Research Articles: Systems/Circuits Prefrontal α7nAChR signaling differentially modulates afferent drive and trace fear conditioning behavior in adolescent and adult rats https://doi.org/10.1523/JNEUROSCI.1941-20.2020 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.1941-20.2020 Received: 27 July 2020 Revised: 29 November 2020 Accepted: 23 December 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 © 2021 the authors 1 Prefrontal α7nAChR signaling differentially modulates afferent drive 2 and trace fear conditioning behavior in adolescent and adult rats 3 4 5 6 7 Running title: Prefrontal α7nAChR control of afferent drive 8 9 10 11 Anabel M. M. Miguelez Fernandez, Hanna M. Molla, Daniel R. Thomases, and Kuei Y. Tseng* 12 13 Department of Anatomy and Cell Biology, University of Illinois at Chicago, IL 14 15 16 17 *Corresponding Author: Kuei Y. Tseng, MD, PhD 18 Department of Anatomy and Cell Biology 19 University of Illinois at Chicago – College of Medicine 20 Chicago, IL 60612, USA 21 Email: [email protected] 22 23 24 Number of figures: 8 25 Number of tables: 0 26 Abstract: 250 27 Main text: 4,030 words (Introduction: 451; Methods: 1,205; Results: 979; Discussion: 1,395) 28 29 30 31 32 Acknowledgements 33 Supported by NIH Grants R01-MH086507 and R01-MH105488 to KYT, and UIC College of Medicine 34 funds to KYT. We thank Dr. Adriana Caballero for thoughtful comments on the manuscript. This study 35 was initiated at Rosalind Franklin University – Chicago Medical School. 36 Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 1 37 Abstract 38 Increased level of kynurenic acid is thought to contribute to the development of cognitive deficits in 39 schizophrenia through an α7nAChR-mediated mechanism in the prefrontal cortex (PFC). Yet, it 40 remains unclear to what extent disruption of PFC α7nAChR signaling impacts afferent transmission and 41 its modulation of behavior. Here we found that PFC infusion of methyllycaconitine (MLA, α7nAChR 42 antagonist) shifts ventral hippocampal-induced local field potential (LFP) suppression to LFP facilitation, 43 an effect only observed in adult male rats. Hippocampal stimulation can also elicit a GluN2B-mediated 44 LFP potentiation (when PFC GABAAR is blocked) that is insensitive to MLA. Conversely, PFC infusion 45 of MLA diminished the gain of amygdalar transmission, which is already enabled by postnatal day 30. 46 Behaviorally, the impact of prefrontal MLA on trace fear-conditioning and extinction was also age- 47 related. While freezing behavior during conditioning was reduced by MLA only in adults, it elicited 48 opposite effects in adolescent and adult rats during extinction as revealed by the level of reduced and 49 increased freezing response, respectively. We next asked if the late-adolescent onset of α7nAChR 50 modulation of hippocampal inputs contributes to the age-dependent effect of MLA during extinction. 51 Data revealed that the increased freezing behavior elicited by MLA in adult rats could be driven by a 52 dysregulation of the GluN2B transmission in the PFC. Collectively, these results indicate that distinct 53 neural circuits are recruited during the extinction of trace fear memory in adolescents and adults, likely 54 due to the late-adolescent maturation of the ventral hippocampal-PFC functional connectivity and its 55 modulation by α7nAChR signaling. 56 57 58 Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 2 59 Significance statement 60 Abnormal elevation of the astrocyte-derived metabolite kynurenic acid in the prefrontal cortex is thought 61 to impair cognitive functions in schizophrenia through an α7nAChR-mediated mechanism. Here we 62 found that prefrontal α7nAChR signaling is recruited to control the gain of hippocampal and amygdalar 63 afferent transmission in an input-specific, age-related manner during the adolescent transition to 64 adulthood. Behaviorally, prefrontal α7nAChR modulation of trace fear memory was also age-related, 65 likely due to the late-adolescent maturation of the ventral hippocampal pathway and its recruitment of 66 PFC GABAergic transmission enabled by local α7nAChR signaling. Collectively, these results reveal 67 that distinct α7nAChR-sensitive neural circuits contribute to regulate behavior responses in adolescents 68 and adults, particularly those requiring proper integration of hippocampal and amygdalar inputs by the 69 prefrontal cortex. 70 Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 3 71 Introduction 72 Several neural processes contributing to prefrontal cortex (PFC) maturation undergo major remodeling 73 during adolescence (Caballero et al., 2016; Caballero and Tseng, 2016) to enable the acquisition of 74 adult cognitive abilities (Casey et al., 2000; Best and Miller, 2010). As PFC processing and integration 75 of inputs matures through adolescence (Caballero et al., 2016), any disruption that compromises the 76 protracted trajectory of prefrontal development is expected to confer vulnerability to the onset of mental 77 disorders (Caballero and Tseng, 2016) that display cognitive deficits and associated dysregulation of 78 affect (Paus et al., 2008; Gogtay et al., 2011; Volk and Lewis, 2014). Thus, elucidating which signaling 79 mechanisms are recruited to strengthen the functional connectivity of PFC afferent transmission is key 80 to reveal how cognitive impairments could emerge in psychiatric disorders when such recruitment fails 81 to occur (Caballero et al., 2016; Caballero and Tseng, 2016). 