Prior studies from this laboratory found that vapor inhalation of THC using an e-cigarette based system decreases body temperature, with an observed nadir similar to the effects of 10-20 mg/kg THC, i.p. but with a duration of action that was much shorter . These studies also found anti-nociceptive effects of inhaled THC that were comparable in magnitude to those produced by THC injection. Although we’ve previously shown efficacy of the THC inhalation procedure independently in SD and WI strains, there appeared to be a slight difference in thermoregulatory sensitivity with SD rats reaching a lower nadir body temperature . Since our prior studies were not designed as direct strain-comparisons , additional investigation is needed. It continues to be important to connect results of the more translationally relevant inhalation model to a broader previous literature involving THC injection. This study was therefore designed to determine if responses to THC inhalation and THC injection differed across two common laboratory rat strains. Rats were implanted with sterile radiotelemetry transmitters in the abdominal cavity as previously described at 10 weeks of age. For experiments, starting at 12 weeks of age, rats were evaluated in clean standard plastic home cages in a dark testing room, separate from the vivarium, during the dark cycle. Radiotelemetry transmissions were collected via telemetry receiver plates placed under the cages as described in prior investigations . Test sessions for inhalation studies started with a 15 minute interval to ensure data collection, then a 15 minute interval for baseline temperature and activity values followed by initiation of vapor sessions in a separate chamber; animals were returned to the recording chamber after cessation of the inhalation interval. The 15 minute baseline was omitted for the studies with intraperitoneal injection of THC due to the delayed onset of hypothermia . Pre-treatment drugs or vehicle for the antagonist studies were injected prior to THC administration as specified in the following experimental descriptions. ∆9-tetrahydrocannabinol was administered by vapor inhalation with doses described by the concentration in the propylene glycol vehicle and by the duration of inhalation.
Our prior reports show that 30 minutes of inhalation of vapor produced from THC concentrations of 50-200 mg/mL produce plasma THC levels similar to those produced by 3-10 mg/kg THC, i.p. . THC was also administered intraperitoneally in doses of 5, 10, 20 or 30 mg/kg. SR141716 was administered intraperitoneally in a dose of 4 mg/kg. The dose was selected from precedent literature,grow rack including our prior study , as well as pilot work in the laboratory. For injection, THC or SR were suspended in a vehicle of 95% ethanol, Cremophor EL and saline in a 1:1:8 ratio. The THC was provided by the U.S. National Institute on Drug Abuse; SR141716 was obtained from ApexBio . Blood samples were collected via jugular needle insertion following anesthesia with an isoflurane/oxygen vapor mixture . Plasma THC content was quantified using fast liquid chromatography/mass spectrometry adapted from . 50 µl of plasma were mixed with 50 µl of deuterated internal standard , and cannabinoids were extracted into 300 µL acetonitrile and 600 µl of chloroform and then dried. Samples were reconstituted in 100 µl of an acetonitrile/methanol/water . Separation was performed on an Agilent LC1100 using an Eclipse XDB-C18 column using gradient elution with water and methanol, both with 0.2 % formic acid . Cannabinoids were quantified using an Agilent MSD6140 single quadrodpole using electrospray ionization and selected ion monitoring [CBD , CBD-d3 , THC and THC-d3 ]. Calibration curves were conducted daily for each assay at a concentration range of 0-200 ng/mL and observed correlation coefficients were 0.999. A study was conducted in which rats were injected with the vehicle or SR141716 fifteen minutes prior to the start of 30 minute inhalation sessions of PG or THC vapor in a counterbalanced order. The goal was to determine if a dose of this cannabinoid receptor antagonist/inverse agonist that blocks the hypothermia induced by injected THC also blocks hypothermia induced by inhaled THC. The dose was selected based on prior experiments showing efficacy against THC when injected . Telemetry data were only available from 7 animals per group in this study due to experimental exigencies. Approximately half of the test days were completed prior to Experiment 5 and half after the second plasma experiment.
The telemeterized body temperature and activity rate were collected on a 5- minute schedule in telemetry studies, but are expressed as 30 minute averages for analysis . The time courses for data collection are expressed relative to the THC injection time or the initiation of vapor inhalation, and times in the figures refer to the end of the interval . Any missing temperature values were interpolated from the values before and after the lost time point. Activity rate values were not interpolated because 5-minute to 5-minute values can change dramatically, thus there is no justification for interpolating. Data were analyzed with Analysis of Variance including repeated measures for the Drug treatment condition and the Time after vapor initiation or injection. Any significant main effects were followed with post-hoc analysis using Tukey or Sidak correction. All analysis used Prism for Windows . This study shows that the inhalation of THC vapor using an e-cigarette approach induces hypothermia in both Wistar and Sprague-Dawley male rats, with the latter strain exhibiting a significantly greater reduction in temperature after identical inhalation conditions. This work was motivated by an indirect observation of a hypothermic insensitivity of SI rats relative to Sprague-Dawley rats in our prior vapor inhalation studies as well as a similar outcome of preliminary pilot studies for an investigation of the impact of CBD co-administration with THC . The difference in the present study was observed in age-matched, identically treated groups and the difference was present in the initial THC inhalation exposure, in the intraperitoneal injection studies and in the final vapor inhalation investigation across an ~one year interval. The data therefore confirm a quantitative, but not qualitative, strain difference in the thermoregulatory response to inhaled THC without a difference in the anti-nociceptive effect. Since the Sprague-Dawley rats were significantly smaller in size, it was critical to determine if THC dose differed between strains under the inhalation conditions. The plasma data show that identical plasma concentrations of THC were produced in each group after inhalation of THC vapor at the 50-200 mg/mL concentrations across different phases of the study and therefore did not depend on strain body weight. Thus, the strain difference in body temperature response cannot be attributed to the delivery of a different THC dose and the strain differences must therefore be attributable to other factors. The consistency of THC plasma levels following vapor inhalation across rats that differed in body size, due to strain, echoes the result of a prior study which showed approximately equivalent plasma concentrations of THC after inhalation in male and female WI rats that differed significantly in body weight by sex .
