This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Systems/Circuits A critical role for the nucleus reuniens in long-term, but not short-term associative recognition memory formation G.r.i. Barker and E.c. Warburton School of Physiology, Pharmacology and Neuroscience, University of Bristol, BS8 1TD, UK. DOI: 10.1523/JNEUROSCI.1802-17.2017 Received: 28 June 2017 Revised: 30 October 2017 Accepted: 29 November 2017 Published: 15 February 2018 Author contributions: G.R.B. and E.C.W. designed research; G.R.B. and E.C.W. performed research; G.R.B. analyzed data; G.R.B. and E.C.W. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This research was supported by a project grant from the Biotechnology and Biological Sciences Research Council ( BBSRC) [ BB/L02134X/1]. We thank Jane Robbins for assistance with histology. Correspondence should be addressed to Author for correspondence: Dr EC Warburton, School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, Tel +44 (0) 117 3311936, email [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1802-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 A critical role for the nucleus reuniens in long-term, but not short-term associative recognition 2 memory formation 3 Abbreviated title: The nucleus reuniens and recognition memory 4 5 Barker, G.R.I. and Warburton, E.C.* 6 School of Physiology, Pharmacology and Neuroscience 7 University of Bristol 8 BS8 1TD, UK. 9 *Author for correspondence: Dr EC Warburton 10 School of Physiology, Pharmacology and Neuroscience, 11 University of Bristol 12 Biomedical Sciences Building 13 University Walk 14 Bristol BS8 1TD 15 Tel +44 (0) 117 3311936 16 email [email protected] 17 Section and Senior Editor 18 No. of figures: 7 19 No. of tables: 4 20 No. of pages: 49 21 No. of words in abstract: 246 22 No. of words in Introduction: 512 23 No of words in Discussion: 1505 24 1 25 Keywords: nucleus reuniens; associative recognition memory, familiarity discrimination, rat, medial 26 prefrontal cortex 27 28 Acknowledgements: This research was supported by a project grant from the Biotechnology and 29 Biological Sciences Research Council ( BBSRC) [ BB/L02134X/1]. We thank Jane Robbins for 30 assistance with histology. 31 32 Conflict of Interest: The authors declare no competing financial interests 33 34 35 2 36 ABSTRACT 37 Recognition memory for single items requires the perirhinal cortex (PRH), while recognition 38 of an item and its associated location, requires a functional interaction between the PRH, 39 hippocampus (HPC) and medial prefrontal cortex (mPFC). While the precise mechanisms 40 through which these interactions are effected are unknown, the nucleus reuniens (NRe) has 41 bidirectional connections with each regions and hence may play a role in recognition 42 memory. Here we investigated, in male rats, whether specific manipulations of NRe function 43 affected performance of recognition memory for single items, object location or object-in- 44 place associations. Permanent lesions in the NRe significantly impaired long-term but not 45 short-term object-in-place associative recognition memory, while single item recognition 46 memory and object location memory were unaffected. Temporary inactivation of the NRe 47 during distinct phases of the object-in-place task, revealed its importance in both the 48 encoding and retrieval stages of long-term associative recognition memory. Infusions of 49 specific receptor antagonists showed that encoding was dependent on muscarinic and 50 nicotinic cholinergic neurotransmission, in contrast, NMDA receptor neurotransmission was 51 not required. Finally, we found that long-term object-in-place memory required protein 52 synthesis within the NRe. These data reveal a specific role for the NRe in long-term 53 associative recognition memory through its interactions with the HPC and mPFC, but not the 54 PRH. The delay-dependent involvement of the NRe, suggests that it is not a simple relay 55 station between brain regions, but rather during high mnemonic demand, facilitates 56 interactions between the mPFC and HPC a process which requires both cholinergic 57 neurotransmission and protein synthesis. 58 3 59 60 Significance statement 61 Recognising an object and its associated location, fundamental to our everyday memory 62 requires specific hippocampal-cortical interactions, potentially facilitated by the nucleus 63 reuniens (NRe) of the thalamus, yet the role of the NRe itself in associative recognition 64 memory is unknown. Here we reveal the crucial role of NRe in encoding and retrieval of 65 long-term object-in-place memory, but not for remembrance of an individual object or 66 individual location and such involvement is cholinergic receptor and protein synthesis 67 dependent. This is the first demonstration that the NRe is a key node within an associative 68 recognition memory network and is not just a simple relay for information within the 69 network. Rather, we argue, the NRe actively modulates information processing during long- 70 term associative memory formation. 