G2 e mitter

For telemetry, G2 emitters (Starr Life Sciences) were implanted into the IP cavity of anesthetized mice. CBT was recorded in Vitalview (Respironics) and analyzed in Clocklab (Actimetrics). Wheel running was assayed as described [17 (link)]. After pre-conditioning with 1X LPS in 12:12 LD during entrainment, mice were moved to DD for 2-3 weeks, then given 3X LPS, followed by 1X LPS 2-3 weeks later. See Supplement for LPS protocol details.

To examine the locomotor-stimulant effects of α-PPP, its effects on core body temperature, and its pharmacokinetic profile, an additional 6 mice were prepared with Starr Life Sciences (Oakmont, PA) G2 E-Mitters using similar procedures to those described previously (Gannon et al. 2016 (link)). After achievement of an appropriate level of anesthesia (inhaled isoflurane 1–3% induction, maintenance to effect), abdominal hair was removed and the surgical area cleansed with soap and water. Subjects were placed in a supine position and the surgical site disinfected isopropyl alcohol, and at least three min later, a midline abdominal incision of less than two cm was made, approximately one cm caudal to the diaphragm. The probes were sterilized using Benz-All (benzalkonium chloride 12.9%, diluted 1:50; Moore Medical, Farmington, CT) according to the manufacturer’s instructions. The probe was implanted sagittally, ventral to the caudal arteries and veins, and dorsal to the digestive organs, and the incision was closed with interrupted mattress absorbable sutures (5–0 Vicryl). Cephazolin (10 mg/kg, IP) was given at the end of surgery for antibiotic prophylaxis, and ketoprofen (2 mg/kg, IP) was at the beginning of the surgery as well as once daily for 3 days to relieve any pain/inflammation.

Tb and activity were measured continuously by telemetry (Starr Life Sciences, Oakmont, PA) using ER4000 energizer/ receivers, G2 E-mitters implanted intraperitoneally, and VitalView software with data collected each minute. Invalid E-mitter data points (defined as a change of ≥ 1 °C in one minute) were replaced by interpolation using flanking data. Any Tb ≤ 24 °C were scored as 24 °C. Unless noted otherwise, the average Tb response was calculated using the first 60 min after agonist dosing. Hypothermia duration is the duration of the interval between dosing and 300 min during which core temperature is <35 °C. Activity is the sum of counts from 10–60 min after A₁AR agonist administration. Inhibitors were dosed 15 min before agonists. Indirect calorimetry was performed as described (Carlin et al, 2016 ). Experiments were performed at ~22 °C. Occasionally a mouse did not become hypothermic with a treatment that generally caused hypothermia. In these cases, the same mouse was retested ≥ 7 days later and routinely became hypothermic and the second data set was used.

As some literature suggests that hyperthermia may contribute to METH-induced lethality, and we have consistently observed hyperthermia in METH-treated animals (Murnane et al., 2012b (link)), we examined whether 5-HT_2A or σ₁ receptors played a role in METH-induced hyperthermia and whether there is an association between hyperthermia and lethality. Mice were prepared with Starr Life Sciences G2 E-Mitters (N = 6 per group). Given the practical necessity of surgically implanting telemetry probes (see section below) to study core body temperature, we choose to initially study modulation of METH-induced hyperthermia at a dose that did not induce lethality (18 mg/kg). In a limited set of additional experiments, we examined modulation of hyperthermia induced by 78 mg/kg of METH, as this was the same dose used in the lethality experiments. These mice were housed individually over receivers designed to collect signals from the probes. Core body temperature was collected over 5 minute intervals and then averaged into 15 minute bins. Pretreatments were administered 30 minutes prior to METH, M100907 and BD 1047 challenges, and core temperature was recorded for 3 hours following all administrations, as this captures the entire time course of each treatment following IP injection.

Core body temperature (T_b) was recorded using a surgically implanted telemetry system into the peritoneal cavity as previously described [82 (link)] (G2 E-Mitters, Starr Life Sciences or TA10TA-F10, Data Sciences, Inc.) and analyzed using the software provided (VitalView for the G2 E-Mitters).

Electronic telemetry devices (G2 E-mitters, Starr Life Sciences, Oakmont, PA) were surgically implanted into the peritoneal cavity using an aseptic technique as previously described [35 (link)]. Mice recovered from surgery with in-cage running wheels (Starr-Life Sciences) with a 120.7 mm diameter and 50.8 mm width for 1 week prior to data collection. Following this recovery period, cages were placed on receiving platforms (ER4000, Starr Life Sciences) to continuously record core body temperature and total locomotion. Glass probes recorded voluntary wheel running at 1-min sampling intervals using VitalView 5.1 software (Starr Life Sciences). ClockLab Analysis 6 (Actimetrics, Wilmette, IL, USA) generated 24-h graphs of wheel running, locomotion, and body temperature. All wheel running, locomotion, and body temperature graphs were double-plotted to better visualize trends in chemotherapy-induced fatigue (average dark phase running wheel revolutions and locomotor emitter counts) and fever (°C). In Experiment 2, a recording error prevented data acquisition on days 25-27 (11-13 d following chemotherapy) but did not affect statistical differences in recovery from fatigue measures. VitalView software was used to ensure that voluntary wheel running occurred when analyzing effects of exercise on body composition and central and peripheral inflammation.