Anterograde transneuronal viral tract tracing reveals central sensory circuits from brown fat and sensory denervation alters its thermogenic responses

Animals.

Male Siberian hamsters (Phodopus sungorus; ∼3–3.5 mo old; n = 29) were single housed after being selected from our breeding colony (16 h:8 h light:dark cycle; lights on at 0200 h). Room temperature was maintained at 21 ± 2°C. The lineage of this colony has been described previously (7). Hamsters were single housed for at least 6 days before virus injections.

H129 virus injections.

Siberian hamsters were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL) and IBAT pads were exposed. All hamsters received a unilateral injection into the left IBAT pad with the attenuated strain of HSV-1, H129 (0.75 × 10⁸ pfu/ml), at five areas (150 nl each; 750 nl total) across the length of the IBAT pad. Preliminary studies demonstrated equal CNS labeling from left or from right injected IBAT lobes (Song CK and Bartness TJ, unpublished observations), thereby justifying our injections only into the left IBAT pad. The syringe was kept in place for 60 s to prevent efflux of virus after each injection. The syringe needle entry site was then wiped with sterile saline-soaked gauze. The incision was closed with sterile sutures and wound clips, and nitrofurozone powder (nfz Puffer; Hess & Clark, Lexington, KY) was applied to minimize the risk of bacterial infection. The above precautionary procedures (small volumes, needle held in place to minimize efflux) helped assure against leakage of virus to neighboring peripheral tissues. After the H129 injections, the animals were placed back into single housing in fresh cages and allowed to survive for 24, 48, 72, 96, or 114 h (n = 5, 72 h; n = 6, other groups). The parameters for viral injections, including the optimal postinoculation survival times for infection of the most rostral forebrain areas and the virus titer/load, were determined in pilot studies to mimic the intensity of labeling seen in our demonstration of central sensory circuits from WAT (54).

Histological tissue preparation.

Hamsters were overdosed with pentobarbital sodium (300 mg/kg ip) and perfused transcardially with heparinized (0.02%) saline and paraformaldehyde (4%) in 0.1 M phosphate buffer (PB; pH 7.4) after their respective survival periods. Brains and spinal cords were harvested and stored in fresh paraformaldehyde in PB solution overnight at 4°C and then transferred to a sucrose solution (30%) with 0.1% sodium azide and stored at 4°C until they were sectioned on a freezing stage sliding microtome at 35 μm into three sets for each hamster. Sections were stored in 0.1 M phosphate-buffered saline (PBS) solution with 0.1% sodium azide until immunohistochemical processing.

HSV-1 immunohistochemistry.

One set of brain sections for each hamster was rinsed in PBS (0.1 M; pH 7.4) and then incubated in 5% normal goat serum (Vector Laboratories, Burlingame, CA) with 1% H₂O₂ to minimize nonspecific labeling. Next, sections were incubated with the primary HSV-1 antibody (1:750,000; DakoCytomation, Carpinteria, CA) overnight, then biotinylated secondary antibody (goat anti-rabbit; 1:750; Vector Laboratories) for 2 h, and then avidin-biotin complex (ABC; 1.75 μl/ml each of avidin and biotin, Vector Laboratories) for 1 h with each step followed by PBS rinses. Finally, diaminobenzidine (0.1 mg/ml; Sigma Chemicals, St. Louis, MO) was used as a peroxidase substrate in the presence of 0.0025% H₂O₂ to produce a chromogen for visualization. The reaction was ended by a final set of PBS rinses. All steps were performed at room temperature. The sections were mounted onto gelatin-coated slides, counterstained with cresyl violet, dehydrated in ethanols, delipidated in xylenes, and then coverslipped. As a control for nonspecific labeling, some sections were processed without the addition of the primary antibody.

Data analysis and imaging.

All brain sections processed for HSV-1 immunoreactivity (ir) were observed using light microscopy. Manual counts of HSV-1-infected neurons in the brain were made from the level of the hypothalamic preoptic area to the brain stem using an Olympus BX41 microscope. Images were captured digitally with an Olympus DP70 and labeled using Adobe Photoshop (v7.0, San Jose, CA). Two mouse atlases were used (1, 46) in identifying brain and spinal cord regions, because there is no Siberian hamster brain atlas commercially available, and the size and shape of most mouse brain areas are similar to that of Siberian hamsters but not Syrian hamsters (Mesocricetus auratus). Manual counts of infected neurons were then averaged across the animals for each group at the different infection times. Counts are presented as means ± SE. In the cases where only one animal exhibited HSV-ir (H129) there is no standard error reported.

