The ability to efficiently learn associations that lead to positive outcomes and flexibly change those associations in response to new environmental conditions is essential for an organism’s survival. Associative learning and flexible action are sub-served by neural circuitry that is highly conserved across species with topographically-organized loops originating in cortical subregions that project through structures in the basal ganglia before returning to the cortex (Schilman et al., 2008, Voorn et al., 2004). It is well established that circuits connecting the lateral orbitofrontal cortex (lOFC) and dorsal striatum (dS) mediate the acquisition of well-learned associations and the ability to flexibly change these associations when required (Schilman et al., 2008, Voorn et al., 2004). Studies in primates and rodents using an array of associative stimuli and responses have demonstrated that the OFC is not essential for initial choice learning, but is functionally necessary for optimal behavioral flexibility (Chudasama and Robbins, 2003, Dias et al., 1996, Moore et al., 2009, Rudebeck and Murray, 2008). In contrast, the dorsal striatum (dS) mediates choice behavior by balancing bottom up stimulus preference and action-outcome learning (Featherstone and McDonald, 2004, Palencia and Ragozzino, 2005, Yin et al., 2004). Reversal learning tasks recruit both of these systems, by requiring a subject to learn a choice or response pattern that leads to reward and then shift to the previously unrewarded choice when reward contingencies are reversed. We have previously demonstrated that mouse touchscreen reversal recruits these corticostriatal circuits, as dS is selectively recruited during discrimination learning and OFC is activated when flexible behavior is highly taxed during early reversal (Brigman et al., 2013a, Graybeal et al., 2011, Marquardt et al., 2017). However, the specific mechanism by which OFC-dS cortico-striatal circuits balance efficient learning and flexible behavior is not fully understood.

It is well established that both learning and reversal require the induction of synaptic plasticity via the activation of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and N-methyl-D-aspartate receptors (NMDAR). AMPAR play a critical role in post-synaptic depolarization, and are required to release the voltage-sensitive magnesium block and allow glutamate binding to active NMDAR. NMDAR are heteromeric complexes composed of an obligatory GluN1 subunit together with one or more GluN2 subunits (GluN2A-2D) which confer distinct physiological and molecular properties to the receptor. The predominant subunits in the adult forebrain, GluN2A and GluN2B, have been widely studied in order to understand the role of NMDAR in the synaptic plasticity required for learning and flexible choice. The ratio of GluN2A to GluN2B-containing NMDAR is hypothesized to play a crucial role in the plasticity required to form, and flexibly alter new associations. Multiple learning paradigms increase the GluN2A/GluN2B ratio and increase the threshold for inducing plasticity (Holehonnur et al., 2016, Philpot et al., 2001, Quinlan et al., 2004). GluN2A and GluN2B expression has been directly implicated in efficient reversal learning. Loss of GluN2A has been shown to globally impair associative learning (Brigman et al., 2008), while loss of GluN2B function spares associative learning but impairs behavioral flexibility (Brigman et al., 2013b). In addition, loss of GluN2B also significantly alters spike-firing activity and reduces and delays coherence of OFC pyramidal neurons during early reversal (Marquardt et al., 2019).

The role of AMPAR subunits in various forms of experience-dependent plasticity is less well-studied outside the area of substance use disorders. AMPAR are heterotetramers, composed of 4 subunits GluA1-4 (Lu et al., 2009; Malenka, 2003), with GluA1 and GluA2 predominantly expressed in the hippocampus and the cortex (Lu et al., 2009). The trafficking of the AMPAR in and out of the synapse plays a central role in LTP and LTD respectively (Malenka, 2003). AMPAR subunits, specifically the subunit specific trafficking is critical for spatial reversal learning. Impaired GluA1 endocytosis in MAPK-activated protein kinases 2 and 3 (MK2/3) knock out mice showed deficits in hippocampal dependent spatial reversal task(Eales et al., 2014). As with NDMAR, different subunits confer different electrophysiological property as GluA2 containing AMPAR are calcium impermeable whereas GluA2 lacking are calcium permeable (Keifer and Zheng, 2010, Malenka, 2003).

While there is strong evidence from genetic and pharmacological studies that NMDAR and AMPAR subunits are involved in specific aspects of learning and reversal, how subunit synaptic expression may be dynamically expressed or altered during these behaviors is not well understood. In order to address this issue, we tested cohorts of mice to previously validated learning stages of discrimination and reversal known to recruit activation of corticostriatal circuits specifically. We then examined the expression of AMPA and NMDA receptor subunits in the synaptic fraction in brain regions known to be involved in regulation of learning and reversal: the lateral OFC, dS (both medial and lateral aspects) as well as brain regions known to be involved in behavioral flexibility including the medial prefrontal cortex (infralimbic/prelimbic; mPFC) basolateral amygdala (BLA) and a control region (piriform cortex; Pir). By examining how NMDA and AMPA receptor subunits are expressed in the synapse we sought to understand how synaptic levels of both NMDAR and AMPA subunits are changed across states of learning and reversal behavior during the touchscreen task. Our results suggest that visual discrimination reversal learning is associated with significant alterations in synaptic expression of NMDAR and AMPAR subunits in corticostriatal subregions.