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Blunted Amphetamine-induced Reinforcing Behaviors and Transporter Downregulation in Knock-in Mice Carrying Alanine Mutations at Threonine-258 and Serine-259 of Norepinephrine Transporter

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Abstract

Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.

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Acknowledgements

The authors thank the VCU Massey Cancer Center Transgenic/Knockout Mouse Core supported in part by NIH-NCI Cancer Center Support Grant P30 CA016059 for providing the services in generating NET-T258A/S259A knock-in mice. The authors also thank Mutant Mouse/Viral Vector Core of NIH-NIDA Central Virginia Center on Drug Abuse Research Grant P30DA033934 for providing part of the services in creating the NET-T258A/S259A knock-in mouse model.

Funding

This work was supported by the National Institutes of Health grant DA045888 (LDJ).

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298, USA

    Durairaj Ragu Varman, Padmanabhan Mannangatti, Sammanda Ramamoorthy & Lankupalle D. Jayanthi

  2. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, 23298, USA

    Mark A. Subler & Jolene J. Windle

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  1. Durairaj Ragu Varman
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  2. Padmanabhan Mannangatti
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  3. Mark A. Subler
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  5. Sammanda Ramamoorthy
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  6. Lankupalle D. Jayanthi
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Contributions

Mark A. Subler and Jolene J. Windle created the NET-T258A/S259A knock-in mice using CRISPR/Cas9 strategy. Durairaj Ragu Varman conducted NET and DAT transport analyses and surface expression experiments and acquired data, performed data analysis, created figures, and cowrote the manuscript. Padmanabhan Mannangatti conducted locomotor activity and CPP experiments and acquired data. Lankupalle D. Jayanthi conceived the project, planned the experiments, performed data analysis, created figures, and drafted the manuscript. Sammanda Ramamoorthy critically reviewed and cowrote the manuscript.

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Correspondence to Lankupalle D. Jayanthi.

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All procedures involving the animal experiments were conducted according to National Institutes of Health guide for the Care and Use of Laboratory animals. Virginia Commonwealth University Institutional Animal Care and Use Committee has approved the protocols (AD10000476) of this study. The study does not involve human subjects and hence consent to participate is not applicable.

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Ragu Varman, D., Mannangatti, P., Subler, M.A. et al. Blunted Amphetamine-induced Reinforcing Behaviors and Transporter Downregulation in Knock-in Mice Carrying Alanine Mutations at Threonine-258 and Serine-259 of Norepinephrine Transporter. J Mol Neurosci 72, 1965–1976 (2022). https://doi.org/10.1007/s12031-022-01988-x

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