Animals.

Male Siberian hamsters (Phodopus sungorus; ∼3–3.5 mo old; n = 61) were single housed after being selected from our breeding colony (16 h:8 h light:dark cycle; lights on at 0200 h). Hamsters were moved to single-housed cages (16 h:8 h light:dark cycle; lights on at 0500 h) for at least 1 wk before capsaicin injections.

Surgical procedures.

Capsaicin injections, IBAT transponder implantation (for direct T_IBAT measures), and iButton (DS1922T; Embedded Data Systems, Louisville, KY) implantation (for direct T_c measures) were done in a single surgery. iButtons were calibrated and programmed before implantation to collect data every 30 min at an 11-Bit (0.0625°C) resolution, and data recording began the day before handling (see Acute cold exposure section below). iButtons were then securely tied with string and dipped in Paraffin/Elvax (Mini Mitter, Bend, OR) heated to 80°C. The wax was allowed to dry and iButtons were dipped twice more and when dry, and excess string was cut.

On the day of surgery, hamsters were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL) and then shaved. A midline incision was made along the upper dorsal surface of the body to expose both IBAT pads. Hamsters were body weight matched into two groups and were injected with 30 microinjections (1 μl per injection) of 20 μg/μl of capsaicin (Sigma Chemical) or vehicle (1:10, ethanol:olive oil) to cover the full extent of both IBAT pads. The dose of capsaicin was based on the lowest effective dose that significantly decreased IBAT CGRP-ir in pilot studies (Vaughan CH and Bartness TJ, unpublished observations) and used previously to effectively sensory denervate capsaicin-sensitive nerves in WAT (20, 49, 50). Reflux of the solutions up the needle from the injection site was minimized by holding the needle at each injection site for 60 s before removing the Hamilton microsyringe.

After the capsaicin injections, a temperature transponder [Implantable Programmable Temperature Transponder (IPTT) 300, BioMedic Data Systems, Seaford, DE] that was gas sterilized was tied to sterile silk sutures and then secured under the left IBAT pad as we described previously (8, 35, 56). The dorsum was closed with sterile wound clips (Stoelting, Wood Dale, IL), and then a ventral incision was made through the skin and peritoneal cavity for iButton implantation. After iButton implantation, the wall of the peritoneal cavity was sutured closed with sterile Vicryl (Ethicon, Sommerville, NJ), and then the skin was closed with wound clips. Hamsters recovered in home cages for 3 wk before the acute cold exposure test.

Acute cold exposure.

On the day before acute cold exposure, hamsters were handled and weighed and then separated further into two groups: cold-exposed (4°C) or room temperature (RT; 22°C). The following day all hamsters were moved to another housing room with the same light cycle and a cold chamber, and all were then scanned for IBAT temperature (−2 h). A portable hand-held reader programmer (DAS-5002 Notebook system, BioMedic Data Systems, Seaford, DE) was used to scan T_IBAT as we described previously (8, 35, 56, 60). Nestlets, water, and food from the home cage and cheek pouches were removed at −2 h to eliminate cold-induced hyperphagia and consequent increased thermogenesis. Two hours later, half the hamsters were placed into the cold chamber or left at room temperature (0 h). IBAT temperature was assessed at 0, 2, 4, 8, 12, 16, 18, 22, and 24 h. Hamsters were euthanized within an hour following the 24-h reading.

Transponder verification and tissue harvesting.

The same procedures were used as in Experiment 1 for transcardial perfusions except these hamsters were deeply anesthetized with pentobarbital sodium (260 mg/kg ip; Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA) for tissue harvesting. iButtons and IBAT transponders were removed, and a visual check was done to make sure the copper portion of the IBAT transponder was still situated under the medial border of the left IBAT pad. IBAT was then removed and weighed, and hamsters were perfused. IBAT was minced on ice for immediate homogenization in 0.2 M acetic acid for the CGRP assay.