71 72 INTRODUCTION 73 Successful recognition memory depends upon networks of distributed brain regions, with 74 the nature of to-be-remembered information determining which brain regions are 75 recruited. Thus recognition of single items or objects depends on the perirhinal cortex 76 (PRH) (Diana et al., 2007; Kim, 2011; 2013; Wan et al., 1999; Albasser et al., 2010; Ennaceur 77 et al., 1996; Bussey et al., 1999), while recognition of an object and its associated spatial 78 information (object-in-place memory) requires the PRH (Bachevalier and Nemanic, 2008; 79 Bussey et al., 2001; Barker et al., 2007), hippocampus (HPC) and prefrontal cortex (PFC) 80 (Browning et al., 2005; Kesner and Ragozzino, 2003; Brincat and Miller, 2015; Barker et al., 4 81 2007; Barker and Warburton, 2008; Barker et al., 2017). Importantly during object-in-place 82 memory these three brain regions appear to operate cooperatively forming an associative 83 recognition memory network (Barker et al., 2007, Barker and Warburton, 2011). However, 84 as these regions are connected by both direct and indirect anatomical pathways the precise 85 routes by which these neural interactions are effected are largely unknown. One brain 86 region, the nucleus reuniens of the thalamus (NRe) has reciprocal connections with the PRH 87 (Agster et al., 2016; Pereira et al., 2016) mPFC (Vertes, 2002; Vertes et al. 2006; Hoover and 88 Vertes 2006) and HPC (Herkenham, 1978; Wouterlood et al. 1990; Dolleman-van der Weel 89 and Witter, 1996; Vertes et al. 2006). In addition, a proportion of NRe neurons project to 90 both mPFC and HPC, thus NRe may simultaneously influence both HPC and mPFC processing 91 (Hoover and Vertes, 2012) as well as provide a route through which these three brain 92 regions may interact. 93 There is evidence that the NRe plays a specific role in memory processing under certain 94 conditions. Permanent or temporary inactivation of the ventral midline thalamic nuclei (i.e. 95 NRe and neighbouring rhomboid nucleus (Rh)) impaired a spatial win-shift task (Hembrook 96 and Mair, 2011), a spatial strategy shifting task (Cholivin et al., 2013) and a delayed non- 97 matching to position (Hembrook et al., 2012) task, although such lesions had no effect on 98 simple spatial learning (but see Dolleman-van der Weel et al., 2009). NRe disruption was 99 also found to impair long-term, but not short-term spatial memory (Loureiro et al., 2012). 100 Thus, the NRe plays a key role in spatial memory processing either when the demands on 101 the HPC are high, or interactions between the HPC and mPFC are required. Whether the 102 NRe participates in PRH dependent tasks has not, to our knowledge, been investigated. 5 103 In the present study, we hypothesised that the NRe will have a key role in recognition 104 memory formation, either through interactions with the PRH, mPFC, HPC. To test this 105 hypothesis we examined the effects of specific manipulations of the NRe on a series of 106 object recognition memory tasks. We examined: 1. the necessity of the NRe for single item 107 and associative recognition memory using permanent lesions placed in the midline 108 thalamus; 2. the necessity of the NRe separately during encoding and retrieval; 3. the role 109 of NMDA receptor and cholinergic neurotransmission in the NRe for object-in-place 110 encoding; 4. whether long-term memory maintenance was dependent on protein synthesis 111 in the NRe. 112 113 METHODS 114 Subjects 115 Experiment 1 used 20 male rats and Experiments 2-5 used a single cohort of 12 male rats, 116 (Lister Hooded strain; Harlan, UK) weighing 300-350g at the start of the experiments. All 117 animals were housed in groups of four, under a 12 h light/dark cycle (light phase, 6.00 P.M 118 to 6.00 A.M) with ad libitum access to food and water. Behavioural testing was conducted 119 during the dark phase of this cycle. All animal procedures were performed in accordance 120 with United Kingdom Animals Scientific Procedures Act (1986) and associated guidelines. All 121 efforts were made to minimize any suffering and the number of animals used. 122 Surgical Procedure 123 Bilateral excitotoxic lesion of the ventral midline nuclei (NRE/Rh) (Experiment 1) 6 124 Before surgery all rats were anesthetized (isoflurane: induction 4%; maintenance 2-4%) and 125 placed in a stereotaxic frame with the incisor bar set so as to achieve flat skull.