Research Article: New Research | Cognition and Behavior Syn3 gene knockout negatively impacts aspects of reversal learning performance https://doi.org/10.1523/ENEURO.0251-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0251-21.2021 Received: 4 June 2021 Revised: 4 August 2021 Accepted: 15 August 2021 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 Moore 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. Manuscript Title: Syn3 gene knockout negatively impacts aspects of reversal learning performance Abbreviated Title: Syn3 regulates behavioral flexibility Authors: Alyssa Moore, Department of Psychology, Binghamton University, Binghamton, NY 13902 Jérôme Linden, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles CA 90095 James D. Jentsch, Department of Psychology, Binghamton University, Binghamton, NY 13902 Author Contributions: JDJ and JL designed research; JL and AM performed research; AM analyzed data; AM and JDJ wrote the paper Correspondence should be addressed to: J. David Jentsch, Department of Psychology, State University of New York at Binghamton, Binghamton NY, 13902. Email: [email protected] Conflict of Interest: Authors report no conflict of interest Funding Sources: These studies were supported, in part, by Public Health Service grants R21- DA038377 (JDJ), T32-AA025606 (JDJ and AM) and P50-DA039841 (JDJ). 1 1 2 Abstract 3 Behavioral flexibility enables the ability to adaptively respond to changes in contingency 4 requirements to maintain access to desired outcomes, and deficits in behavioral flexibility have been 5 documented in many psychiatric disorders. Previous research has shown a correlation between 6 behavioral flexibility measured in a reversal learning test and Syn3, the gene encoding synapsin III, which 7 negatively regulates phasic dopamine release. Syn3 expression in the hippocampus, striatum, and 8 neocortex is reported to be negatively correlated with reversal learning performance, so here, we 9 utilized a global knockout line to investigate reversal learning in mice homozygous wildtype, 10 heterozygous null, and homozygous null for the Syn3 gene. Compared to wildtype animals, we found a 11 reversal specific effect of genetic Syn3 deficiency that resulted in a greater proportional increase in trials 12 required to reach a preset performance criteria during contingency reversal, despite no observed 13 genotype effects on the ability to acquire the initial discrimination. Behavioral flexibility scores, which 14 quantified the likelihood of switching subsequent choice behavior following positive or negative 15 feedback, became significantly more negative in reversal only for Syn3 homozygous null mice, 16 suggesting a substantial increase in perseverative behavior in the reversal phase. Syn3 ablation reduced 17 the number of anticipatory responses made per trial, often interpreted as a measure of waiting 18 impulsivity. Overall, Syn3 expression negatively affected behavioral flexibility in a reversal specific 19 manner but may have reduced waiting impulsivity. 20 2 21 Significance Statement 22 Adaptations to changes in the environment are facilitated by behavioral flexibility, and inflexible 23 behavior is observed in several mental health disorders. The Syn3 gene encodes synapsin III, a protein 24 that negatively regulates phasic dopamine release by sequestering vesicles away from the ready- 25 releasable pool. Previous research has shown a positive genetic correlation between Syn3 expression in 26 brain and behavioral flexibility in a reversal learning task. Here, we show that mice carrying null alleles 27 of the Syn3 gene exhibit less flexible responding following contingency reversal. These data reveal novel 28 information about genetic mechanisms that may contribute to the impaired flexibility observed in 29 multiple psychiatric conditions. 30 3 31 Introduction 32 Behavioral flexibility relates to an individual’s ability to modify behavioral patterns in changing 33 environmental conditions. Deficits in behavioral flexibility have been characterized in several 34 psychiatric disorders (Uddin, 2021), including schizophrenia (Waltz, 2017; Waltz & Gold, 2007), autism 35 spectrum disorder (Kelly & Reed, 2020), obsessive-compulsive disorder (Gruner & Pittenger, 2017; 36 Vaghi et al., 2017), and substance use disorders (Winstanley et al., 2010; Isten et al., 2017; Robbins et 37 al., 2012; Ersche et al., 2010). 