In that prior study, however, male and female WI rats exhibited a similar magnitude of hypothermia and anti-nociception produced by the THC, indicating minimal sex-differences. Together this indicates that the differential effects of THC across strain in this study cannot be uniquely associated with rat body size. The difference in plasma THC across the first two observation time points in this study was unexpected, particularly given the similarity of the temperature response to the THC 100 mg/mL condition across the first and second vapor inhalation experiments . However, this is the first time we have explicitly compared plasma levels of THC across significant intervals of time in the same rats following inhalation. Additional experimentation would be required to determine if this is due to different respiration / weight, different drug distribution, different metabolism or possibly differential behavior in the vapor exposure chamber. With respect to this latter,microgreens shelving animals could vary where they put their nose, relative to the vapor delivery location and thereby change the exposure; inspection of the individual distributions in Figure 3 supports this possibility. Also of interest is that plasma THC concentrations reached following inhalation of THC at the 200 mg/mL concentration were not substantially higher compared with plasma concentrations after the 100 mg/mL dose which may reflect a ceiling on the rate of intrapulmonary uptake as drug concentration is increased. For the present purposes, however, it is most critical that there was no significant difference across the strains at any time of plasma collection. There was no strain difference in the anti-nociception assay, which shows that the influence of strain on hypothermia is dissociable from the effects on anti-nociception. This dissociation combines with the similar plasma concentrations across strain to further support the interpretation that the strain-related hypothermia difference was not due to different THC dose. It suggests instead that WI rats are more resistant to body temperature dysregulation than are Sprague-Dawley rats, either in response to THC or more generally. Consistent with this interpretation, one prior report shows a differential hypothermia in response to challenge with the serotonin receptor 1a agonist 8-OH-DPAT in different breeding colony sources of Sprague-Dawley rats and in WI rats . In contrast, our prior report showed that, if anything, a slightly larger temperature change relative to vehicle/baseline in response to mg/kg equivalent injection of 8-OH-DPAT in our study . Thus it appears most likely the strain difference in this study is specific to THC-induced hypothermia. The present data also appeared to suggest a more pronounced strain difference in thermoregulatory sensitivity after i.p. injection compared with the difference after inhalation of THC. This may possibly reflect differences in THC distribution throughout the larger body mass of the WI rats, in part or in whole due to differences in body fat percentage; indeed the strain differences in THC-induced hypothermia appeared to grow more pronounced as the animals aged and the body size difference increased.
This conclusion seems unlikely, however, given that the strains did not differ in plasma concentrations of THC when exposed to identical inhalation conditions or when administered an equivalent mg/kg dose by i.p. injection. One alternate hypothesis is that WI rats became more tolerant from a pharmacodynamic standpoint, compared with the Sprague-Dawley rats, in the course of the repeated exposure. Recent work with the inhalation model shows that adult WI male rats do not become tolerant to THC after 4 sequential days of twice-daily vapor inhalation , thus it seems unlikely that there would be significant pharmacodynamic tolerance with the present dosing history. Together the evidence suggests the apparent strain difference in thermoregulatory response to THC that interacted with route of administration is not directly related to THC exposure but may be related to non-cannabinoid mechanisms involved in temperature regulation. There was no evidence of any strain differences in locomotor suppression caused by THC in this study but conclusions must be tempered by the general failure to observe THC-related effects on activity. This is somewhat expected since activity rate assessed by radiotelemetry was only inconsistently changed by THC inhalation in our prior studies using these methods and it required a 30 mg/kg, i.p. injection dose in male Sprague-Dawley rats to suppress locomotion consistently in another study . One curious finding in this study was that a dose of the CB1 antagonist/inverse agonist SR141716 which was sufficient to block or attenuate the body temperature response to THC administered intraperitoneally did not significantly alter the response to vapor inhalation of THC. SR141716 did not appear to alter the initial nadir in body temperature observed 60 min after the start of inhalation but may perhaps have slightly facilitated the return to normative temperature after about 90 or 120 minutes after the initiation of vapor inhalation. Interestingly the body temperature of rats only started to diverge from that observed with vehicle pre-injection 60 or 150 minutes after THC i.p. injection. This finding warrants additional followup study.Identifying individuals at heightened risk for developing alcohol-related problems remains an important goal of medical practitioners. One important risk factor for alcohol misuse is one’s own genetic liability. Twin and family studies indicate that genetic influences on alcohol use disorders account for ~50% of the variation in the population. Genome-wide association studies have identified multiple variants associated with AUD, alcohol consumption, and maximum alcohol intake. Using information from these GWASs, we are now able to aggregate risk across the genome by creating polygenic risk scores in independent samples.