Rapid synaptic potentiation within the anterior cingulate cortex mediates trace fear learning

Molecular Brain Rapid synaptic potentiation within the anterior cingulate cortex mediates trace fear learning Giannina Descalzi 0 2 Xiang-Yao Li 0 1 2 Tao Chen 0 2 Valentina Mercaldo 0 2 Kohei Koga 0 2 Min Zhuo 0 1 2 0 Department of Physiology, Faculty of Medicine, University of Toronto , 1 King's College Circle, Toronto, Ontario M5S 1A8 , Canada 1 Center for Neuron and Disease, Frontier Institute of Science and Technology, Xi'an Jiaotong University , 28 Xianning West Road, Xian, Shaanxi 710049 , China 2 Department of Physiology, Faculty of Medicine, University of Toronto , 1 King's College Circle, Toronto, Ontario M5S 1A8 , Canada Although the cortex has been extensively studied in long-term memory storage, less emphasis has been placed on immediate cortical contributions to fear memory formation. AMPA receptor plasticity is strongly implicated in learning and memory, and studies have identified calcium permeable AMPA receptors (CP-AMPARs) as mediators of synaptic strengthening. Trace fear learning engages the anterior cingulate cortex (ACC), but whether plastic events occur within the ACC in response to trace fear learning, and whether GluN2B subunits are required remains unknown. Here we show that the ACC is necessary for trace fear learning, and shows a rapid 20% upregulation of membrane AMPA receptor GluA1 subunits that is evident immediately after conditioning. Inhibition of NMDA receptor GluN2B subunits during training prevented the upregulation, and disrupted trace fear memory retrieval 48 h later. Furthermore, intra-ACC injections of the CP-AMPAR channel antagonist, 1-naphthylacetyl spermine (NASPM) immediately following trace fear conditioning blocked 24 h fear memory retrieval. Accordingly, whole cell patch clamp recordings from c-fos positive and c-fos negative neurons within the ACC in response to trace fear learning revealed an increased sensitivity to NASPM in recently activated neurons that was reversed by reconsolidation update extinction. Our results suggest that trace fear learning is mediated through rapid GluN2B dependent trafficking of CP-AMPARs, and present in vivo evidence that CP-AMPAR activity within the ACC immediately after conditioning is necessary for subsequent memory consolidation processes. fear learning; memory consolidation; ACC; GluA1; NMDA; Ca2+ permeable AMPARs - Background Long term potentiation (LTP) of central synapses is believed to be the basic mechanism that drives memory storage within the brain [1,2]. Although a critical role for the cerebral cortex in remote fear memory recall has been established [3], little is known regarding immediate cortical contributions to fear memory formation. Much effort instead has focused on the amygdala, where animal studies revealed that associative fear conditioning, which pairs an arbitrary conditioning stimulus (CS) with a noxious one (US), induces changes in excitatory glutamatergic transmission [4-6], and requires postsynaptic GluA2 expression for memory maintenance [7]. Evidence suggests however that in addition to the amygdala, cortical structures also mediate fear learning. In humans, trace fear conditioning, which introduces a time interval between the CS and the US, activates several brain areas including the amygdala, hippocampus, medial prefrontal cortex (mPFC), and the anterior cingulate cortex (ACC) [8,9]. The ACC is involved in the processing of pain, emotion, and threat related stimuli [10,11], and we recently found a trace fear memory enhancement in mice overexpressing Ca2+ calmodulindependent protein kinase IV (CaMKIV), that corresponded with enhancements of ACC LTP in layer II/III pyramidal neurons [12]. In rats, trace fear conditioning induces ACC c-fos expression, and visual distraction during the time interval separating the CS and US prevents fear memory and c-fos expression [13]. Glutamatergic synapses in the ACC are plastic [14-16], and the N-methyl-D-aspartate (NMDA) receptors are critical for LTP induction within the ACC [17]. The GluN2B subunit in particular has been found to be a critical mediator of pain induced alterations within the ACC [18], and forebrain overexpression of GluN2B in mice enhances contextual and auditory fear memory [19]. We previously showed that LTP induction within the ACC corresponds with postsynaptic upregulation of AMPA receptor GluA1 subunits [15,20]. Interestingly, AMPA receptor plasticity is strongly implicated in learning and memory [5,21], and several studies suggest that calcium permeable AMPA receptors (CP-AMPARs) mediate synaptic strengthening [22-24]. In particular, in the CA1 region of the hippocampus, transient increases of CP-AMPARs were observed in response to LTP induction through theta burst stimulation [23] and pairing protocols [24]. However whether such rapid plastic events occur within the cortex in response to trace fear learning, and whether GluN2B subunits are required remains unknown. In the present study, we used integrative methods, including behavioral, pharmacological, biochemical, and electrophysiological, to determine if plasticity related events occur within the ACC during trace fear learning. Results Trace fear learning upregulates membrane AMPA receptor GluA1 subunits within the ACC In order to investigate trace fear learning induced alterations within the ACC, we analyzed the ACC of mice following exposure to a trace fear conditioning paradigm that pairs an auditory conditioning tone (CS) with a foot shock (US), with a 30 sec interval (trace) separating the CS from the US (Figure 1A). This paradigm reliably induces freezing behaviour in mice exposed to the CS in a novel context 48 h later (Figure 1B). To determine if membrane bound AMPA receptor expression is altered in the ACC in response to trace fear conditioning, we extracted the ACC of mice immediately after conditioning, and performed Western blot analysis (Figure 1C). We compared the expression levels of membrane AMPA receptor GluA1 subunits in the ACC of adult (812 wks) C57 mice exposed to one of four conditions: trace fear conditioning (10 CS-trace-US), shock only (10 US), delay fear conditioning (10 CS-US), or exposure to the conditioning chamber. Remarkably, trace fear conditioning induced a rapid, significant upregulation of membrane bound GluA1subunits in the ACC (chamber: 1 0.02; trace fear: 1.19 0.05 times the chamber alone value; shock: 1.00 0.07 times the chamber alone value; delay: 1.05 0.07 times the chamber alone value; one-way ANOVA, P = 0.002; Figure 1D). Importantly, total GluA1 levels within the ACC remained unchanged in mice exposed to trace fear conditioning (Figure 1E), and we observed similar levels of membrane bound GluA2/3 (Figure 1F), suggesting a membrane upregulation that was limited to the GluA1 subunit of AMPA receptors. These results suggest that trace fear learning may be mediated through an upregulation of synaptic ACC AMPA receptors containing the GluA1 subunit. Activation of NMDA receptor GluN2B su (...truncated)