Reversible inactivation of amygdala and cerebellum but not perirhinal cortex impairs reactivated fear memories Benedetto Sacchetti, 1,2 Tiziana Sacco 1 and Piergiorgio Strata 1,2,3 1 Rita Levi-Montalcini Center for Brain Repair, Department of Neuroscience, University of Turin, Corso Raffaello 30, I-10125 Turin, Italy 2 Istituto Nazionale di Neuroscienze, Turin, Italy 3 Rehabilitation Hospital and Research Institute Santa Lucia Foundation, Rome, Italy Keywords: fear conditioning, learning and memory, rat, reversible inactivation Abstract The cerebellum, amygdala and perirhinal cortex are involved in fear learning but the different roles that these three structures play in aversive learning are not well defined. Here we show that in adult rats amygdala or cerebellar vermis blockade causes amnesia when performed immediately, but not 1 h, after the recall of fear memories. Thus, the cerebellum, as well as the amygdala, influences long- term fear memories. These effects are long lasting, as they do not recover over time, even after a reminder shock administration. However, all of the subjects were able to form new fear memories in the absence of inactivation. By increasing the strength of conditioning, we observed that stronger fear memories are affected by the combined but not independent amygdala and cerebellar blockade. These results demonstrate that the cerebellum supports the memory processes even in the absence of a crucial site for emotions like the amygdala. Furthermore, they suggest that the amygdala is only one of the neural sites underlying long-term fear memories. Finally, the inactivation of the perirhinal cortex never alters retrieved fear traces, showing important differences between the amygdala, cerebellum and perirhinal cortex in emotional memories. Introduction Despite years of progress, there is still no universal consensus regarding the location of fear memory storage. Aversive traces have been proposed to be stored in the amygdala (Fanselow & LeDoux, 1999; Schafe et al., 2005). However, the amygdala may not be unique but rather one of many regions of the wider fear network (McGaugh, 2000, 2004; Kim & Jung, 2006). In addition to the amygdala, the perirhinal cortex (Corodimas & LeDoux, 1995; Suzuki, 1996; Sacchetti et al., 1999; Maren, 2001) and the cerebellar vermis (Sacchetti et al., 2002a, 2005) may participate in this process. One difficulty in searching for the location of memory storage is due to the fact that the blockade of a selected site during memory retention can also affect both spontaneous behavior as well as some motor responses. As the amygdala and perirhinal cortex are also critically involved in innate fear behavior (Blanchard & Blanchard, 1972; Schulz-Klaus et al., 2005), their inactivation will affect innate fear performance as well as memory. Cerebellar inactivation can affect motor responses related to fear behavior, e.g. the freezing response (Sacchetti et al., 2005). As consolidated fear memories can be altered when amnesic agents are applied immediately after memory retrieval (Misanin et al., 1968; Schneider & Sherman, 1968; Nader et al., 2000), an alternative way of studying the critical structures involved in the maintenance of fear memories is to inactivate a selected neural site after memory recall. Despite some negative results (Dawson & McGaugh, 1969; Squire et al., 1976; Biedenkapp & Rudy, 2004; McGaugh, 2004), recent evidence confirms the fragility of retrieved memory (Przybyslawski & Sara, 1997; Przybyslawski et al., 1999; Anokhin et al., 2002; Bozon et al., 2003; Salinska et al., 2004; Alberini, 2005). These effects have been interpreted as being caused by interference with storage (Nader et al., 2000; Duvarci & Nader, 2004) or retrieval (Judge & Quartermain, 1982; Millin et al., 2001; Vianna et al., 2001; Anokhin et al., 2002; Lattal & Abel, 2004; Prado-Alcala et al., 2006) of the memory trace. Thus, post-retrieval manipulations can help to locate neural sites involved in emotional memory maintenance, independent of their involvement in motor responses or innate fear behavior. Therefore, in the present study we reversibly inactivated the amygdala, cerebellum or perirhinal cortex during post-retrieval fear processes in order to identify the neural network that underlies long- term fear memories. Materials and methods Subjects Male Wistar rats (age 65–75 days, weight 250–320 g, Harlan Italy, Verona, Italy) were used. The animals were housed in plastic cages, four to a cage, with food and water available ad libitum, under a 12 h light ⁄ dark cycle at a constant temperature of 22 ± 1°C. All animal care and experimental manipulations were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC) and approved by the Bioethical Committee of the University of Turin. Behavioral procedures In fear conditioning training, the rats were gently placed in a basic Skinner box module (rat test cage, Coulbourn Instruments, Correspondence: Dr Benedetto Sacchetti, 1 Rita Levi-Montalcini Center for Brain Repair, as above. E-mail: benedetto.sacchetti@unito.it Received 3 January 2007, revised 28 February 2007, accepted 5 March 2007 European Journal of Neuroscience, Vol. 25, pp. 2875–2884, 2007 doi:10.1111/j.1460-9568.2007.05508.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd