Generation Of GABA Transporter Subtype I Gene-knockout Mice By Homologous Recombination And Analysis Of Their Phenotypes

(Part I and Ⅱ) Mice lacking GAT1 and the behavioral effects of ethanolIt is widely accepted that the GABAergic system plays an important role in theaction of ethanol in vivo. GABA transporter subtype 1 (GAT1) constructs highaffinity reuptake sites in the CNS and regulates GABAergic transmissions. In ourpresent work, mice lacking GAT1 were developed by homologous recombination asdemonstrated by the PCR and Southern-blot analysis, and there is no expression ofGAT1 in GAT1^-/- mice as confirmed by the RT-PCR and Western blot analysis.Also, GABA reuptake activity is significant decreased in the GAT1^-/- mice brainand spinal cord. Both hetero-and homozygous GAT1 mutant mice were tested forethanol, saccharin or quinine consumption, ethanol-conditioned place preference,ethanol-conditioned taste aversion, ethanol-simulated motor activity andethanol-induced sedation/hypnosis. The GAT1^-/- mice showed decreased ethanolaversion and ethanol reward, and insensitivity to both the sedative/hypnotic and themotor stimulant effects of ethanol, along with increased avoidance of quininepreference and consumption. Interestingly, the GAT1^-/- mice show anxiolytic-likebehavior in the EPM test. In addition, ethanol still can have obvious anxiolytic effectin the GAT1^-/- mice. However, GAT1^-/- mice showed significantly increasedconsumption of ethanol and saccharin, enhanced the rewarding and preference effectof ethanol, increased avoidance of quinine, and higher sensitivity to the motorstimulant effect of ethanol. These results firstly demonstrated that GAT1, perhaps in abi-directional way, modulates some behavioral effects of ethanol. Hence, theGAT1mutant mice provided us a very useful model to investigate the mechanisms ofethanol action in vivo.(Part Ⅲ) Mice lacking GAT1 and the antinociceptive effectsTiagabine, one of antiepileptic drugs, is effective in the treatment of differentpain. Its pharmacological mechanism is thought to inhibit the activity of GABAtransporter subtype 1(GAT1) and thus elevate the extracelluar concentration of GABA,which cause activation of GABA receptors and increase the GABA-mediatedinhibition on the CNS. The GAT1 knockout mice (both heterozygous andhomozygous mice) are regarded as a model of lifelong treatment with a GAT1inhibitor. Here, using four kinds of nociceptive tests: the tail-flick test, the hot-platetest, the acetic acid-induced writhing test and the formalin test, we demonstratedhomozygous mice show significant analgesia in nearly all nociceptive tests except forthe second phase in the formalin test. Heterozygotes also show analgesia in two acutethermal pain models. When treated with two GAT1 inhibitors (No-711 and tiagabine,10 mg/kg, i.p.), wild-type and heterozygous mice exhibit significant analgesia in allfour nociceptive tests, whereas no antinociceptive action of these two inhibitors inhomozygous mice. The antinociceptive effect of morphine (5mg/kg, s.c.) is similar inall three genotypes in two thermal pain tests, however, in the tail-flick test morphinehas significantly enhanced antinociceptive effect in the GAT1 mutant mice. Comparedwith wild-type mice, the GAT1 mutant mice also show increased stress-inducedanalgesia (SIA) in the forced-swim tests in both cold water (4℃) and warmer water(33℃). Furthermore, we found the GAT1 mutant mice show altered responses tobaclofen-induced hyperemia, which indicated the normal functions of GABA(B)receptors in GAT1 mutant mice may be changed. The present results indicate thatGAT1 plays an important role in regulating nociceptive responses in the CNS ofrodents. Besides, these results also suggest that long-term treated with high doses ofthe GAT1 inhibitors as anodyne may cause tolerance for the persistent pain.(Part Ⅳ) Mice lacking GAT1 and drug rewarding, cognitionMany studies have demonstrated that the GABAergic system is involved inregulating processes of cognition and drug addiction. GABA transporter subtype1(GAT1) that regulates GABA concentration in synaptic cleft by its GABA uptakeactivity is one of key player in the GABAergic system. In our previously studies,using GAT1-overexpressing transgenic mice (the Tg mice), we demonstrated thatup-regulation of GAT1 in brain lead to decrease the rewarding and dependence effectsof morphine, and impair the cognitive ability in the Tg mice. Here, we find that theGAT1 (-/-) mice reduce but GAT1 (+/-) mice enhance the rewarding properties ofmorphine and cocaine as shown in the CPP test when compared with their wild-typelittermates. Also, GAT1 (-/-) mice show significant decrease in one of main morphinewithdrawal symptom-jumping, but there is no difference in the other withdrawalsymptoms between the GAT(-/-) mice and wild-type mice. In two different learningand memory tests: a spatial learning test (Morris water maze) and an avoidancelearning test (passive avoidance), the GAT1 (-/-) mice exhibit impaired memoryperformance, whereas the GAT1 (+/-) exhibit enhanced memory performance.Consistent with these findings in the behavioral tests, the GAT1 (-/-) show reducedbut the GAT1 (+/-) mice show enlarged the amplitude of theta-burst stimulation(TBS)-induced long term potentiation (LTP) in hippocampal CA1 region in vitrowhen compared with wild-type mice. All these results suggest GAT1 can bidirectionalregulate not only the rewarding effects of many drugs (ethanol, morphine and cocaine)but also the cognitive ability in mice. These findings also confirm a strong positivecorrelation between the drug rewarding and the learning and memory.