Inhibition of Cav2.2-mediated signaling disrupts conditioned fear extinction

Neuronal voltage gated calcium channels (VGCCs) are well known for its importance in synaptic transmission and signaling pathway in the specific circuits. On the other hand, the role of different VGCCs in the cognitive functions has not been studied. Although it has been reported that fear extinction requires Cav2.1-regulated N-methyl-D-aspartate (NMDA) receptor signaling, Cav2.2-regulated synaptic function in extinction remains unknown. This study examined whether Cav2.2mediated signaling plays role in consolidation of extinction. Mice received intracerebroventricular injection of Cav2.2 blocker (ω-conotoxin GVIA, 5 pg/side) showed impaired extinction behavior and decreased expression of CREB-dependent gene Arc in medial prefrontal cortex (mPFC). Intra-mPFC injections of ω-conotoxin GVIA (5 pg/midline) blocked extinction. These results indicate that Cav2.2-mediated signaling is critical in the mPFC-dependent fear extinction. Introduction Extinction procedures do not erase the original fear memory, but yield a new safety memory that inhibits fear under certain conditions [1]. Animals learn to associate a previously neutral or conditioned stimulus (CS) with an aversive or unconditioned stimulus (US) during fear conditioning. Subsequent re-exposure to the CS alone triggers two competing processes. Brief re-exposure to the CS initiates a reconsolidation process that serves to stabilize or maintain the original CS-US memory [2]. In contrast, more prolonged re-exposure to the CS leads to the formation of an inhibitory extinction (CS-no US) memory [3]. It is interesting to understand the neural circuit mechanism underlying fear extinction. Ca2+ signaling through voltage gated calcium channels (VGCCs) mediates Ca2+ entry into cells in response to membrane depolarization and thus transduces electrical signals into chemical signals [4]. Neuronal VGCCs including Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) channels mediate a number of neuronal functions including neuronal excitation, neurite outgrowth, synaptogenesis, neurotransmitter release, neuronal survival, differentiation, plasticity, and regulation of gene expression [4-6]. On the other hand, the role of different VGCCs in the neural circuits underlying fear extinction has not been studied. Cav2.1 channels mediate the presynaptic machinery for glutamate release [7]. Cav2.1 and NMDA receptors and express broadly in the central nervous system [8-11]. A previous study reported that fear extinction requires a distributed neural circuit in the brain, especially the medial prefrontal cortex (mPFC) [12,13]. Blocking protein synthesis in the mPFC prevents the formation of extinction memory, and activation of cAMP-responsive element-binding protein (CREB)mediated transcription is induced in the mPFC in the consolidation phase of extinction [14]. Our previous study has showed that Cav2.1regulated NMDA receptor signaling in the mPFC regulates the CREB cascade involved in extinction memory [15]. The administration of a Cav2.1 blocker induced dysfunctional fear extinction [15]. However, the physiological role of Cav2.2-regulated extinction has not yet been determined. Cav2.2 also express broadly in the central nervous system [8]. Thus, administration of a Cav2.2 blocker should result in dysfunctional fear extinction, because precise regulation of neurotransmitter release through Cav2.2 also plays an important role in neuronal circuits [4-6]. There has been a growing interest in the neural mechanisms of fear extinction [16,17], as extinction may be a potential target for the treatment of neuropsychiatric diseases. In the present study, to examine the relationship between Cav2.2-mediated signaling in the mPFC and extinction memory formation, we investigated the extinction of conditioned fear using the wild-type mice received intracerebroventricular or intra-mPFC injection of Cav2.2 blocker (ω-conotoxin GVIA). We used immunohistochemical analysis to characterize the relationship between Cav2.2-mediated signaling and the expression of the CREB-dependent gene activity-regulated cytoskeleton-associated protein (Arc) after prolonged re-exposure. The studies presented here demonstrate the importance of mPFCdependent Cav2.2-mediated signaling in the consolidation of fear extinction. Correspondence to: Eiki Takahashi, DVM, PhD, Research Resources Center, RIKEN Brain Science Institute 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan, Tel:+81-48-467-5871; Fax:+81-48-467-9692; E-mail: [email protected]