Project Summary A major challenge for recovering methamphetamine (Meth) users is persistence of vulnerability to craving and relapse even after long periods of abstinence. We study plasticity underlying this persistent vulnerability using the `incubation of craving' model, in which rats exhibit progressive intensification (incubation) of cue-induced craving after withdrawal from extended-access drug self-administration (SA). Incubation of craving occurs in humans, so the model has translational relevance. We previously showed that high conductance Ca2+-permeable AMPARs (CP-AMPAR) accumulate in synapses on medium spiny neurons (MSN) of the nucleus accumbens core (NAcc) after withdrawal from Meth SA, strengthening these synapses, and that blocking CP-AMPAR or removing them from synapses prevents expression of Meth incubation. Thus, glutamate inputs onto NAcc MSN must be required for Meth incubation, but there have been no previous pathway-specific studies to identify them. We propose to test the role of the two MSN subtypes in NAcc (D1 and D2 MSN) and glutamate inputs to NAcc originating from basolateral amygdala (BLA), prelimbic prefrontal cortex (PL/PFC) and paraventricular nucleus of the thalamus (PVT); these inputs are strongly implicated in regulating drug seeking. We hypothesize that CP- AMPAR plasticity occurs in D1 MSN, but that both MSN subtypes and glutamate inputs from all three upstream regions contribute to expression of Meth incubation. To distinguish D1 and D2 MSN, we will use well validated transgenic rats (male and female) expressing Cre recombinase in neurons that express the D1 or the adenosine 2a (A2a) receptor. In the rat NAc, D2 and A2a receptors are colocalized. Thus, these rat lines enable selective manipulation of D1 or A2a/D2 MSN and, when crossed with reporter lines, enable their visualization. Aim 1 will use whole-cell patch-clamp recordings to assess excitatory synaptic transmission in BLA, PL/PFC, and PVT inputs to D1 and A2a MSN before and after Meth incubation, focusing on, but not limited to, CP-AMPAR plasticity. Aim 2 will use fiber photometry to measure Ca2+ responses time-locked to active hole nose-pokes during cue- induced seeking tests before and after incubation. First, we will measure Ca2+ in D1 and A2a MSN by expressing Cre-dependent GCaMP in NAcc of D1-Cre and A2a-Cre rats. Then, to obtain a presynaptic measure, we will record from BLA, PL/PFC and PVT axon terminals in NAcc by expressing axon-GCaMP in the upstream regions and allowing time for its transport to NAcc terminals. Aim 3 will use chemogenetics to determine if D1 or A2a MSN and BLA-NAcc, PL/PFC-NAcc or PVT-NAcc pathways are necessary for expression of incubation. First, we will express the inhibitory DREADD hM4Di in D1 or A2a MSN and administer the DREADD ligand J60 systemically before an early or late withdrawal (incubated) seeking test. Then, we will express the inhibitory DREADD in BLA, PL/PFC or PVT, allow time for its transport to NAcc termin...