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Research Article: New Research | Disorders of the Nervous System
PICK1-Deficient Mice Exhibit Impaired Response to Cocaine and Dysregulated Dopamine Homeostasis 1
1,2
Kathrine Louise Jensen , Gunnar Sørensen 1
2
3
, Ditte Dencker , William Anthony Owens , Troels Rahbek-
1
1
1
2
Clemmensen , Michael Brett Lever , Annika H. Runegaard , Nikolaj Riis Christensen , Pia Weikop , 2
2
1
3
1
Gitta Wörtwein , Anders Fink-Jensen , Kenneth L. Madsen , Lynette Daws , Ulrik Gether and Mattias Rickhag
1
1
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DK-2200, Denmark 2
Laboratory of Neuropsychiatry, Psychiatric Center Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DK-2200, Denmark 3
Department of Cellular and Integrative Physiology, University of Texas Health Science Center at San Antonio, Texas USA DOI: 10.1523/ENEURO.0422-17.2018 Received: 5 December 2017 Revised: 18 April 2018 Accepted: 27 April 2018 Published: 14 May 2018
Author contributions: KLJ, GS, GW, AFJ, KLM, LD, UG and MR Designed research; KLJ, GS, DD, WAO, TRC, MBL, AHR, NRC, PW, GW and MR Performed research; KLJ, GS, DD, WAO, TRC, MBL, AHR, NRC, PW, GW, KLM, LD, UG and MR Analyzed data; KLJ, GS, GW, LD, UG and MR Wrote the paper. Funding: Lundbeck Foundation (MR); Danish Medical Research Council (UG, MR, KLJ); University of Copenhagen BioScaRT programme (UG, GS); Lundbeck Foundation Center for Biomembranes in Nanomedicine (UG); Købmand I Odense Johan og Hanne Weimann Født Seedorffs Legat (MR); NIH R21 DA038504 (LD); Conflict of Interest: Author’s declare no conflict of interest. This work was supported by the Lundbeck Fondation (M.R.), the Danish Medical Research Council (U.G., M.R., K.L.J.), University of Copenhagen BioScaRT Program of Excellence (G.S., U.G.), Lundbeck Foundation Center for Biomembranes in Nanomedicine (U.G.), the Weimann Foundation (M.R.), and National Institutes of Health R21 DA038504 (L.C.D.). Animal experiments were performed in accordance with the guidelines of the Danish Animal Experimentation Inspectorate (permission number 2012-15-293-00279 and 2012-15-2934-00038) in a fully AAALAC accredited facility under the supervision of a local animal welfare committee. Chronoamperometric experiments carried out at UTHSCSA were performed in accordance with Institutional Animal Care and Use Committee approved protocols, in a fully AAALAC accredited facility. K.L.J. and G.S. contributed equally to this work. Corresponding authors: Mattias Rickhag, Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark, E-mail:
[email protected] or Ulrik Gether, Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark, Tel: +45 2384 0089; E-mail:
[email protected] Cite as: eNeuro 2018; 10.1523/ENEURO.0422-17.2018
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 Jensen et al.
