Neuropharmacology 62 (2012) 457e463 Contents lists available at SciVerse ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm A time-dependent role of midline thalamic nuclei in the retrieval of fear memory Nancy Padilla-Coreano1, Fabricio H. Do-Monte1, Gregory J. Quirk* Departments of Psychiatry and Anatomy & Neurobiology, University of Puerto Rico School of Medicine, P.O. Box 365067, San Juan 00936, Puerto Rico article info abstract Article history: Increasing evidence indicates that the medial prefrontal cortex (mPFC) and the amygdala mediate Received 6 June 2011 expression and extinction of conditioned fear, but few studies have examined the inputs to these Received in revised form structures. The dorsal part of the midline thalamus (dMT) contains structures such as the mediodorsal 21 August 2011 nucleus, paraventricular nucleus, and paratenial nucleus that project prominently to mPFC, as well as to Accepted 22 August 2011 basal (BA) and central (Ce) nuclei of the amygdala. Using temporary inactivation with GABA agonist muscimol, we found that dMT was necessary for retrieving auditory fear memory that was 24 h old, but Keywords: not 2e8 h old. However, pre-training infusions did not impair fear acquisition or extinction. To determine Thalamus Central amygdala the possible targets of dMT that might modulate fear retrieval, we combined dMT inactivation with Fos Fos immunohistochemistry. Rats with inactivation-induced impairment in fear retrieval showed increased Prefrontal cortex Fos in the lateral division of Ce (CeL), and decreased Fos in the medial division of Ce. No differences in Fos Extinction expression were observed in the mPFC or BA. We suggest that the projections from the paraventricular nucleus to CeL are involved in retrieval of well consolidated fear memories. This article is part of a Special Issue entitled ‘Anxiety and Depression’. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction modulate fear retrieval and/or extinction, either via the mPFC or the amygdala. In recent years, there has been renewed interest in the neural Despite anatomical as well as physiological evidence for an circuits mediating the regulation of conditioned fear memories. The influence of dMT on mPFC and amygdala neurons (Ferron et al., medial prefrontal cortex (mPFC) has emerged as a key structure in 1984; Gigg et al., 1994; Pirot et al., 1994; Vives and Mogenson, emotion regulation (Etkin et al., 2011; Quirk and Beer, 2006; Sotres- 1985), relatively few studies have examined dMT’s role in condi- Bayon et al., 2006), and can modulate fear expression bidirection- tioned fear. Regarding fear acquisition, MD lesions reportedly had ally via projections to the amygdala (McDonald, 1991; Quirk and no effect on acquisition or expression of tone conditioning (Garcia Beer, 2006; Sotres-Bayon and Quirk, 2010; Vertes, 2004). Pharma- et al., 2006), but impaired acquisition and expression of context cological inactivation and stimulation studies indicate that the conditioning (Li et al., 2004). Regarding extinction, MD lesions did prelimbic (PL) subregion of the mPFC is essential for fear expression not impair extinction learning or retrieval (Garcia et al., 2006), even (Corcoran and Quirk, 2007; Sierra-Mercado et al., 2011; Vidal- though extinction increases mPFC potentials evoked by MD stim- Gonzalez et al., 2006), whereas the infralimbic subregion (IL) is ulation (Herry and Garcia, 2002; Herry et al., 1999). essential for fear extinction (Kim et al., 2009; Laurent and The lesion approach often underestimates the role of a given Westbrook, 2009; Milad and Quirk, 2002; Sierra-Mercado et al., structure, due to recovery of function by other areas (Anglada- 2011). PL and IL both receive strong input from the dorsal midline Figueroa and Quirk, 2005; Poulos et al., 2010). In light of this, we thalamic nuclei (dMT), including the mediodorsal nucleus recently used the GABAA agonist muscimol to evaluate the contri- (MD) and paraventricular nucleus (PV) (Hoover and Vertes, 2007; butions of prefrontal, hippocampal, and amygdalar areas to the Vertes, 2006). dMT nuclei also project to the basal (BA) and expression and extinction of tone-induced fear (Sierra-Mercado central (Ce) nuclei of the amygdala (Turner and Herkenham, 1991; et al., 2011). In the present study, we used the same technique to Vertes and Hoover, 2008). Thus, dMT nuclei are well situated to evaluate the role of the dMT in fear acquisition, retrieval and extinction. We found that while dMT was not necessary for acquisition or extinction, it was necessary for retrieval of fear memories that were 24 h old, but not 2e8 h old. We extended our * Corresponding author. Tel.: þ787 999 3058; fax: þ787 999 3057. E-mail address: [email protected] (G.J. Quirk). approach with Fos immunohistochemistry (neural activity marker) 1 Contributed equally to this study. to evaluate the effect of dMT inactivation on target areas involved in 