Research Article: New Research | Neuronal Excitability Noradrenergic suppression of persistent firing in hippocampal CA1 pyramidal cells through cAMP-PKA pathway https://doi.org/10.1523/ENEURO.0440-20.2020 Cite as: eNeuro 2021; 10.1523/ENEURO.0440-20.2020 Received: 12 October 2020 Revised: 3 December 2020 Accepted: 20 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.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Valero-Aracama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Noradrenergic suppression of persistent firing in hippocampal CA1 2 pyramidal cells through cAMP-PKA pathway 3 4 Abbreviated Title: Noradrenaline suppress persistent firing 5 6 1Ɨ 2,3Ɨ 2 3 7 Maria Jesus Valero-Aracama , Antonio Reboreda , Alberto Arboit , Magdalena Sauvage , Motoharu 2,3 * 8 Yoshida 9 1 10 Faculty of Psychology, Mercator Research Group - Structure of Memory, Ruhr-University Bochum, 11 44780 Bochum, Germany 12 Current affiliation: Institute for physiology and pathophysiology, University of Erlangen, Germany 13 2 14 German Center for Neurodegenerative Diseases (DZNE), 39120 Magdeburg, Germany 15 3 16 Leibniz Institute for Neurobiology (LIN) 39118 Magdeburg, Germany 17 Ɨ Equal contributions ∗ 18 To whom correspondence should be addressed 19 20 Author contributions: 21 MY, AR, MJ and MS designed research; MJ, AR and AA performed research and analyzed data; 22 MJ, AR and MY wrote the paper. 23 24 Correspondence should be addressed to: 25 Dr Motoharu Yoshida 26 German Center for Neurodegenerative Diseases (DZNE) Magdeburg 27 Leipziger Str. 44 / Haus 64, 39120, Magdeburg, Germany 28 E-mail: [email protected] 29 30 Number of figures: 5 31 Number of tables: 2 32 Number of Multimedia: 0 33 Number of words for Abstract: 164 34 Number of words for Significance Statement: 35 Number of words for Introduction: 418 36 Number of words for Discussion: 1740 37 38 Acknowledgments: We thank Dr. Menno Witter for kindly sharing experimental procedures with us. 39 Conflict of Interest: Authors report no conflict of interest 40 Funding sources: This work was supported by the Mercator Stiftung and the German Research 41 Foundation (DFG) project YO177/4-1 and YO177/7-1. 42 1 43 Abstract 44 Persistent firing is believed to be a cellular correlate of working memory. While the effects of 45 noradrenaline (NA) on working memory have widely been described, its effect on the cellular 46 mechanisms of persistent firing remains largely unknown. Using in vitro intracellular recordings, we 47 demonstrate that persistent firing is supported by individual neurons in hippocampal CA1 pyramidal 48 cells through cholinergic receptor activation, but is dramatically attenuated by NA. In contrast to the 49 classical theory that recurrent synaptic excitation supports persistent firing, suppression of persistent 50 firing by NA was independent of synaptic transmission, indicating that the mechanism is intrinsic to 51 individual cells. In agreement with detrimental effects of cyclic adenosine monophosphate (cAMP) on 52 working memory, we demonstrate that the suppressive effect of NA was through cAMP-PKA pathway. 53 In addition, activation of β1 and/or β3 adrenergic receptors, which increases cAMP levels, 54 suppressed persistent firing. These results are in line with working memory decline observed during 55 high levels of NA and cAMP, which are implicated in high stress, aging and schizophrenia. 56 2 57 Significance Statement 58 While cholinergic modulation supports working memory, high concentrations of noradrenaline (NA), 59 which occurs under high stress for example, are detrimental for working memory. However, cellular 60 and molecular mechanisms underlying such working memory deficit remain largely unclear. In this 61 paper, we studied the effect of these two neuromodulators on persistent firing, the cellular correlate 62 of working memory. We demonstrate that a cholinergic receptor activation supports, while a 63 noradrenergic activation strongly inhibits persistent firing due to the PKA activation through specific 64 NA receptor types which upregulate cAMP. These data are in line with working memory deficits in 65 aging and schizophrenia in which cAMP levels are altered, and indicate potential intrinsic cellular 66 mechanism of working memory impairment. 