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1. PICK1-deficient mice exhibit impaired response to cocaine and dysregulated dopamine homeostasis 2. Abbreviated title: Attenuated cocaine response in PICK1 KO mice 3. Author list: Kathrine Louise Jensen1*, Gunnar Sørensen1,2*, Ditte Dencker2, William Anthony Owens3, Troels Rahbek-Clemmensen1, Michael Brett Lever1, Annika H. Runegaard1, Nikolaj Riis Christensen1, Pia Weikop2, Gitta Wörtwein2, Anders Fink-Jensen2, Kenneth L. Madsen1, Lynette Daws3, Ulrik Gether1 and Mattias Rickhag1 *equal contribution 1
Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark; 2Laboratory of Neuropsychiatry, Psychiatric Center Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark; 3 Department of Cellular and Integrative Physiology, University of Texas Health Science Center at San Antonio, Texas, USA 4. Author contributions: KLJ, GS, GW, AFJ, KLM, LD, UG and MR Designed research; KLJ, GS, DD, WAO, TRC, MBL, AHR, NRC, PW, GW and MR Performed research; KLJ, GS, DD, WAO, TRC, MBL, AHR, NRC, PW, GW, KLM, LD, UG and MR Analyzed data; KLJ, GS, GW, LD, UG and MR Wrote the paper 5. Corresponding authors: Mattias Rickhag, Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark,E-mail:
[email protected] or Ulrik Gether, Molecular Neuropharmacology and Genetics Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark, Tel: +45 2384 0089; E-mail:
[email protected] 6. Number of Figures: 6 7. Number of Tables: 2 8. Number of Multimedia: 0 9. Number of words for Abstract: 239 10. Number of words for Significance Statement: 115 11. Number of words for Introduction: 630 12. Number of words for Discussion: 1500 13. Acknowledgements: The PICK1 KO mouse strain (Gardner et al., 2005) was kindly provided by Dr. Richard Huganir (Johns Hopkins University, Baltimore, USA). The lentiviral vectors (Citri et al., 2010) were kindly provided by Prof. Robert C. Malenka at Stanford University (Palo Alto, California, USA). We also thank Anna Mai Jansen for providing the dual promoter lentiviral constructs used to knock-down PICK1 in dopaminergic cultures. 14. Conflict of interest: Author’s declare no conflict of interest 15. This work was supported by the Lundbeck Fondation (M.R.), the Danish Medical Research Council (U.G., M.R., K.L.J.), University of Copenhagen BioScaRT Program of Excellence (G.S., U.G.), Lundbeck Foundation Center for Biomembranes in Nanomedicine (U.G.), the Weimann Foundation (M.R.), and National Institutes of Health R21 DA038504 (L.C.D.).
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Animal experiments were performed in accordance with the guidelines of the Danish Animal Experimentation Inspectorate (permission number 2012-15-293-00279 and 2012-15-293400038) in a fully AAALAC accredited facility under the supervision of a local animal welfare committee. Chronoamperometric experiments carried out at UTHSCSA were performed in accordance with Institutional Animal Care and Use Committee approved protocols, in a fully AAALAC accredited facility.
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Abstract
82
Protein Interacting with C-Kinase 1 (PICK1) is a widely expressed scaffold protein known to
83
interact via its PDZ domain with several membrane proteins including the dopamine (DA)
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transporter (DAT), the primary target for cocaine’s reinforcing actions. Here, we establish the
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importance of PICK1 for behavioral effects observed after both acute and repeated
86
administration of cocaine. In PICK1 knockout (KO) mice, the acute locomotor response to a
87
single injection of cocaine was markedly attenuated. Moreover, in support of a role for
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PICK1 in neuroadaptive changes induced by cocaine, we observed diminished cocaine
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intake in a self-administration paradigm. Reduced behavioral effects of cocaine were not
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associated with decreased striatal DAT distribution and most likely not caused by the ~30%
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reduction in synaptosomal DA uptake observed in PICK1 KO mice. The PICK1 KO mice
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demonstrated preserved behavioral responses to DA receptor agonists supporting intact
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downstream DA receptor signaling. Unexpectedly, we found a prominent increase in striatal
94
DA content and levels of striatal tyrosine hydroxylase (TH) in PICK1 KO mice.
95
Chronoamperometric recordings showed enhanced DA release in PICK1 KO mice,
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consistent with increased striatal DA pools. Viral-mediated knockdown of PICK1 in cultured
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dopaminergic neurons increased TH expression, supporting a direct cellular effect of PICK1.
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In summary, in addition to demonstrating a key role of PICK1 in mediating behavioral effects
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of cocaine, our data reveal a so far unappreciated role of PICK1 in DA homeostasis that
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possibly involves negative regulation of striatal TH levels.