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.08.037 458 N. Padilla-Coreano et al. / Neuropharmacology 62 (2012) 457e463 fear regulation. Impaired fear retrieval was correlated with changes 2.6. Immunohistochemistry in Fos expression in Ce, but not in PL, IL or BA. Ninety minutes after fear retrieval, muscimol or saline infused rats were deeply anesthetized with sodium pentobarbital (450 mg/kg i.p.) and perfused transcardially 2. Materials and methods with 100 ml saline (0.9%), followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post fixed for 3 h in the same fixative solution 2.1. Subjects and transferred to a solution of 30% sucrose in 0.1 M phosphate buffer at 4 C for 2 nights. Brains were then frozen and a series of 40 mm sections were cut with A total of 108 male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) a cryostat (Leica, CM 1850) in the frontal plane and collected at different levels of the weighing 270e320 g were housed and handled as described previously (Quirk et al., medial prefrontal cortex and amygdala. One complete series of sections was pro- 2000). Rats were restricted to 18 g/day of standard laboratory rat chow, followed by cessed for immunohistochemistry with anti-Fos serum raised in rabbit (Ab-5, training to press a bar for food on a variable interval schedule of reinforcement Oncogene Science) at a dilution of 1:20,000 overnight. The primary antiserum (VI-60). Pressing a bar for food ensures a constant level of activity against which was localized using a variation of the avidinebiotin complex system. Sections were freezing behavior can be reliably measured during long training sessions, and incubated for 120 min at room temperature in a solution of biotinylated goat anti- provides an additional measure of fear (suppression ratio) that is more sensitive rabbit IgG (Vector Laboratories) and then placed in the mixed avidinebiotine than freezing (Quirk et al., 2000). All procedures were approved by the Institutional horseradish peroxidase complex solution (ABC Elite kit, Vector Laboratories) for Animal Care and Use Committee of the University of Puerto Rico, School of Medicine 90 min. Black immunoreactive nuclei labeled for Fos were visualized after 10 min of in compliance with National Institutes of Health guidelines for the care and use of exposure to a chromogen solution containing 0.02% 3,30 diaminobenzidinetetrahy- laboratory animals. drochloride with 0.3% nickel-ammonium sulfate (DAB-Ni) in 0.05 M Tris buffer (pH 7.6) followed by incubation for 5 min in a chromogen solution with glucose oxidase 2.2. Surgery (10%) and D-Glucose (10%). The reaction was stopped using potassium PBS (pH 7,4). Sections were mounted in coated-gelatin slides and then dehydrated and cover After bar-press training, rats were anesthetized with intraperitoneal injections slipped. Counter sections were collected, stained for Nissl bodies, cover slipped and of a mixture of ketamine (80 mg/kg)-xylazine (10 mg/kg) and were stereo- examined in an optical microscope to determine the anatomical boundaries of each taxically implanted with a double 26-gauge guide cannula (Plastics One, Roanoke, structure analyzed. VA, USA) targeting the medial dorsal nucleus of the thalamus (coordinates: ante- À þ À roposterior, 2.5 mm from bregma; mediolateral, / 0.60 mm from midline; 2.7. Immunoreactivity quantification dorsoventral, À4.5 mm from skull surface) (Paxinos and Watson, 1997). Cannulas fi were xed to the skull with dental cement and two stainless steel screws. After Counting of Fos positive cells was carried out at 20Â magnification of an surgery, a triple antibiotic was applied and an analgesic (Ketofen; 2 mg/kg) was Olympus microscope (Model BX51) equipped with a digital camera. Images were injected intramuscularly. Stainless steel obturators (33-gauge) were inserted into generated for PL, IL, basal nucleus of the amygdala (BA), lateral/central nucleus of the the guide cannulas to avoid obstructions until infusions were made. After surgery, central amygdala (CeL/CeC) and medial nucleus of the central amygdala (CeM). To be e animals were allowed to recover 5 7 days before initiating experiments. considered positive for Fos-like immunoreactivity, the nucleus had to be of appro- priate size (area ranging from 100 to 500 mm2) and shape (at least 50% of circularity), 2.3. Histology and be distinct from the background. Fos positive cells were automatically counted and averaged across 2 distinct rostro-caudal sections for each brain structure Upon completion of experiments, rats were transcardially perfused with 0.9% analyzed (Metamorph software version 6.1). The density of Fos positive cells in the saline followed by 10% buffered formalin. Brains were extracted and stored in a 30% CeL/CeC and CeM was calculated by dividing the number of Fos positive cells by the sucrose/10% formalin solution. Coronal sections were cut 40 mm thick, mounted on total area of each region.