The Experimental Study Of Methamphetamine Effects On Electrophysiological Properties Of Mesolimbic Dopaminergic Neurons

In the midbrain of mammals, dopaminergic neurons (DA nerurons) mainly distribute in substantia nigra (SN) and ventral tegmental area(VTA). They innervate to wide and discrete brain areas and are involved in series important functions, e.g. motivation, emotion and addiction. Mesolimbic DA pathway is the dopaminergic projections from VTA to nucleus accumbens (NAc) and has been proposed to be the neural structural basis of addiction and also served as the main target for psychostimulants. Recent studies show that DA neurons in VTA possess different molecular, electrophysiological and pharmacological reaction characteristics. Therefore identify mesolimbic DA neurons is a critical premise for further researches.Methamphetamine (MA) is a widely abused and highly addictive pyschostimulant. Recent researches implicate that MA not only inhibits DA neurons, but also excites them through DAT. So MA can impact DA neurons, but the various effects and underlying mechanisms remain unclear and need further research.Most of DA neurons display hyperpolarization-activated cation current (Ih) which not only directly participates in setting membrane potential, generating spontaneous action potential, releasing neurotransmitter, but also connects with alcoholic addiction, Parkinson’s disease, and even antidepressants mechanism and Ih has been also proposed as an electrophysiological marker. Ih can be regulated by many second messengers, such as PIP2, cAMP, PKc, and can be also affected by extracellular matters. MA could increase the extracellular DA mainly by combining with vesicular monoamine transporters2(VMAT2) and dopamine transporter (DAT) and the increased DA can inhibit DA neurons through activating D2receptor. But we still do not know much about the effects of MA on Ih of DA neurons, and the mechanism of the effects.Therefore we designed the following experiments to observe the effects of MA on mesolimbic DA neurons.(1) In order to label the mesolimbic DA neurons, fluorescent retrograde tracer was injected into NAc after fixing two weeks old rats on stereotaxic apparatus. Tyrosine hydroxylase (TH) immunohistochemistry was employed to identify the labeled cells24hours later.(2) The brain slices were prepared from the rat brain which had been injected24hours ago and the thickness was300μm. Using infrared phase contrast microscope we could observe the labeled neurons. Whole-cell patch-clamp recordings were made to observe the effects of different concentrations of MA on labeled neurons and a proper concentration would be decided for the following experiments. Then with the treatments of10μmol/L concentration of MA, we tested the changes of basic characteristics of spontaneous action potentials, voltage-current response.(3) Ih was identified with ZD7288and CsCl. And the effect of MA on Ih was also observed.(4) For the investigation of regulating mechanism of MA on Ih, we used sulpiride, a D2receptor blocker, for preincubation, and observed the effects of MA.The results showed:(1) The neurons projected to NAc were successfully labeled with fluorescence tracer. Immunohistochemical staining showed that labeled cells were TH positive which mainly distributed in VTA. These results implicated that the labeled neurons were mesolimbic DA neurons.(2)10and100μmol/L MA reduced the spontaneous action potential frequency of labeled cells obviously and rapidly and even stop them, while1.0and0.1μmol/L MA had no effect.10μmol/L MA was used for the further experiments. We found that MA evidently reduced spontaneous action potential frequency to(0.63±0.15)%versus without MA, prolonged action potential duration, enlarged after hyperpolarization potential, inhibited the "sag", and all these changes were statistically significant (p<0.05). (3) Hyperpolarizing potentials could elicit typical Ih and it could be blocked by ZD7288and CsCl. MA inhibited Ih, but had no effect on its voltage and time dependence.(4) Meanwhile after the use of sulpiride MA show a excite effect and had no inhibition on Ih.In conclusion MA inhibits the mesolimbic DA neurons and its Ih, and these effects may mainly through the activating of D2receptor.