Research Articles: Systems/Circuits Ventral hippocampal input to the prelimbic cortex dissociates the context from the cue association in trace fear memory https://doi.org/10.1523/JNEUROSCI.1453-19.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1453-19.2020 Received: 19 June 2019 Revised: 2 March 2020 Accepted: 5 March 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Twining et al. 1 Title: Ventral hippocampal input to the prelimbic cortex dissociates the context from the cue 2 association in trace fear memory 3 4 Running Title: VH-PL communication mediates contextual fear learning 5 6 Authors: Robert C. Twining*, Katie Lepak, Adam J. Kirry, Marieke R. Gilmartin* 7 Affiliation: Department of Biomedical Sciences, Marquette University, Milwaukee, WI 53233, USA. 8 *Corresponding Authors: 9 Marieke R. Gilmartin, Ph.D. 10 E-mail: [email protected] 11 12 Robert C. Twining, Ph.D. 13 E-mail: [email protected] 14 15 16 Number of pages: 44 17 Number of figures: 6 18 Number of words: Abstract: 238 Introduction: 682 Discussion: 1916 19 20 Conflict of Interest: The authors declare no competing financial interests. 21 22 Author Contributions: RCT and MRG designed the experiments and wrote the manuscript. RCT 23 performed the surgeries and analyzed the data. RCT and KL ran the experiments. KL and AJK 24 performed histological analysis. AJK provided critical feedback on the manuscript. 25 26 Acknowledgments: This work was supported by the Whitehall Foundation (2014-8-67 to MRG) and 27 the National Science Foundation (IOS:1558121 to MRG). 28 29 30 Twining et al. 31 Abstract 32 The prefrontal cortex, through its high degree of interconnectivity with cortical and subcortical 33 brain areas, mediates cognitive and emotional processes in support of adaptive behaviors. This 34 includes the formation of fear memories when the anticipation of threat demands learning about 35 temporal or contextual cues, as in trace fear conditioning. In this variant of fear learning, the 36 association of a cue and shock across an empty trace interval of several seconds requires sustained 37 cue-elicited firing in the prelimbic cortex (PL). However, it is unknown how and when distinct PL 38 afferents contribute to different associative components of memory. Among the prominent inputs to PL, 39 the hippocampus shares with PL a role in both working memory and contextual processing. Here we 40 tested the necessity of direct hippocampal input to the PL for the acquisition of trace cued fear memory 41 and the simultaneously acquired contextual fear association. Optogenetic silencing of ventral 42 hippocampal (VH) terminals in the PL of adult male Long-Evans rats selectively during paired trials 43 revealed that direct communication between the VH and PL during training is necessary for contextual 44 fear memory, but not for trace cued fear acquisition. The pattern of the contextual memory deficit and 45 the disruption of local PL firing during optogenetic silencing of VH-PL suggest that the VH continuously 46 updates the PL with the current contextual state of the animal, which when disrupted during memory 47 acquisition is detrimental to the subsequent rapid retrieval of aversive contextual associations. 48 49 50 51 Keywords: memory formation, fear conditioning, optogenetics, working memory 52 53 2 Twining et al. 54 Significance Statement 55 56 Learning to anticipate threat from available contextual and discrete cues is crucial for survival. 57 The prelimbic cortex is required for forming fear memories when temporal or contextual complexity is 58 involved, as in trace fear conditioning. However, the respective contribution of distinct prelimbic 59 afferents to the temporal and contextual components of memory is not known. We report that direct 60 input from the ventral hippocampus enables the formation of the contextual, but not trace cued, fear 61 memory necessary for the subsequent rapid expression of a fear response. This finding dissociates the 62 contextual and working memory contributions of prelimbic cortex to the formation of a fear memory and 63 demonstrates the crucial role for hippocampal input in contextual fear learning. 64 3 Twining et al. 65 Introduction 66 The prefrontal cortex (PFC) is an integral component of a neural system that mediates cognitive 67 and emotional processes and regulates a wide range of adaptive behaviors (Euston et al., 2012; Godsil 68 et al., 2013; Hiser and Koenigs, 2018). Through its interconnectivity with cortical and subcortical areas, 69 the PFC integrates previously learned information with current emotional and motivational states of the 70 animal to guide behavior. However, it is increasingly clear that the PFC is crucial for the initial encoding 71 of some memories, including associative fear memories. The prelimbic (PL) and anterior cingulate 72 cortices of the rodent are necessary for fear learning when temporal or contextual processing is needed 73 (Han et al., 2003; Zhao et al., 2005; Gilmartin and Helmstetter, 2010; Guimarais et al., 2011; Gilmartin 74 et al., 2013b; Gilmartin et al., 2014; Heroux et al., 2017; Robinson-Drummer et al., 2017; Kirry et al., 75 2019). The acquisition of trace fear memory includes both temporal and contextual components for 76 which the PL is necessary, but how these components are regulated by afferent input to PL is 77 unknown. Trace fear conditioning requires subjects to associate an auditory conditional stimulus (CS) 78 and an aversive footshock unconditional stimulus (UCS) that are separated in time. The presence of 79 this temporal gap recruits episodic memory systems, including the hippocampus and PFC. Functional 80 disruption of either area severely impairs the acquisition of fear to the trace-CS and also the 81 background context-shock association formed at the same time (Yoon and Otto, 2007; Czerniawski et 82 al., 2009; Esclassan et al., 2009; Gilmartin and Helmstetter, 2010; Czerniawski et al., 2011; Guimarais 83 et al., 2011; Gilmartin et al., 2012, 2013a). In PL, a subset of neurons exhibit sustained increases in 84 firing in response to a trace-CS and throughout the trace interval, similar to cue-elicited delay-cell 85 activity in primate dorsolateral PFC during working-memory tasks (Baeg et al., 2001; Gilmartin and 86 McEchron, 2005). Optogenetic inhibition of PL firing selectively during the trace interval prevented 87 animals from learning the predictive cue-shock relationship while leaving contextual learning intact 88 (Gilmartin et al., 2013b). Thus, a specific PL signal promotes the anticipation of threat through a 89 working-memory-like function, but how specific inputs to the PFC contribute to the nature of this signal 90 and learning is unknown. 4 Twining et al. 91 Among the major afferents to the PFC, the ventral hippocampus (VH) is a likely partner in 92 memory formation for salient events. The VH receives valence-encoded information from both CS- 93 responsive and appetitive UCS-responsive amygdala neurons (Beyeler et al., 2016; Beyeler et al., 94 2018), and VH neurons respond robustly to an aversive UCS (Weible et al., 2006). The PL PFC 95 receives monosynaptic input from the VH, which is glutamatergic (Jay et al., 1989; Jay and Witter, 96 1991; Cenquizca and Swanson, 2007; Hoover and Vertes, 2007; Parent et al., 2010). A functional 97 interaction between the hippocampus and PFC has been implicated, but not demonstrated, in cue 98 encoding during a related associative paradigm, trace eyeblink conditioning. Prefrontal firing was 99 phase-locked to hippocampal theta, and sustained prefrontal firing to the CS was observed only on 100 trials delivered during hippocampal theta (Darling et al., 2011). Disrupted hippocampal-prefrontal 101 connectivity is associated with working-memory deficits in humans diagnosed with schizophrenia 102 (Sigurdsson and Duvarci, 2015; Schneider et al., 2017) and with poor spatial working-memory 103 performance in genetic mouse models of schizophrenia (Sigurdsson et al., 2010). Moreover, 104 optogenetic inhibition of the VH-PL pathway in wild-type mice produced similar working-memory deficits 105 (Spellman et al., 2015). These deficits were specific to the encoding of spatial cues, consistent with 106 recent work highlighting the importance of VH-PL for contextual processing. Inhibition of VH input 107 disrupted prefrontal encoding of aversive locations in anxiety-provoking contexts (Padilla-Coreano et 108 al., 2016), and silencing VH-IL impaired context-modulated fear renewal after extinction (Marek et al., 109 2018). These findings raise the question of whether VH-PL mediates prefrontal encoding of cues more 110 generally to include non-spatial temporal associative learning in addition to spatial working memory or if 111 VH-PL input preferentially conveys contextual information. Here we use projection-specific 112 optogenetics to selectively silence VH terminals in PL during trace fear conditioning and test the 113 hypothesis that both cued and contextual fear memory acquisition depends on VH input to PL. 114 115 Materials & Methods 116 Subjects 5 Twining et al. 117 Adult male Long-Evans rats (325 g; Envigo, Indianapolis, IN) were housed individually in an 118 AAALAC-accredited vivarium and maintained on a 14h:10h light:dark cycle. Rats received food and 119 water ad libitum throughout the experiment. Twenty-eight rats were used in this study: twenty-four for 120 behavior and four for electrophysiological verification of optogenetic terminal silencing. All procedures 121 were in accordance with the National Institutes of Health guidelines and approved by the Marquette 122 University Institutional Animal Care and Use Committee. 123 Optogenetic viral constructs 124 The adeno-associated viral vector containing the light-sensitive archaerhodopsin from 125 Halorubrum strain TP009 (ArchT; rAAV9/CAG-ArchT-GFP) was prepared by Dr.