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3
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Significance Statement
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Cocaine addiction is a major societal problem and better treatments are needed. We
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demonstrate here the importance of the PDZ-domain scaffold protein PICK1 for both the
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acute reinforcing effects of cocaine and the long-term behavioral changes seen upon
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repeated cocaine exposure. Interestingly, our data suggest that these alterations are
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independent of PICK1 binding to the primary target of cocaine, DAT. Moreover, our study
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reveals a novel role of PICK1 in maintenance of DA homeostasis that involves negative
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regulation of the levels of striatal TH, the rate-limiting enzyme in DA synthesis. Summarized,
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these data provide an important framework for further exploring the role of PICK1 in
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addiction and as putative target for treatment of psychostimulant abuse.
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Introduction
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Cocaine is a highly addictive and widely abused psychostimulant (Karila et al., 2012;
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Proctor, 2012; Degenhardt et al., 2013; Karila et al., 2014). Treatment of cocaine abuse,
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however, remains a major challenge and still no pharmacological agents have proven
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therapeutically useful (Kristensen et al., 2011; Shorter et al., 2015). The reinforcing
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properties of cocaine can be attributed primarily to its high-affinity interaction with the
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presynaptic plasma membrane dopamine transporter (DAT). DAT mediates rapid reuptake
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of dopamine (DA) from the extracellular space (Giros et al., 1996; Kristensen et al., 2011)
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and is thereby critical for replenishment of intracellular striatal DA pools (Jones et al., 1998).
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Binding of cocaine to DAT leads to a fast increase in extracellular DA, stimulation of DA
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receptors and subsequent long-lasting changes in synaptic plasticity, which are thought to
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underlie the craving for continuous drug intake (Chen et al., 2006; Thomsen et al., 2009).
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Protein interacting with C-kinase 1 (PICK1) is a scaffold protein implicated in several
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cellular functions (Xu and Xia, 2006; Hanley, 2008; Focant and Hermans, 2013). Scaffold
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proteins constitute a highly diverse family of proteins that serve a key role in orchestrating
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neuronal signaling processes ensuring specificity in intracellular signaling networks (Kim and
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Sheng, 2004; Good et al., 2011). PICK1 is unique by possessing both a PDZ (PSD-
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95/Discs-large/ZO-1)
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(Bin/amphiphysin/Rvs) domain (Hanley, 2008; Madsen et al., 2012). The BAR domain is
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dimeric and thus PICK1 contains in its functional form two separate PDZ domains allowing
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simultaneous binding of two interaction partners (Xu and Xia, 2006; Hanley, 2008; Focant
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and Hermans, 2013). The PDZ-domain of PICK1 interacts with the C-termini of several
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receptors, channels and transporters expressed in the CNS (Xia et al., 1999; Dev et al.,
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2000; Torres et al., 2001; Madsen et al., 2005). These include DAT (Torres et al., 2001;
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Bjerggaard et al., 2004) and the AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
159
acid receptor) subunit GluA2 (ionotropic glutamate receptor 2) (Xia et al., 2000; Kim et al.,
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2001; Jin et al., 2006; Steinberg et al., 2006; Thorsen et al., 2010), and for both it has been
protein
interaction
domain
and
a
lipid
binding
BAR
5
161
suggested that PICK1 modulates their trafficking (Xia et al., 2000; Kim et al., 2001; Torres et
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al., 2001; Jin et al., 2006; Steinberg et al., 2006; Thorsen et al., 2010). Interfering with the
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interaction between PICK1 and AMPA receptors was previously shown to blunt the synaptic
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plasticity induced at glutamatergic synapses onto VTA dopaminergic neurons upon a single
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cocaine exposure (Bellone and Luscher, 2006). Moreover, both cerebellar and hippocampal
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long-term depression is abolished upon disruption of the PICK1-GluA2 interaction (Xia et al.,
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2000; Kim et al., 2001; Jin et al., 2006; Steinberg et al., 2006; Thorsen et al., 2010).
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However, the consequences of disrupting PICK1-DAT interactions for the behavioral effects
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of cocaine are unknown.
