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  • Analgesic activity was evaluated by measuring tail flick lat

    2021-11-29

    Analgesic activity was evaluated by measuring tail flick latencies to the immersion of rat’s tail in water, maintained at ∼51°C [18] after recovery from anesthesia. Typically, rats were immobilized inside a restrainer, leaving only the tail exposed, which was then immersed into the hot water bath, at 5 and 15min post intracisternal agent administration. The latencies to tail withdrawal were measured precisely using a timer. In the absence of any reflex, a 15s period was used as a cutoff to prevent tissue damage. To examine if GABA-C receptors are present presynaptically, in the substantia gelatinosa, their co-localization with presynaptic markers, synaptophysin (SYP) or synaptic vesicle protein (SV2), was tested. In separate experiments, rats were anesthetized with pentobarbital (100mg/kg) and perfused with cold 5% heparinized saline solution, followed by 4% paraformaldehyde. Spinal cords (lumbosacral) were removed and further cryoprotected in 20% and 40% sucrose solution. 40μm thick sections were cut using a vibratome (Leica 1200S), free floating sections collected in phosphate buffer saline (PBS) and processed for doubled labeled immunofluorescence colocalization [29]. Briefly, selected sections were incubated in 0.2% Triton X-100 for 10min. After three subsequent washes, the sections were blocked in 5% normal goat serum for 1h at room temperature and incubated overnight in a cocktail of polyclonal GABA-Cρ2 receptor antibody (1:300) and monoclonal ML133 HCl for presynaptic marker SV2 (1:200) or SYP (1:300) at 4°C in a humid atmosphere. GABA-Cρ2 receptor (sc-21343), SV2 (sc-11944) and SYP (sc-17750) antibodies were purchased from Santa Cruz Biotechnology, Inc. Subsequently, sections were washed and incubated for 1h at room temperature with secondary antibodies, donkey anti-goat Alexa-594 and goat anti-mouse Alexa-488. Sections were then washed with PBS and mounted on the slides, examined and photographed using the Leica DMLB microscope equipped with a Retiga 2000R camera. All photograph composites were generated using NIH ImageJ software program and Adobe Photoshop (San Jose, CA). For statistical analysis, a two-way ANOVA was used to determine the anti-nociceptive effects of GABA-C receptors. Treatment and time were considered as the two factors for variation. Where ANOVA indicated significant effects and/or interactions, post-hoc comparisons were conducted using Sidak’s multiple comparison post-test. The level of significance was taken as <0.05. All values are reported as mean±SEM. GraphPad Prism software was used for analysis.
    Discussion and conclusion GABA-C receptors are widely expressed in the CNS. Although their functional roles are well studied in the visual system [5], [26], much less is known about their involvement in pain. In the present study, CACA, an agonist to GABA-C receptors [16], induced an anti-nociceptive affect, when injected directly into the CNS. This effect was blocked by TPMPA, a GABA-C receptor antagonist [16], [28]. TPMPA, when applied alone, decreased the latency of tail withdrawal, presumably by blocking the effect of endogenous GABA on the ρ type receptors. As expected, injection of IG, a GABA-A receptor agonist, also produced a significant increase in the latency of tail withdrawal [13], [27]. TPMPA did not antagonize this effect, indicating that the analgesic actions of GABA-A and GABA-C receptors are independent from each other. Since drugs were administered directly into the CNS via intracisternal injections, the anti-nociceptive effects are likely due to the activation of centrally located GABA-C receptors, unlike previous studies, where peripheral effects were mainly studied [30]. The mechanisms involved are, however, unclear. GABA-C receptors are distributed across the CNS. While some of the anti-nociceptive effects of CACA could be due to actions on receptors located proximal to the site of injections and/or through ascending/descending control from higher centers [41], it is important to note that analgesic effects were significant at 5 and 15min post-injection, around the time dye-penetration experiments revealed that drugs could indeed redistribute to the lumbar region of the spinal cord.