Abstract Heroin addiction is characterized by compulsive craving, drug seeking and re-occurrence of relapse, and is considered to be one of the most problematic public health concerns. Heroin relapse is significantly affected by stress. Human studies suggest that exposure to life stressors is correlated with compulsive drug abuse and relapse to drugs during periods of abstinence. More importantly, environmental stress during early life is related to bigger risk for developing addiction and increased relapse vulnerability. Early social isolation (during adolescence, ESI), as one of the widely used models for early life stress, causes many behavioral abnormalities that related to mental health issues including increased vulnerability for relapse. My preliminary data confirmed that ESI potentiates cue-induced heroin seeking after forced abstinence from heroin self- administration (SA). However, the underlying neurobiological mechanisms are largely understudied. Prefrontal cortex (PFC) is involved in the regulation of drug relapse. PFC hypofunction has been identified in opioid abusers. Preclinical studies indicate that PFC, which projects to subcortical regions such as NAc and VTA, is critical for heroin relapse. As ESI induces irreversible synaptic dysfunction in the PFC, it is likely that ESI potentiates heroin seeking by exacerbating PFC malfunction. Moreover, due to the heterogeneous of PFC projecting neurons (PFC-VTA and PFC-NAc projection have distinct distribution and molecular signatures), it remains unclear what are the neuronal circuit-specific molecular mechanisms for heroin relapse vulnerability. Therefore, my central hypothesis is that hypofunction of PFC (PFC-VTA/PFC-NAc) projecting neurons is involved in ESI-potentiates heroin seeking, and this effect is accompanied by gene transcriptional changes within the PFC-VTA and/or PFC-NAc projecting neurons. To test my hypothesis, I will incorporate state-of-the-art electrophysiology, chemogenetic strategies (DREADDs), and projection-specific molecular profiling (TRAP [translating ribosome affinity purification]) technologies into my study. I propose to measure excitatory synaptic transmission in PFC-NAc and PFC-VTA projecting neurons after forced abstinence from heroin SA. Chemogenetic tools will be used to test the functional role of PFC-VTA and PFC-NAc projecting neurons in ESI-intensified heroin seeking. TRAP methods (using GFPL10 transgenic mice) will be applied to isolate projection-specific neurons for RNA-seq to identify potential molecular mechanisms for ESI-potentiated heroin seeking. Fulfillment of my Research Plan and Training Plan will allow me to independently pursue my long-term career goals: study neuronal circuit-specific molecular mechanisms for heroin seeking to ultimately contribute to the development of pharmacotherapies for heroin relapse.