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Here, we investigate the importance of PICK1 for the behavioral actions of cocaine. In
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mice with targeted deletion of PICK1 (PICK1 knock-out (KO) mice), the acute locomotor
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response to a single injection of cocaine was markedly attenuated. Moreover, cocaine intake
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was diminished in a self-administration paradigm. These behavioral changes were not
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associated with altered presynaptic DAT expression or aberrant postsynaptic DA receptor
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activation. Intriguingly, we found that PICK1 KO mice displayed increased striatal levels of
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tyrosine hydroxylase (TH) and DA, relative to wildtype littermates. Chronoamperometric
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recordings in striatum revealed enhanced DA release in PICK1 KO mice further supporting
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increased striatal DA pools. The importance of PICK1 specifically in DA neurons was
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confirmed by lentiviral knockdown of PICK1 in cultured midbrain dopaminergic neurons
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resulting in elevated TH expression. However, neither co-immunoprecipitation nor
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fluorescence polarization experiments showed a direct interaction between TH and PICK1.
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Our data demonstrate the importance of PICK1 for regulating behavioral effects of cocaine,
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and reveal a so far unknown role of PICK1 in maintenance of DA homeostasis.
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Materials and Methods
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Subjects. Male PICK1 knockout (KO) mice (Gardner et al., 2005), DAT+Ala knock-in mice
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(Rickhag et al., 2013) and wildtype littermates of at least 10 weeks of age at the beginning of
191
an experiment were used, unless otherwise specified. Mice were group-housed in a
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temperature-controlled room maintained on a 12:12 light:dark cycle (lights on at 7 AM) with
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access to standard rodent chow and water ad libitum. Animals were allowed to habituate to
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the facility for at least 7 days prior to initiation of experiments. Animal experiments were
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performed in accordance with the guidelines of the Danish Animal Experimentation
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Inspectorate (permission number 2012-15-293-00279 and 2012-15-2934-00038) in a fully
197
AAALAC accredited facility under the supervision of a local animal welfare committee.
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Chronoamperometric experiments carried out at UTHSCSA were performed in accordance
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with Institutional Animal Care and Use Committee approved protocols, in a fully AAALAC
200
accredited facility. All efforts were made to minimize pain and discomfort as well as the
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number of animals used during the experiments.
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Drugs. SKF82958 hydrobromide and quinpirole hydrochloride was purchased from Sigma-
204
Aldrich. Cocaine hydrochloride was obtained from the Copenhagen University Hospital
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Pharmacy. All drugs were prepared in 0.9% aq. NaCl, with a final pH of 6 - 7 and injected
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i.p. in a volume of 10 mL/kg.
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Drug-induced hyperlocomotion without habituation Locomotor activity was measured in
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activity boxes as previously described (Sorensen et al., 2012; Rickhag et al., 2013). Mice
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(wildtype/PICK1 KO/DAT+Ala mice) were injected with either 0, 5, 10 or 30 mg/kg cocaine
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dissolved in saline i.p. 3-7 min before testing. The animals were then placed in a cage and
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the activity was measured for 1 hour. Each mouse only received one dose of cocaine and
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cages were cleaned between tests.
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Drug-induced hyperlocomotion with habituation. PICK1 KO and wildtype mice were
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habituated to the experimental room for a minimum of 30 min prior to the experiment. The
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mice were placed in the open field (40x40x80 cm) for a 120-min habituation period followed
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by i.p. administration of the DA D1 receptor (D1R) agonist, SKF 82958 (0.3 or 1 mg/kg),
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cocaine (10 or 30 mg/kg), DA D2 receptor (D2R) agonist, quinpirole (0.1 or 10 mg/kg) or
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vehicle. With a minimum of 7 days washout period mice were retested in a counterbalanced
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Latin square design so that each animal only received the same drug once. A video camera
222
placed above the open field recorded the animals’ behavior and the distance travelled by the
223
animal was analysed using the video-tracking software EthoVision (Noldus, Wageningen,
224
The Netherlands). For best possible comparison to the effect of cocaine on the unhabituated
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mice, locomotion was analyzed for the 60 min pre and post drug injection (injection at
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t=120).
