Investigation of the effects of focused ultrasound pulsation of nucleus accumbens on opioid conditioned place preference in opioid dependent rats

Introduction and Objectives: As we know for today, Deep Brain Stimulation (DBS) is the only modality proven to be effective on selective stimulation of the deep brain structures. It was previously reported that by using DBS, stimulation of nucleus accumbens (NA), a region that plays a pivotal role in the pathogenesis of substance addiction, is effective for the treatment of substance addiction. In this study, we used focused ultrasonography, a non-invasive modality, in a place conditioning model to stimulate NAC in rats which have been administered morphine during the experiment, in order to investigate the effect of the stimulation on the conditioned place preference. Material and Method: In this study 26 Sprague Dawley rats ranging between 300 to 490 gr were used. The study was designed in two stages. In the first stage, rats were divided into two groups: serum physiologic (SF) and morphine. Reinforcement and rewarding effects of morphine were assessed by using a 14-days Conditioned Place Preference (CPP) protocol. CPP protocol was divided into 3-days pre-testing, 10-days conditioning and 1-day preference testing phases. In preference testing, morphine seeking behavior and the rewarding effect were assessed. Magnets are placed surgically on the calvaria of each rat. At the second stage, morphine group (conditioned by the administration of morphine) was divided into two groups: SHAM group, which receive fake stimuli and US group which receive low intensity focused ultrasonographic stimulation. During the 10-days stimulation phase, SF group did not receive any stimulation and CPP procedure was being held simultaneously with SF injections during this phase. SHAM group received fake ultrasound stimulation, whereas US group received low intensity focused ultrasonographic stimulation and both groups were conditioned with SF and morphine injections. We adjusted the ultrasound parameters as follows: fundamental frequency: 2,8 MHz, DTF / PRF: 250 kHz, KO / DC: %50, sonication duration: 300 msn, delay: 1700 msn, Isppa (spatial-peak pulse-average intensity) :4.06 W/cm2, Ispta (spatial-peak temporal-averageintensity): 305 mW/cm2. One day after the stimulation procedure was finished, morphine seeking behavior and rewarding effect were assessed in rats which had been and were being administered morphine. In statistical analyses, if more than two groups were compaired, Kruskal Wallis non-parametic tests were performed and in sub groups comparisons Dunn Tests were performed. In dual comparisons, two different groups were analysied by non-parametric Man Whitney U test and in-group changes analysied by non-parametric Wilcoxon test. In all tets, 0,05 determined as the statistical significance level. Results: At the initial stage of our study all rats preferred the black spot significantly (p<0.001). It was reported that morphine induced significant place preference (p<0.05). At the second stage, it was observed that application of low intensity focused ultrasound to NAC, did not reduce the morphine-induced place preference in US group rats which already developed morphine-induced place preference and were still being conditioned by morphine, on the other hand it also did not increase the preference significantly. There were no significant difference between US and SHAM groups with regards to place preference after ultrasonographic stimulation (p>0.05). On the other hand, morphine-induced place preference increased significantly in SHAM group after ultrasonographic stimulation (p<0.05). Before the ultrasonographic stimulation morphine-induced place preference was significantly higher in US group in comparison with SF group (p<0.05), whereas after the ultrasonographic stimulation the difference between these groups were insignificant (p>0.05). Conclusion: To the best of our knowledge, our study is the first study that investigates the effects of low density focused ultrasonographic stimulation on the addiction behavior. In this study, the animals were conscious and were not anesthetized during the low density focused ultrasonographic stimulation; therefore it is unique in this manner. We designed and used a fixing material in order to keep the animals in order during the stimulation. Since most of the studies based on focused ultrasound only held for a day, our study is also different in the way ultrasonographic stimulation was performed for 10 days. In this study we observed that the increase in morphine seeking behavior was avoided by ultrasonographic stimulation in rats in which morphine-induced seeking behavior was established and kept being conditioned with morphine. On the other hand, it did not reduce morphine seeking behavior. Since morphine and other opiate addictions are very important social problems, we state there is a need for future studies to investigate the effects of low density focused ultrasound as an alternative treatment modality in addiction. Key Words: Conditioned Place Preference, Low Density Focused Ultrasound, Morphine Addiction, Neuromodulation