82 Of particular interest is the increased level of kynurenic acid in the PFC and its potential link to 83 the onset of cognitive deficits in schizophrenia (Erhardt et al., 2007; Wonodi and Schwarcz, 2010; 84 Myint, 2012). In addition to disrupting NMDAR function (Kessler et al., 1989; Parsons et al., 1997), 85 nanomolar concentrations of kynurenic acid can elicit a state of excitatory-inhibitory imbalance in the 86 PFC through a presynaptic α7nAChR-mediated mechanism (Flores-Barrera et al., 2017). Certainly, 87 prefrontal regulation of cognitive behavior, such as working memory, behavioral flexibility and attention, 88 requires proper levels of α7nAChR and NMDAR function (Alexander et al., 2012; Alexander et al., 89 2013; Phenis et al., 2020) and integration of hippocampal and amygdalar inputs by the PFC (Floresco 90 et al., 1997; Ishikawa and Nakamura, 2003; Tse et al., 2015). Since α7nAChRs are well-positioned to 91 regulate glutamate release in the PFC (Bortz et al., 2016), any disruption of local α7nAChR signaling is 92 expected to impact the gain of afferent glutamatergic transmission and the recruitment of postsynaptic 93 NMDAR-mediated plasticity by hippocampal and amygdalar inputs (Flores-Barrera et al., 2014). 94 The aim of the present study is to determine how disruption of α7nAChR signaling in the PFC 95 impacts afferent information processing and its control of behavioral responses in adolescent and adult Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 4 96 rats. To this end, local field potential recordings were combined with PFC infusions of antagonists to 97 reveal whether α7nAChR modulation of ventral hippocampal and basolateral amygdalar transmission is 98 input-specific and age-regulated between. Similar pharmacological manipulations were implemented to 99 assess the contribution of prefrontal α7nAChR signaling in modulating behavior using a trace fear 100 conditioning paradigm. Such a behavioral construct was preferred because proper processing of 101 hippocampal and amygdalar afferent information by the PFC is needed for the learning and extinction 102 of conditioned fear memories (Ishikawa and Nakamura, 2003; Sierra-Mercado et al., 2011; Gilmartin et 103 al., 2012; Sotres-Bayon et al., 2012; Gilmartin et al., 2014). Miguelez Fernandez et al_Tseng 2020: α7nAChR_paper 5 104 Materials and Methods 105 All experimental procedures were approved by the University of Illinois at Chicago Institutional Animal 106 Care and Use Committee and met the National Institutes of Health guidelines for care and use of 107 laboratory animals. Male Sprague Dawley rats were purchased from Envigo. Upon arrival, rats were 108 allowed to habituate for at least 7 days before being subjected to any surgical procedures. They were 109 group housed (2-3 rats/cage), maintained under constant temperature (21-23°C) and light/dark cycle 110 (14/10hs) with food and water available ad libitum. All chemicals were obtained from Sigma, except for 111 methyllycaconitine (MLA) and Indiplon that were obtained from Tocris. 112 113 In vivo recordings of local field potential (LFP) responses in the prefrontal cortex (PFC). All recordings 114 and PFC infusions procedures were conducted as previously described (Cass et al., 2013; Thomases 115 et al., 2013; Caballero et al., 2014b; Thomases et al., 2014). Briefly, rats were anesthetized with 8% 116 chloral hydrate (400mg/kg, i.p.), placed in a stereotaxic frame, and maintained at 37-38°C with a steady 117 supplement of 300-400 μL/hour of 8% chloral hydrate. After exposing the skull, two burr holes were 118 drilled to place the recording electrode in the medial PFC and the stimulating electrode within the 119 ventral hippocampus or the basolateral amygdala. All LFP recordings were obtained using a concentric 120 bipolar electrode attached to a 28G cannula (PlasticsOne), amplified (Cygnus Technology), filtered (1– 121 100 Hz bandwidth), and digitized (Digidata 1440A, Molecular Devices) at a sampling rate of 10 kHz. 122 The intensity of stimulation was chosen from the 0.2–0.8 mA range (mean intensity: ~0.6mA) using 300 123 μs duration square pulses delivered every 15 s through a computer-controlled pulse generator (Master 124 8, A.M.P.I.). Typically, a 10 min LFP baseline recording was collected prior PFC infusions of 0.8 μL (0.1 125 μL/min) artificial cerebrospinal fluid (aCSF)-containing vehicle, picrotoxin (50μM in 0.1% DMSO), MLA 126 (300nM) or picrotoxin + MLA.