On the requirement of nitric oxide signaling in the amygdala for consolidation of inhibitory avoidance memory Carolina G. Zinn, Lia R. Bevilaqua, Janine I. Rossato, Jorge H. Medina, Iván Izquierdo, Martín Cammarota * Centro de Memória, Instituto de Pesquisas Biomédicas and Instituto do Cérebro, Pontifícia Universidade Católica de Rio Grande do Sul, Av. Ipiranga 6690, RS 90610-000 Porto Alegre, Brazil article info Article history: Received 14 August 2008 Revised 18 September 2008 Accepted 30 September 2008 Available online 31 October 2008 Keywords: Consolidation Nitric oxide Amygdala Inhibitory avoidance abstract Evidence suggests that the NO/sGC/PKG pathway plays a key role in memory processing but the actual participation of this signaling cascade in the amygdala during memory consolidation remains unknown. Here, we show that when infused in the amygdala immediately after inhibitory avoidance training, but not later, the NO synthase inhibitor L-NNA hindered long-term memory retention without affecting loco- motion, exploratory behavior, anxiety state or retrieval of the avoidance response. The amnesic effect of L-NNA was not state-dependent and was mimicked by the soluble guanylyl cyclase inhibitor LY83583 and the PKG inhibitor KT-5823. On the contrary, post-training intra-amygdala infusion of the NOS substrate L- Arg, the NO-releasing compound SNAP or the non-hydrolysable analog of cGMP 8Br-cGMP increased memory retention in a dose-dependent manner. Co-infusion of 8Br-cGMP reversed the amnesic effect of L-NNA and LY83583 but not that of KT-5823. Our data indicate that the NO-induced activation of PKG in the amygdala is a necessary step for consolidation of inhibitory avoidance memory. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Nitric oxide (NO) is a labile and highly diffusible gaseous rad- ical synthesized from L-arginine (L-Arg) by a family of enzymes known as NO synthases. NO modulates different physiological processes, including progression of the immune response (Cole- man, 2001), inflammation (Sharma, Al-Omran, & Parvathy, 2007), sodium homeostasis (Pollock & Pollock, 2008) as well as blood flow and oxygen delivery to tissue (Buerk, 2007). In the nervous system, NO acts as a retrograde messenger (Son et al., 1996) regulating neurotransmitter and neuropeptide release in an activity-dependent manner (Hanbauer, Wink, Osawa, Edel- man, & Gally, 1992). NO signaling is mostly mediated by soluble guanylyl cyclase (sGC) (Arnold, Mittal, Katsuki, & Murad, 1977; Marsault & Frelin, 1992; but see also Davis, Martin, Turko, & Murad, 2001 for other possible biochemical targets of NO). Bind- ing of NO to the heme group of sGC increases the activity of this enzyme several hundredfold to produce the second messenger cGMP (Murad, 2004), which upregulates cyclic nucleotide-gated cation channels (Barnstable, Wei, & Han, 2004; Thompson, 1997) and stimulates cGMP-dependent protein kinase (PKG). In turn, PKG promotes mobilization of synaptic vesicles in the pre- synaptic terminal leading to enhanced transmitter release (Haw- kins, Kandel, & Siegelbaum, 1993; Hawkins, Son, & Arancio, 1998) and induces activation of the neuronal transcription machinery (Lu, Kandel, & Hawkins, 1999) as well as assembly of the spliceosome (Wang et al., 1999), two processes required for memory consolidation (Antunes-Martins, Mizuno, Irvine, Lepicard, & Giese, 2007; Beffert et al., 2005; Colombo, 2004; Gold, 2008; McClung & Nestler, 2008; Pepeu & Giovannini, 2004; Powell, 2006; Ule & Darnell, 2006). In fact, several reports suggest that NO plays an essential role in synaptic plasticity and memory formation (Baratti & Kopf, 1996; Bernabeu, de Stein, Fin, Izquierdo, & Medina, 1995; Böhme et al., 1993; Hölscher & Rose, 1992; Mogensen, Wörtwein, Hasman, Nielsen, & Wang, 1995; Rickard, Ng, & Gibbs, 1994; Stevens & Wang, 1993; Susswein, Katzoff, Miller, & Hurwitz, 2004; Telegdy & Kokavszky, 1997). Extensive evidence indicates that the baso-lateral amygdala (BLA) modulates memory consolidation of emotionally arousing or stressful experiences (McGaugh, 2004). Indeed, it is known that aversive stimuli such as those utilized in fear learning experiments activate stress-related hormonal systems and modify the release of different neurotransmitters in the amygdala (Delaney, Crane, & Sah, 2007; Ma & Morilak, 2005; Morilak et al., 2005). However, although it was recently shown that pre-training inhibition of NOS blocks acquisition of auditory fear conditioning memory (Schafe et al., 2005), little else is known about the role played by NO signaling within the amygdala during the consolidation of aversive memory. Here, we analyzed the effect of the post-training intra-amygdala infusion of different compounds acting on the NO/ cGMP/PKG signaling pathway on inhibitory avoidance memory retention. 1074-7427/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2008.09.016 * Corresponding author. Fax: +55 51 3320 3312. E-mail addresses: mcammaro@terra.com.br, martin.cammarota@pucrs.br (M. Cammarota). Neurobiology of Learning and Memory 91 (2009) 266–272 Contents lists available at ScienceDirect Neurobiology of Learning and Memory journal homepage: www.elsevier.com/locate/ynlme