A Study On The Role And Neural Circuit Mechanism Of NAc GABAergic Neurons In The Cocaine-context-associated Memory

The nucleus accumbens (NAc) as the main regulating brain nucleus of the addictive drugs, is consisted of>95 GABAergic medium-sized spiny neurons (MSNs) and defined by differential expression of D1 and D2 dopamine receptors. Both D2+ and D1+MSNs in the NAc form projections into the ventral pallidum, whereas only D1+MSNs directly project into midbrain neurons, such as substantia nigr (SNr) and ventral tegmental area (VTA), etc. They are critical in rewarding and aversive learning, and understanding the function of these NAc efferents and the alteration of their targeted brain regions in responding to a reward-associated context is important for exploring the expression of motivational effects induced by cocaine-context-associated cues.Optogenetics combines tissue-and cell type-specific expression of light-sensitive proteins named opsins and advanced optical methods to reach, record, and control the activity of a specific cell population. Compared with the traditional research methods in the nervous system such as electrophysiological techniques and neuropharma-cology methods, optogenetics has the high spatial selectivity and specificity, and high temporal precision. Using optogenetics may timely manipulate the activity of pecific neurons in the certain brain area of freely moving experimental animals, and brings breakthrough for the research of neural circuit mechanism in the rewarding-behaviour. Using optogenetics to timely and in vivo manipulate the activity of NAc GABAergic neurons within drug-context-associated memory behavior in mice, is needed in researching the function and neural mechanism of NAc GABAergic neurons in the drug-context-associated memory.In this study we used the VGAT-ChR2(H134)-EYFP mice expressing channelrhodopsin-2 and fusion protein EYFP under the control of the vesicular GABA transporter promoter to research, and foud NAc had a moderate level of ChR2-EYFP expression in VGAT-ChR2-EYFP mice. The localization of ChR2-EYFP fluorescence in GAD67-positive cells could be observed throughout the NAc, indicating these ChR2- EYFP expressing cells are mostly GABAergic neurons. And the GABAergic neurons expressing ChR2 in the NAc responded with high fidelity and sustainability to prolonged blue laser up to 10 Hz, in strings of 473nm light flashes lasting for 1 s and 2 min. Thes results indicate optogenetics could specifically manipulate the activity of NAc GABAergic neurons of VGAT-ChR2-EYFP mice.Based on the establishment of optogentic manipulating behavior in mice, we used cocaine conditioned place preference model to research the function of NAc GABAergic neurons in the expression of cocaine-context-associated memory. After training of cocaine conditioned place preference, Day 4 under the condition of no laser delivery both WT and VGAT-ChR2(H134)-EYFP mice showed significant preference for the cocaine-paired chamber, indicating the successful expression of the acquired cocaine-context-associated memory. However, laser delivery to the bilateral NAc abolished the cocaine CPP in VGAT-ChR2(H134)-EYFP mice, but had no effect on WT mice.These results suggest that optogenetic activation of GABAergic neurons in the NAc produces a rapid inhibitory effect on the expression of cocaine-context-associated memory. Mice that acquired cocaine-CPP were tested for drug-induced CPP with laser delivered into bilateral NAc and a second CPP test was carried out 24 h later without laser stimulation, found the optogenetic activation of GABAergic neuron significantly reduced the cocaine preference score in VGAT-ChR2(H134)-EYFP mice as compared with WT mice. However, when tested 24 h later, no significant difference in the expression of cocaine-induced CPP was observed between the VGAT-ChR2(H134)-EYFP and WT mice. These results demonstrate the activation of GABAergic transmission in the NAc upon re-exposure to reward-associated context transiently and reversibly inhibits the expression of cocaine-induced CPP. Futhermore the low-dose cocaine-induced behavioural sensitization was significantly decreased by laser stimulation of ChR2-expressing GABAergic neurons in the NAc of VGAT-ChR2(H134)-EYFP mice, as compared with WT mice. These results show the activation of NAc GABAergic neurous inhibits the expression of motivational effects induced by cocaine-context-associated cues.To explore how the cocaine-context-associated information was processed and integrated, we used the immunofluorescence to assessed the activity of NAc MSN-targeted brain nuclei during the cocaine-context-associated memory and found that the activation of NAc GABAergic neurons during CPP expression resulted in a decrease of c-Fos+cells in the ventral palladium, but not SNr or VTA neurons. Our data suggested that the NAc GABAergic efferents inhibit the ventral palladium activity and negatively regulate the expression of motivational effects induced by cocaine-context-associated cues.In the present study, we firstly demonstrated that optogenetic activation of NAc GABAergic neurons transiently and reversibly inhibits the expression of cocaine-context-associated memory; optogenetic activation of NAc GABAergic neurons attenuates the expression of cocaine-induced behavioral sensitization; optogenetic activation of NAc GABAergic neurons may regulate the expression of the cocaine-context-associated memory by down-regulation the activity of VP neurons. This study show that NAc GABAergic neurons play important roles in the drug-context-associate memory and might be a potential target for the motivational behaviour induced by drug-context-associated cues.