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Cocaine-induced locomotor sensitization. After administration of saline on the initial day
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(day 0), PICK1 KO and wildtype mice were divided into two groups that received either
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cocaine (10 mg/kg, i.p.) or saline for six days (day 1–6). All mice were given cocaine (10
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mg/kg, i.p.) on day 12 and day 20 and retested in the open field. On day 21, all mice were
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injected with saline and re-exposed to the open field to examine whether context
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conditioning had any effect on the observed response. Mice spent 60 minutes in the open
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field box on all testing days, and locomotion was recorded and analyzed using EthoVision
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(Noldus, Wageningen, The Netherlands).
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Intravenous self-administration of cocaine or liquid food. Equipment, training, and
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evaluation procedures were performed as previously described for chronic self-
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administration of cocaine or liquid food (Schmidt et al., 2011; Sorensen et al., 2015).
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Cocaine (1.0 mg/kg/infusion) was available under a fixed ratio 1:1 (FR1) schedule in daily 3
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hour sessions, 5–6 days/week, until baseline criteria was met (≥ 20 reinforcers earned, with
8
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≤ 20% variation over two consecutive sessions and ≥ 70% responses in the active hole). In
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consecutive sessions, saline was substituted for cocaine until extinction criteria were met
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(< 80% of the baseline responding for cocaine self-administration). This was followed by re-
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introduction of the training dose until previously established baseline criteria were met again
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or a new baseline was established with similar requirements as above. Subsequently, dose–
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effect functions (saline, 0.03, 0.1, 0.3, 1.0 mg/kg/infusion of cocaine) were determined for
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each mouse according to a Latin-square design twice in each mouse. To prevent
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overdosing, total drug intake was limited to 30 mg/kg per session.
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Another set of experimentally naïve mice was used for self-administration of a non-drug
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reinforcer under a FR1 schedule. The mice were food deprived for 18-20 hours (with ad
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libitum water) before the first presentation of liquid food (5 mL of Nutridrink, vanilla flavor,
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Nutrica, Denmark) in the operant chamber. When ≥1.5 mL of the 5 mL available was
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consumed per 2 hours session, mice were placed in the operant chamber with one active
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and one inactive nose-poke hole for daily 2 hours sessions similar to cocaine FR1 self-
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administration. Acquisition lasted until criteria were met (≥20 reinforcers earned, with ≤20%
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variation over two consecutive sessions and ≥70% responses in the active hole).
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Subsequently, water was substituted until responding was extinguished to < 80% of food-
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maintained responding. This was followed by re-introduction of the training concentration of
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liquid food until previously established baseline criteria were met again or a new baseline
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was established with similar requirements as above. Then, a range of liquid food dilutions
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(Nutridrink: water; 3%, 10%, 32%, and 100%) was presented according to a Latin-square
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design, determined twice in each mouse.
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Immunoblotting. Equal amounts of samples, prepared as described in the following
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sections, were run on a 10/15-well any kD, prestacked gel (Biorad, CA, USA) and
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transferred to PDVF membranes (Millipore, MA, USA). Membranes were blocked in either
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5% bovine serum albumin in 0.05% PBS/Tween-20 or 2% PVP-40 in 0.05% PBS/Tween-20
9
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(phospho-proteins) or in 5% dry milk in 0.05% PBS/Tween-20 (unphoshorylated proteins)
271
and incubated overnight with antibodies against flotillin (1:1000, SAB2500404, rabbit
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polyclonal, Sigma), DAT (1:1000, MAB369, rat monoclonal, Millipore), D1R (1:1000, D2944,
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rat polyclonal, Sigma), TH (1:1000, MAB318, mouse monoclonal, Millipore), phospho-TH
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(1:1000, 2791S, rabbit polyclonal, Cell Signaling), CREB (1:1000, 48H2, rabbit polyclonal,
275
Cell Signaling), anti-phospho-Ser133 CREB (1:1000, p1010-133, PhospoSolutions) or
276
VMAT2 (1:1000, Ab191121, rabbit polyclonal, Abcam) depending on the experiment.