CGRP enzyme immunoassay.

CGRP levels were measured using a commercially available EIA kit (SPI Bio, Massy, France) according to the manufacturer's instructions. For the CGRP assays, the overall correlation coefficient was 98.9%.The assay was previously analytically validated by determination of dilutional parallelism.

Preliminary experiments.

As a peripheral anatomical control, in some animals the same (titer and volume) virus was placed on the surface of exposed IBAT resulting in no infection of cells in the dorsal root ganglia (DRG), spinal cord, or brain (data not shown; Song CK and Bartness TJ, unpublished observations). This is in contrast to the direct localized injections of H129 into IBAT that resulted in infection of these components of the neuroaxis reported below. As an additional peripheral anatomical control, we surgically isolated IBAT from the surrounding tissues before H129 injection. This resulted in a pattern of infection indistinguishable from that of IBAT pads injected in their natural in situ position/condition indicating that the results reported below are from IBAT not from the surrounding tissues (data not shown; Song CK and Bartness TJ, unpublished observations).

24 h post-H129 infection.

Processing of the tissue without the addition of the primary antibody resulted in no labeling (data not shown).

One of the six hamsters showed no signs of H129 infection in cell nuclei. Overall counts of infected cells were relatively low with just a few positive cells in the central autonomic nucleus of the dorsal horn in the spinal cord. Infected cells were first seen in the lateral funiculus, dorsal horn, and lamina 10 of the spinal gray of the spinal cord (10 Sp) in the lower cervical, upper thoracic region (∼C6-T1) in one of six hamsters (see Supplemental Table S1). In the brain stem, the largest number of H129-positive neurons was in the parvicellular and gigantocellular reticular nuclei and the vestibular nuclei. In the midbrain, there were H129-positive neurons in pontine nuclei and the peduncles (inferior and middle cerebellar). In two of the six hamsters, virus infection occurred as far anterior as the caudate putamen and median preoptic nucleus (see Supplemental Table S2).

48 h post-H129 infection.

All six hamsters had H129-infected cells in the CNS. Only four of six had infected cells in dorsal, ventromedial, and lateral funiculi and 10Sp in the spinal cord in the thoracolumbar region (see Supplemental Table S1). In one hamster, the most rostral sign of infection was in the medial vestibular nucleus in the brain stem. The other five hamsters had sparse infected neurons as far rostral as the granular insular cortex and caudate putamen in forebrain.

72 h post-H129 infection.

Four of six hamsters showed infection in spinal cord, and the cells were sparse and localized to laminae 2 and 5 of the spinal gray of the dorsal horn and the intermediolateral column (IML) in the thoracic and lumbar regions. One of the hamsters that showed infection in the spinal cord (5 cells total; see Supplemental Table S1) did not show signs of infection in forebrain or midbrain. The most rostral sign of infection was seen in the caudate putamen in three of five hamsters and in the medial amygdaloid nucleus in one of five hamsters.

96 h post-H129 infection.

HSV-1-ir was present in the upper thoracic level of the spinal cord (Fig. 1, A and B; Supplemental Table S1). In the brain stem (Fig. 1, C and D), five of six hamsters had H129 infection in raphe pallidus, vagal, facial, and hypoglossal nuclei, area postrema, cuneate nucleus, cochlear regions, intercalated nucleus of the medulla, inferior cerebellar peduncles, pyramids, raphe areas, solitary tract, and ventrospinocerebellar tract. Five of six hamsters had cells positive for H129-ir in the midbrain in cerebellar lobules and peduncles, central gray of the pons (CGPn), lemniscal areas, olivary areas, rubral areas, tectospinal tract, and the substantia nigra (Supplemental Table S2). Two of six hamsters showed signs of infection in the forebrain, specifically in the hypothalamus (anterior, dorsomedial, arcuate, lateral, periventricular, paraventricular, and posterior nuclei) and cortex (somatosensory, motor, visual, prelimbic, piriform, retrosplenial granular, insular, cingulate and ectorhinal) (Supplemental Table S3).

114 h post-H129 infection.

