Indiana University-Purdue University Indianapolis (IUPUI) / Mu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.

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Generation of free radicals upon reperfusion has been cited as one of the major causes of ischaemia/reperfusion injury. The following series of experiments was designed to study the effect of manganese superoxide dismutase (MnSOD) overexpression in transgenic mice on ischemia/reperfusion injury. A species of 1.4 kb human MnSOD mRNA was expressed, and a 325% increase in MnSOD activity was detected in the hearts of transgenic mice with no changes in the other antioxidant enzymes or heat shock proteins. Immunocytochemical study indicated an increased labeling of MnSOD mainly in the heart mitochondria of the transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x left ventricular developed pressure, was 52 ± 4% in the transgenic hearts as compared to 31 ± 4% in the non-transgenic hearts. This protection was accompanied by a significant decrease in lactate dehydrogenase release from the transgenic hearts. Overexpression of MnSOD limited the infarct size in vivo in a left coronary artery ligation model. Our results demonstrate that overexpression of MnSOD renders the heart more resistant to ischemia/reperfusion injury.

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In order to optimize the overall efficiency of pronuclear microinjection, we designed experiments to: 1) test the best developmental stage for transferring injected embryos to obtain pregnancies and transgenic pups; 2) determine the optimum number of injected embryos transferred to obtain pregnancies and transgenic pups; 3) investigate whether addition of non-injected embryos with injected embryos increased pregnancy rate (PR) and transgenic pups; and 4) establish the time during pregnancy of highest embryonic or fetal loss. Mice (CD1; 3 to 4 wk old), were superovulated with 10 iu PMSG and 5 iu hCG 48 h apart. One-cell embryos were collected for microinjection 20 to 24 h after hCG. The gene used was the whey acidic protein promoter linked to a coding sequence of the human protein C gene (WAP-hPC). Embryos were cultured in CZB at 37°C in 5% CO₂ in air. All the live pups born and embryos and fetuses recovered were analyzed by the polymerase chain reaction to detect the presence of the transgene. Experiment one consisted of nine transfer treatments (TRT) which included all the combinations of three developmental stages (1-cell, 2-4 cell and morula/blastocyst) with three quantities of embryos per transfer (15-24, 25-34 and 35-44). Ten transfers were performed for each TRT. The highest PR and total pups born (TOTP) were obtained after transferring 25 to 34 2-4 cell embryos (PR=90% TOTP=3.5/ pregnancy). However, overall analysis indicated that the percentage of transgenic pups born (%TRS) was highest using 1-cell embryos [33.9%, 20.0% and 11.1% for 1-cell, 2-4 cell and morula/blastocyst (mor/bl), respectively]. The second experiment consisted of six transfer TRT: 20-0, 16-4, 12-8,30-0, 26-4, and 22-8 injected - non-injected embryos, respectively (10 transfers/TRT). Data showed that PR and TOTP can be improved by addition of non-injected embryos. However, the percentage of transgenic pups was significantly (p< .05) higher when all the embryos transferred were injected (53.6 % vs 46.4 % for transfers without and with non-manipulated embryos, respectively). Additionally, 30 embryos per transfer yielded a significantly higher percentage (p< .05) of transgenic pups than 20 embryos per transfer (67.9 % vs 32.1 % for 30 and 20 embryos per transfer, respectively). In experiment three 45 transfers of microinjected embryos were performed (30 embryos per transfer). Fifteen recipients were sacrificed on day 4, 12, and 18 of gestation. On each day all embryos and fetuses were counted and analyzed for the presence of the transgene. The percentage of transgenic embryos or fetuses was not statistically different at any recovery day (45.8%, 35.5%, and 34.6% for days 4, 12, and 18, respectively). However, the number of viable embryos at day 4 was significantly greater than the number of viable fetuses on days 12 or 18 (10 ± 1.1,,5.1 ± 1.6, and 2.4 ± 1.3 for days 4,,12 and 18, respectively). Collectively, the results indicate that: 1) transfer of 20 to 30 1-cell embryos was the best method to obtain transgenic mice, 2) addition of non-injected embryos decreased the number of transgenic pups obtained per pregnancy, and 3) although most of the embryonic losses after microinjection happen before day 4 of gestation, additional losses occurred between days 4 and 18 of pregnancy. / Ph. D.

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