277
Following incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit/anti-
278
mouse/anti-rat antibodies (1:2000), the blots were visualized by chemiluminescence (ECL
279
kit, Amersham Biosciences for un-phosphorylated proteins and SuperSignal ELISA Femto
280
Maximum Sensitivity Substrate, Thermo scientific for phosphorylated proteins) using
281
AlphaEase (Alpha Innotech, USA). To verify equal protein loading, the membranes were
282
probed with antibodies against Na+/K+ ATPase (1:1000, Ab7671, mouse monoclonal,
283
Abcam) or HRP-conjugated β-actin (1:10000, A3854, mouse monoclonal, Sigma). In surface
284
biotinylation experiments, band intensities of biotinylated DAT and D1R were normalized to
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the band intensity of total DAT and D1R in the respective input lysates.
286 287
Preparation of striatal lysates for surface biotinylation experiments. Surface
288
biotinylation was performed as previously described (Runegaard et al., 2017).
289 290
Preparation of striatal and midbrain lysates for immunoblotting. Adult mice were
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sacrificed by decapitation and brains rapidly removed. Striatum and midbrain were rapidly
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dissected and homogenized in RIPA lysis buffer (1% Triton X-100, 1 mM EGTA, 1 mM
293
EDTA, 150 mM NaCl, 1% NP-40, 10 mM TRIS-HCl (pH 7.4) supplemented with protease
294
inhibitor cocktail (Roche Diagnostics, USA) and phosphatase inhibitor cocktail 3 (Sigma
295
Aldrich, USA). Tissue was incubated on ice for 20 min followed by centrifugation at 16.000 x
296
g for 20 min at 4oC. Supernatant was transferred to new tubes and an aliquot was removed
10
297
to determine protein concentration using a BCATM Protein Assay kit (Pierce). Samples were
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eluted in SDS loading buffer and left at 37C for 30 min followed by immunoblotting.
299 300
Preparation of striatal lysates for sucrose gradient centrifugation experiments. Adult
301
mice were sacrificed by decapitation and brains rapidly removed. Striatum from PICK1
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wildtype and KO mice were rapidly dissected and homogenized in homogenization buffer
303
(0.32 M Sucrose, 4 mM HEPES, pH 7.4). Samples were centrifuged at 1.000 x g for 10 min
304
at 4°C to remove cell nuclei followed by centrifugation at 16.000 x g for 20 min at 4°C to
305
pellet the membranes. The pellet was homogenized in 1 mL lysis buffer (1% Brij56 (w/V),
306
protease inhibitor, and 0.2 mM PMSF in the gradient buffer (25 mM HEPES, 150 mM NaCl,
307
pH 7.4) for 15 min on ice. 1 mL sample was mixed with 1 mL sucrose (80% w/V) in gradient
308
buffer to create 2 mL with final 40% (w/V) sucrose content in a 15 mL centrifuge tube
309
(Beckman Coulter, California, USA). On top of this, a 12 mL 35-15% continuous gradient
310
was assembled using a SG15 gradient maker (Hoefer, Massachusetts, USA). Subsequently
311
the gradient was ultracentrifuged at 100.000 x g for 18 hours in a Beckman SW28.1 rotor
312
head (Beckman Coulter, USA) and fractionated into 9 fractions using a P1 pump. Each
313
fraction was incubated for 30 min at 37°C in SDS loading buffer with a final concentration of
314
66 mM DTT followed by immunoblotting.
315 316
Preparation of striatal and midbrain lysates for co-immunoprecipitation experiments.
317
Adult male C57BL/6 mice (age 2-3 months) were sacrificed and brains immediately placed
318
in ice-cold artificial cerebrospinal fluid (aCSF) (124 mM NaCl, 3 mM KCl, 26 mM NaHCO3,
319
1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4, and 10 mM D-glucose) while striatum and
320
midbrain was dissected. Tissue was homogenized in lysis buffer (50 mM Tris pH = 7.4, 150
321
mM NaCl, 0.1% SDS, 0.5% NaDeoxycholate, 1% Triton X-100, 5 mM NaF and 1× Roche
322
protease inhibitor cocktail). Lysates were centrifuged at 20.000 x g for 15 min and
323
supernatants pre-cleared with streptavidin beads (Dynabeads® MyOne™ Streptavidin T1,
11
324
Thermo). 500 g protein lysate was incubated with 5 g TH/PICK1 Ab (TH, MAB318
325
Millipore; PICK1 rabbit, a kind gift from Dr. Richard Huganir, Johns Hopkins University,
326
Baltimore, U.S.A.) and incubated at 4C, rotating overnight. Protein-G beads were added
327
and samples left for an additional 3 hours. Beads were washed in lysis buffer and proteins
328
were eluted in SDS loading buffer and left at 100C for 6 min followed by immunoblotting.