67 68 Introduction 69 Persistent firing is a cellular response characterized by repetitive spiking that outlasts triggering 70 stimulus, and is believed to be a neural base of working memory (Goldman-Rakic, 1995; Major & 71 Tank, 2004). Persistent firing observed in humans and animals in vivo during working memory tasks 72 correlate with the task performance (Bukalo et al., 2004; Colombo & Gross, 1994; Funahashi, Bruce, 73 & Goldman-Rakic, 1989; Hampson & Deadwyler, 2003; Kamiński et al., 2017). In contrast to the 74 classical hypothesis that persistent firing is supported by recurrent excitatory synaptic connections, 75 recent studies have shown that persistent firing can also be supported in individual cells (Egorov, 76 Hamam, Fransén, Hasselmo, & Alonso, 2002; Jochems & Yoshida, 2013; Knauer, Jochems, Valero- 77 Aracama, & Yoshida, 2013). These studies in general used cholinergic receptor activations to induce 78 persistent firing, which is in agreement with the supportive role of acetylcholine on working memory 79 (Reboreda et al., 2018). However, roles of other neuromodulators such as the noradrenaline (NA) on 80 this type of persistent firing largely remain to be studied. 81 While cholinergic modulation generally supports working memory (Kaneko & Thompson, 82 1997; Weiss et al., 2000), NA could be detrimental for working memory (Arnsten, 2009; Roozendaal 83 & McGaugh, 2011). High levels of NA are usually present during states of stress (Bremner, Krystal, 84 Southwick, & Charney, 1996), which are known to impair working memory (Arnsten, Wang, & 85 Paspalas, 2012; Diamond, Ingersoll, Fleshner, & Rose, 1996; Qin, Hermans, van Marle, Luo, & 86 Fernández, 2009; Schoofs, Preuß, & Wolf, 2008). At the receptor subtype levels, β1 and α2 87 adrenoreceptors, which are positively and negatively coupled to cAMP signaling, impair and facilitate 88 working memory, respectively (Arnsten & Li, 2005; Jäkälä et al., 1999; Li, Mao, Wang, & Mei, 1999; 3 89 Ma, Qi, Peng, & Li, 2003; Ramos et al., 2005). In addition, elevated and decreased cAMP levels impair 90 and enhance, respectively, working memory performance (Arnsten, Mathew, Ubriani, Taylor, & Li, 91 1999; Runyan, Moore, & Dash, 2005; Taylor, Birnbaum, Ubriani, & Arnsten, 1999; M. Wang et al., 92 2007). Moreover, upregulated cAMP levels are associated with working memory impairment in aging 93 and schizophrenia (Barch & Ceaser, n.d.; Millar, 2005; M. Wang et al., 2011). Raising cAMP levels 94 would increase PKA activation that also is negatively correlated to working memory performance 95 (Kobori, Moore, & Dash, 2015), together supporting the importance of NA and cAMP-PKA in working 96 memory. 97 At the cellular level, a supportive role of NA on persistent firing through a reduction of cAMP 98 levels has been observed in the prefrontal cortex (PFC, Zizhen Zhang, Cordeiro Matos, Jego, 99 Adamantidis, & Séguéla, 2013). In addition, elevated cAMP levels caused a suppression of the 100 calcium-activated nonspecific cation (CAN) current and persistent firing in the PFC and hippocampus 101 (El-Hassar, Hagenston, D’Angelo, & Yeckel, 2011; Sidiropoulou et al., 2009; Zizhen Zhang et al., 2013). 102 However, it remains unclear whether NA has a supportive or detrimental effect on persistent firing in 103 the hippocampus. 104 We therefore tested the effects of cholinergic and noradrenergic receptor activation in both 105 induction and modulation of persistent firing in individual hippocampal CA1 pyramidal cells in mice. 106 We find that a noradrenergic stimulation strongly inhibits persistent firing while a cholinergic 107 activation supports it. In addition, the noradrenergic suppression of persistent firing is independent 108 of ionotropic synaptic transmission, but is through the cAMP-PKA pathway. We further demonstrate 109 specific NA receptor subtypes involved in this suppression. These observations indicate that high NA 110 and cAMP conditions, which often suppress working memory through PKA activation, also suppress 111 cellular mechanisms for persistent firing in the hippocampus. 112 113 Methods 114 115 All the experimental designs were approved by the local ethic committee (Der Tierschutzbeauftragte, 116 Ruhr-Universität Bochum, and Deutsches Zentrum für Neurodegenerative Erkrankungen) and carried 117 out in accordance with the European Communities Council Directive of September 22nd, 2010 118 (2010/63/EU). 119 120 Slice preparation 121 Acute hippocampal slices were obtained from adult (2 - 4 month) C57BL6 female mice (Charles 122 River). Animals were deeply anesthetized (intraperitoneal injection of 120 mg/Kg of ketamine and 16 123 mg/Kg of xylazin) and intracardiac perfusion with ice-cold modified artificial cerebrospinal fluid 4 124 (mACSF) was conducted