to still dominate the autonomic balance in mice housed at 20°C as assessed by HR, BP, and BAT activation. The clinical relevance of BAT activation with a cool T_a in mice should be explored given that BAT activation is a primary factor in clearance of plasma triglycerides in mice (4) and that activation of brown fat by cool conditions in humans leads to thermogenesis and lower body fat (10, 44, 46). Our data support the concept that the current system imposes a mismatch between housing of mice and housing of humans that makes translation of studies from mouse to human difficult, and this TNZ mismatch is not corrected by either huddling or nest building.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: R.L.M., S.M.B., and S.J.S. conception and design of research; R.L.M., S.M.B., and D.V.L. performed experiments; R.L.M., S.M.B., D.V.L., and S.J.S. analyzed data; R.L.M., S.M.B., and S.J.S. interpreted results of experiments; R.L.M., S.M.B., and S.J.S. prepared figures; R.L.M., S.M.B., D.V.L., and S.J.S. edited and revised manuscript; R.L.M., S.M.B., D.V.L., and S.J.S. approved final version of manuscript; S.J.S. drafted manuscript.