Center for Information and Neural Networks, Suita City,Osaka 565-0871, JapanSummaryThe frontal association cortex (FrA) is implicated in higherbrain function [1]. Aberrant FrA activity is likely to beinvolved in dementia pathology [2–4]. However, the func-tional circuits both within the FrA and with other regionsare unclear. A recent study showed that inactivation of theFrAimpairsmemoryconsolidationofanauditoryfearcondi-tioninginyoungmice[5].Inaddition,dendriticspineremod-eling of FrA neurons is sensitive to paired sensory stimulithat produce associative memory [5]. These findings sug-gest that the FrA is engaged in neural processes criticalto associative learning. Here we characterize stimulus inte-gration in the mouse FrA during associative learning. Weexperimentally separated contextual fear conditioning intocontext exposure and shock, and found that memory forma-tion requiresprotein synthesisassociatedwith bothcontextexposure and shock in the FrA. Both context exposure andshock trigger Arc, an activity-dependent immediate-earlygene, expression in the FrA, and a subset of FrA neuronswas dually activated by both stimuli. In addition, we foundthat the FrA receives projections from the perirhinal (PRh)and insular (IC) cortices and basolateral amygdala (BLA),which are implicated in context and shock encoding [6–8].PRh and IC neurons projecting to the FrA were activatedby context exposure and shock, respectively. Arc expres-sion in the FrA associated with context exposure and shockdepended on PRh activity and both IC and BLA activities,respectively.ThesefindingsindicatethattheFrAisengagedinstimulusintegrationandcontributestomemoryformationin associative learning.ResultsThe Frontal Association Cortex Is Required for MemoryFormation in Contextual Fear ConditioningAsamodelforassociativelearning,weusedacontextualfear-conditioning task, which establishes an association betweencontext and shock. To test whether the frontal associationcortex (FrA) is involved in memory formation in contextualfear conditioning, we infused (2R)-amino-5-phosphonovalericacid(APV),anN-methyl-D-aspartate(NMDA)receptorantago-nist, anisomycin, a protein synthesis inhibitor, or vehicle intothe FrA. APV and anisomycin were infused 30 min prior to, orimmediately after, contextual fear conditioning, respectively(experiment 1; Figures 1A and 1B). Contextual fear memorywas assessed by measuring the percentage of freezing timein the conditioning context 1 day after conditioning. BothAPV and anisomycin infusions disrupted freezing behavior.When anisomycin infusions were administered into the dorso-medial prefrontal cortex, which is close to the FrA (experiment2; Figure S1A available online), freezing behavior was compa-rable to that of mice administered vehicle infusions (Fig-ureS1B).TheseresultsindicatethatNMDAreceptoractivationandproteinsynthesisintheFrAarerequiredforcontextualfearconditioning.ProteinSynthesisintheFrAIsRequiredforEncodingBothContext and ShockWe aimed to determine whether the FrA encodes context,shock, or both. To this end, we separated 10 min of contextexposureandimmediateshockbya1-dayintervalandinfusedanisomycinintotheFrAaftereithercontextexposureorshock(experiment 3; Figures 1C and 1D). Anisomycin infusions intothe FrA immediately after both context exposure and immedi-ate shock disrupted freezing behavior during the test. How-ever, when anisomycin infusions were administered into theFrA 6 hr after context exposure (Figure 1E), freezing behaviorwas comparable to that of mice administered vehicle infu-sions. These results indicate that protein synthesis in the FrAis required for encoding both context and shock.FrA Neurons Receive Convergent Information RegardingContext and Shock during Fear ConditioningBecause protein synthesis in the FrA is required for encodingboth context and shock, we hypothesized that paired stimuliconverge in a subset of FrA neurons to potentially contributeto the memory trace. To visualize stimulus convergence, weanalyzed the temporal dynamics of nuclear versus cyto-plasmic Arc localization by fluorescent in situ hybridization[9]. Arc is an activity-dependent immediate-early gene thatis essential for synaptic plasticity and long-term memory[10–13]. Transcribed Arc mRNA first appears in neuronalnuclei, and processed Arc mRNA then accumulates in thecytoplasm. Thus, an analysis of the subcellular localization ofArc enabled us to identify active neuronal ensembles duringtwo behavioral tasks [9, 14, 15]. We first examined the timecourse of the nuclear and cytoplasmic Arc signal after neuralactivity in the FrA. Mice were exposed to a context for 5 minand sacrificed either immediately or 30 min later (experiment4; Figures S2A and S2B). We observed more nuclear Arc more...