Cells infected in the spinal cord were present in all six hamsters in the column of Clarke, 10Sp, central autonomic nucleus, dorsal funiculi, dorsal horn, lateral funiculi, rubrospinal tract, and the IML (Fig. 2, A–C, Supplemental Table S1). Brain stem areas (Fig. 3, A and B) that showed H129 infection include area postrema, dorsal motor nucleus of the vagus, facial nuclei, parabrachial areas, reticular areas, and trigeminal areas. Midbrain areas that showed H129 infection (Fig. 4, A and B) include CGPn, lemniscal areas, olivary nuclei, rubral areas, and the periaqueductal gray. H129 expression (Fig. 4, C and D) was present in the forebrain in hypothalamus (anterior, arcuate, dorsomedial, periventricular, paraventricular, ventromedial and lateral), thalamus, subincertal nucleus, subzona incerta, zona incerta, amygdalar areas, habenular areas, and medial pretectal nuclei.

IBAT sensory denervation verification.

IBAT-treated with capsaicin had significantly lower CGRP concentrations (CAP, cold: 10.00 ± 3.7 pg/mg IBAT vs. VEH, cold: 22.04 ± 3.9 pg/mg IBAT; CAP, RT: 5.32 ± 1.4 pg/mg IBAT vs. VEH, RT: 18.05 ± 3.2 pg/mg IBAT; P < 0.05) representing ∼55 and ∼72% decreases in CGRP.

IBAT mass.

IBAT masses were significantly smaller in hamsters exposed to cold (cold: 0.18 ± 0.02 g vs. RT: 0.25 ± 0.02 g) regardless of IBAT treatment (P < 0.05).

Body mass.

There were no significant differences in body mass before and 2 wk after surgery for all groups. As expected, body weights were significantly less after 24 h cold exposure (P < 0.01) for both the capsaicin (CAP, postcold: 33.8 ± 1.1 vs. CAP, precold: 38.5 ± 1.3) and vehicle (VEH, postcold: 35.6 ± 1.5 vs. CAP, precold: 41.3 ± 1.8)-treated hamsters.

T_IBAT temperature.

At 2 and 12 h into the test, T_IBAT was significantly lower in hamsters with capsaicin injected into IBAT than in hamsters given the vehicle (Fig. 5A). Data also were analyzed in blocks of time separated into light and dark periods (see inset bar graphs; 0–4 h, light; 4–12 h, dark; 12–23 h light) during the 24-h test to test whether there were dark cycle-related, and thus likely activity-related, differences in thermogenesis in capsaicin-treated hamsters. There were no significant differences in T_IBAT during either the dark or light cycle (bar graph inset of Fig. 5A).

The average T_IBAT across the 24 h in the cold for CAP (35.3 ± 0.2°C) and VEH (35.7 ± 0.1°C). Specifically, T_IBAT was significantly decreased 2 h postcold exposure for capsaicin- versus vehicle-treated hamsters (Fig. 5B; P < 0.05) and then slowly rose until the 12-h time point where it reached control levels. Analysis of data broken into light and dark periods (inset of Fig. 5B) show that only during the first 4 h T_IBAT was significantly lower for CAP-cold hamsters (P < 0.05) demonstrating that the capsaicin treatment decreased T_IBAT in initial acclimation to cold exposure.

Core body temperature.

At RT, animals with capsaicin-injected IBAT had elevated T_c across the 24-h test period. More specifically, capsaicin treatment triggered an overall increase in T_c in hamsters kept at RT, with significant increases seen at various points (5, 11.5, 14, 17.5, and 18 h) after the start of the test (Fig. 6A; P < 0.05), partially due to a less severe drop in T_c for the capsaicin- versus the vehicle-treated animals than typically occurs with lights-on in nocturnal rodents (25). The increase in T_c in capsaicin-treated hamsters during the last 11 h of the test day is reflected in the last light period of the test day (inset of Fig. 6A).

Capsaicin-treated hamsters (CAP-cold) showed low T_c 2–4 h after being placed into 4°C. CAP-cold hamsters showed an exaggerated nycthemeral rhythm in the cold and exhibited a high T_c at the 18-h time point (P < .05; Fig. 6B). This exaggerated rhythm also is seen when light and dark periods are analyzed (inset of Fig. 6B). Specifically, capsaicin-treated hamsters have lower T_c in the cold during the first 4 h in the light and during the last 11h of the test day (inset of Fig. 6B).