We recently demonstrated that vapor inhalation of THC using an e-cigarette based approach produces anti-nociceptive effects and reduces the body temperature and spontaneous activity of male and female rats . These are canonical effects that are observed after injection or oral delivery of THC in laboratory vertebrates including in rats , mice , monkeys and dogs , although the individual behavioral or physiological outcomes may be observed after different doses. Behavioral and physiological effects, and plasma THC levels, of THC delivered by vapor inhalation depend on the exposure duration as well as the dose administered. We have shown that dose can be controlled during inhalation exposure by varying either the concentration of THC in the e-liquid vehicle or the duration of exposure at a fixed concentration . This validated platform is therefore ideal to test the hypothesis that aerosol THC exposure of lobsters has physiological effect. Development of different animal model species, including invertebrates, for the evaluation of drug effects can offer both unique and converging advantages, as recently reviewed by Smith . The lobster is an established model for evaluating neuronal morphology, central pattern generation and synaptic mechanisms in the stomato-gastric ganglion . More practically, the lobster can be studied within institutions that are not equipped to oversee vertebrate animal research, or can be studied at reduced expense in institutions where vertebrate research is supported. A recent review indicates there are no clear data on lobster nociception and decapod crustacean investigations of nociception do not typically involve thermal stimuli ; only one available report shows that crayfish are sensitive to a thermal stimulus delivered by soldering iron .Thus it serves the additional goal of determining if thermal nociception exists in this crustacean species which would add to the evidence for thermoception in decapod crustaceans more generally. There is very limited evidence on whether the lobster would be behaviorally sensitive to THC exposure, however, pipp mobile systems the neuromuscular junction of lobsters appears to be regulated, in part, by cannabinoid mechanisms.

Turkanis and Karler showed that THC had doserelated effects on excitatory neuromuscular junction potential amplitudes, increasing them at moderate concentrations and decreasing amplitude at higher concentrations . This enhances confidence that some cannabinoid-sensitive mechanisms are present in the lobster and that THC might affect locomotor behavior, despite the fact there may not be a homolog or ortholog of the vertebrate endocannabinoid receptor expressed in the lobster . The invertebrate C. elegans expresses a cannabinoid-like ortholog receptor which mediates effects of the endogenous cannabinoid agonists 2-arachidonoylglycerol and anandamide , suggesting that there may be as yet un-identified ortholog receptors in the lobster. It is also possible that THC acts in the lobster via transient receptor potential channels, since THC appears to function as a ligand at TRPV2, TRPV3, TRPV4, TRPA1, and TRPV8, as reviewed . The Caribbean spiny lobster expresses 17 TRP including TRPA and TRPV homologs . Hypolocomotion is a canonical sign of cannabinoid action in rats and mice , and occurs after vapor inhalation of THC . Thus, locomotor activity was selected to assay for evidence of in vivo behavioral effect. Less directly, recent studies in the crayfish, a related aquatic crustacean, have shown locomotor effects of cocaine, morphine and methamphetamine and intravenous self-administration of amphetamine . This further enhances confidence that behavioral pharmacological effects of recreational drugs can be effectively assessed in the lobster. Traditional cooking of lobster is by immersion of live animals in either boiling water or steam, leading to concerns by some that the animal might experience pain. Indeed, live cooking has been banned in Switzerland . There is no available evidence demonstrating clearly that lobsters are sensitive to temperature, however one paper has shown that crayfish respond to a hot metal rod stimulus applied to the claw .

Since TRPA1 homolog receptors in invertebrates are activated by high temperature, e.g., at about 48 °C in one study and over 43 °C in other work , these mechanisms may be the primary thermoreceptor. It is thus of interest to develop assays to determine if lobsters exhibit thermal nociceptive behavioral responses and then to determine if those responses can be altered by THC exposure. The hot-water tail withdrawal assay in rats involves a reflexive tail movement when it is inserted in hot water and has been shown to be altered in rats after vapor inhalation of THC . Thus, one goal was to determine if warm water immersion produces a behavioral response in the lobster and if so, if THC exposure decreased thermal nociception as it does in rodents . As part of a model development, it was important to determine if different responses could be obtained from tail, claw or antenna immersion and if the response depended on the temperature of the water bath, as in . Wild caught female and male Maine lobster were obtained from a local supermarket. When housed longer than several hours in the laboratory, the animals were maintained in chilled , aerated aquariums and fed variously with frozen krill, fish flakes and anacharis. The tissue distribution studies were conducted under protocols approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute due to a decision that a protocol was required for this invertebrate species. The remaining studies were conducted at the University of California, San Diego where the institution does not require protocol supervision/approval for this invertebrate species. Sealed exposure chambers were modified from the 259 mm × 234 mm × 209 mm Allentown, Inc. rat cage to regulate airflow and the delivery of vaporized drug to the chamber using e-cigarette cartridges as has been previously described . The controllers were triggered to deliver the scheduled series of puffs by a computerized controller designed by the equipment manufacturer . The chamber air was vacuum controlled by a chamber exhaust valve to flow room ambient air through an intake valve at ~1 L per minute. This also functioned to ensure that vapor entered the chamber on each device triggering event. The vapor stream was integrated with the ambient air stream once triggered. The chambers were empty of any water or bedding material for these exposures .For these studies, animals were obtained, dosed and euthanized for tissue collection within 4–6 h.