38 Reversal learning is an operant test of behavioral flexibility in which an initial association 39 (stimulus-response or response-outcome) is learned through reinforcement, before the conditions for 40 reinforcement are reversed and the organism is tested for its ability to update behavior (Izquierdo et 41 al., 2017, Izquierdo & Jentsch, 2012). In other words, one response is established as prepotent during 42 initial acquisition through positive feedback and must then be inhibited or changed during reversal 43 testing to procure reward. 44 Laughlin et al. (2011) evaluated reversal learning in a panel of BXD mouse strains and utilized a 45 genome-wide linkage approach to model the impact of genetic variation on the reversal phenotype. A 46 genome-wide quantitative trait locus on mouse chromosome 10 was identified, and Syn3, the gene 47 encoding synapsin III, emerged as a positional candidate expressed from that genomic region. Syn3 48 mRNA expression is regulated in cis, and its expression in the hippocampus, neocortex, and striatum 49 was found to be positively genetically correlated with reversal learning performance in the BXD panel, 50 such that greater Syn3 expression associated with faster reversal learning (Laughlin et al., 2011). 51 Synapsin III is a member of the synapsin family of neuronal phosphoproteins (Kao et al., 1998). 52 Synapsin III can be localized on the cytoplasmic side of synaptic vesicles and is implicated in 53 neurotransmitter release. Feng and colleagues (2002) demonstrated loss of synapsin III led to larger 54 vesicular recycling pools but did not alter vesicular release or quantal dynamics. Synapsin proteins 4 55 regulate a distal reserve pool of vesicles (Greengard et al., 1993; Hilfiker et al., 1999; Hilfiker et al., 56 2005). Functionally, a loss of synapsin III prevents vesicles from being sequestered away from the 57 ready-releasable pool, promoting more sustained release during continued stimulation. In a typical 58 case, sustained release is limited by the rate of transfer of vesicles from the reserve pool to the active 59 zone; this rate-limiting process is theoretically disrupted in cells lacking synapsin III as vesicles are 60 inadequately sequestered in the reserve. 61 Synapsins are differentially expressed in neuronal populations. Bogen and colleagues (2006) 62 found selective deletion of synapsin I and II substantially reduced vesicular uptake of GABA and 63 glutamate but did not alter dopamine (DA) uptake. Deletion reduced concentration of vesicular 64 transporters related to glutamate and gamma-aminobutyric acid (GABA), but not vesicular monoamine 65 transporter 2 (VMAT2), the transporter responsible for packaging dopamine. Further, synapsin I and II 66 colocalized in cells expressing vesicular transporters for GABA and glutamate, but not in dopaminergic 67 terminals. A subsequent study utilized a triple knock-out approach to investigate differential regulation 68 of dopamine and serotonin by synapsins (Kile et al., 2010): serotonin was not altered by deletion of all 69 three synapsins, but DA release was significantly enhanced. Selective deletion of synapsin III also 70 elicited enhanced DA release, demonstrating a distinct role for the synapsins in regulating 71 neurotransmitter release. Because DA is extensively implicated in neuropsychiatric disorders, 72 subcellular proteins which contribute to dopamine dynamics and the genes encoding them are of 73 interest. 74 The action of dopamine on its cognate receptors in corticostriatal systems are functionally 75 implicated in behavioral flexibility and reversal learning. Inactivation of the D1-mediated direct 76 pathway of the basal ganglia interfered with the acquisition of novel and reversed contingencies, while 77 inactivation of the D2-mediated indirect pathway interfered selectively with reversal performance by 78 increasing perseverative errors (Yawata et al. 2012). Selective deletion of presynaptic D2 receptors 5 79 also tended to impair reversal performance and increased the number of attempts required to 80 complete a sustained observing response to initiate a trial (Linden et al., 2018), a deficit related to 81 waiting impulsivity (Dalley & Ersche, 2019). Groman et al (2011) found that D2 receptor availability in 82 the caudate and putamen of vervet monkeys was correlated with reversal learning performance and 83 sensitivity to positive feedback. In a study examining DA in a compulsivity-relevant behavioral 84 phenotype, Barker et al. (2014) found that inhibiting D1 or activating D2 in the infralimbic