Lobsters were exposed to THC vapor for 30 or 60 minutes, then removed from the chamber and rinsed with tap water. Thereafter, they were rapidly euthanized by transection of the thoracic nerve cord using heavy kitchen shears and then transection of the thorax behind the brain by a heavy chef’s knife. Samples included the gills , claw muscle obtained from proximal and distal aspects, anterior and posterior segments of tail muscle, a red membrane surrounding the claw muscle , brain, heart, liver and hemolymph. Hemolymph was allowed to coagulate to facilitate analysis as ng of THC per mg of tissue, as with the other tissues. For N = 2 per exposure-duration group, one claw was cooked immediately after euthanasia by boiling it in water for 10 min, prior to collection of muscle tissue and the red membrane that surrounds it. Tissues were frozen for storage until analysis was conducted. Tissue THC content was quantified using liquid chromatography/mass spectrometry adapted from methods describe previously . THC was extracted from brain tissue by homogenization in chloroform/ACN containing 100ng/mL of THC-d3 as internal standard followed by centrifugation, decanting of the lower supernatant phase, evaporation and reconstitution in acetonitrile for analysis. Specifically, ~200–300 mg of tissue was homogenized in 1.5 mL of chloroform, 0.500 mL of acetonitrile and 0.100 mL of deuterated internal standard . Samples were centrifuged at 3000 RPM for 10 min, followed by decanting of the lower supernatant phase, evaporation using a SpeedVac, and reconstitution in 200 μL of an acetonitrile/methanol/water mixture. Separation was performed on an Agilent LC1100 using a gradient elution of water and methanol at 300 μL/min on an Eclipse XDB-C18 column . THC was quantified using an Agilent MSD6180 single quadrupole with electrospray ionization and selected ion monitoring [THC and THC-d3 ]. Calibration curves were generated each day at a concentration range of 0–200 ng/mL with observed correlation coefficients of 0.9990. For these studies, industrial drying rack animals were maintained in the laboratory for 4–21 days in chilled aquariums. Salt-water baths for the nociception assay were maintained at the target temperature and confirmed by thermometer immediately prior to each test. The investigator held the animal gently by the thorax and the tail or the tip of the antenna was inserted approximately 3 cm; the claws were inserted to a depth of approximately 5 cm. The latency to respond was recorded by stopwatch and a maximum 15 s interval was used as a cutoff for the assay. Homarus genus lobsters exhibit asymmetry of their claws with one larger and one smaller claw that can be on either the left or the right; feral experience appears to be necessary for proper development of the claw asymmetry as it is less pronounced in cultivated lobsters . This asymmetry produces a crusher muscle that is constituted of 100% slow fibers, whereas the cutter muscle exhibits only 90% fast fibers as assessed by ATPase staining and fast and slow motoneuron innervation . Thus, for this study the cutter and crusher claws were assessed independently. The order of assessment was always tail, claw, claw then antenna. The body part was inserted in the ambient water bath for 5–10 s after each warm water assessment.

The temperatures for assessment were “ambient”, and then three warm temperatures ; the order of testing of the warmer temperatures was in a counterbalanced order with at least two hours between assessments and no more than two tests per day. Lobsters were next assessed for the reaction of tail, crusher and cutter claws, and the antenna to insertion in 48 °C water after vapor exposure to PG or THC for 30 or 60 min, with the testing order counterbalanced. Distinct responses of tail, antenna and claw were observed following insertion in warm water but not in the ambient temperature water bath . Sometimes, a reflexive and powerful contraction of the tail muscle was observed first . This appeared to be the caridoid escape response best described in crayfish, but likely present in the lobster , which is a complex behavior mediated by lateral giant and medial giant interneurons . In other cases, the lobster initiated distinct movements of legs and claws, this often preceded the powerful tail contraction. Thus, the tail assay was scored with two latency values, the very first reaction of any type and the tail contraction if it occurred. For analysis, the time of first overt response was used. The post-hoc test failed to confirm any significant difference associated with Vapor Condition for any individual body part. The primary finding of this study was that vapor exposure of Maine lobsters to Δ9-tetrahydrocannabinol , using an e-cigarette based system, produces tissue levels of THC in a dose dependent manner. THC was confirmed in the hemolymph , claw and tail muscle, brain, heart and liver . This wide distribution across body tissues is consistent with respiratory uptake, i.e., via the gills with distribution by the hemolymph circulation of the lobster. This conclusion is further supported by the much higher amount of THC that was associated with the gill tissue, consistent with a limited uptake by the respiration system of the lobster. The THC exposure also had behavioral consequences, since locomotor activity was significantly reduced after exposure to THC vapor compared with exposure to the vehicle vapor . Hypolocomotion is a canonical feature of THC exposure in rats and mice, at least at higher doses, thereby confirming a similarity of effect across the vertebrate and invertebrate organisms in which this has been evaluated. Thus, the assertions of the restaurateur that cannabinoids could be introduced into the lobster by atmospheric exposure , and that this would be in sufficient amount to induce behavioral effect, is supported. The impact of THC on thermal nociception was, however, minimal. The locomotor effects may be specifically related to a report of THC altering the amplitude of excitatory potentials at the lobster neuromuscular junction in a concentration dependent manner . Overall, however, it confirms that the levels of THC achieved by as little as 30 min of vapor exposure were behaviorally significant.