329 330
Striatal DA uptake. Synaptosomal uptake assay was performed as described in (Jensen et
331
al., 2017; Runegaard et al., 2017).
332 333
HPLC determination of striatal DA content. HPLC analysis was performed as described
334
in (Jensen et al., 2017).
335 336
In vivo high-speed chronoamperometry. In vivo high-speed chronoamperometry was
337
conducted using the FAST-12 system (Quanteon; http://www.quanteon.cc) as previously
338
described (Owens et al., 2005; Williams et al., 2007; Daws et al., 2016) with some
339
modification. Recording electrode/micropipette assemblies were constructed using a single
340
carbon-fiber (30 μm diameter; Specialty Materials; http://www.specmaterials.com), which
341
was sealed inside fused silica tubing (Schott, North America). The exposed tip of the carbon
342
fiber (150 μm in length) was coated with 5% Nafion (Aldrich Chemical Co.; 3–4 coats baked
343
at 200°C for 5 min per coat) to provide a 1000-fold selectivity of DA over its metabolite 3,4-
344
Dihydroxyphenylacetic acid (DOPAC). Under these conditions, microelectrodes display
345
linear amperometric responses to 0.25–10 μM DA during in vitro calibration in 100 mM
346
phosphate-buffered saline (pH 7.4).
347
Male wildtype or PICK1 KO mice weighing 26-36 g were anesthetized by intraperitoneal
348
injection (10 mL/kg body weight) of a mixture of urethane (70 mg/mL) and α-chloralose (7
349
mg/mL), then fitted with an endotracheal tube to facilitate breathing, and placed into a
350
stereotaxic frame (David Kopf Instruments). Body temperature was maintained by placing
12
351
the animals on a water-circulated heating pad. To locally deliver potassium chloride (KCl,
352
200 µM, Sigma, pH 7.4) close to the recording site, a glass multi-barrel micropipette (FHC)
353
was positioned adjacent to the microelectrode using sticky wax (Moyco). The center-to-
354
center distance between the microelectrode and the micropipette ejector was ~200 μm. The
355
electrode/micropipette assembly was lowered into the striatum at the following coordinates
356
(in mm from bregma (Franklin, 1997): A/P, +1.1; M/L, ±1.4; D/V, −2.25). To evoke release of
357
endogenous DA, KCl was pressure-ejected using a Picospritzer II (General Valve
358
Corporation) in an ejection volume 43 ± 7 nl, 8.5 ± 1 pmol and 41 ± 6 nl, 8.0 ± 1 pmol, for
359
wildtype and KO respectively. To record KCl-evoked efflux of DA at the carbon fiber
360
electrode, oxidation potentials-consisting of 100-ms pulses of 550 mV, each separated by a
361
900-ms interval during which the resting potential was maintained at 0 mV-were applied with
362
respect to an Ag/AgCl reference electrode implanted into the contralateral superficial cortex.
363
Oxidation and reduction currents were digitally integrated during the last 80 ms of each 100-
364
ms voltage pulse. For each recording session, DA was identified by its reduction/oxidation
365
current ratio, which ranged from 0.50–0.83. Detailed methods can be found in (Daws et al.,
366
2016). Peak signal amplitude was used as an index of striatal tissue pools of DA. At the
367
conclusion of each experiment, an electrolytic lesion was made to mark the placement of the
368
recording electrode tip. Mice were then decapitated while still anesthetized, and their brains
369
were removed, frozen on dry ice, and stored at −80°C until sectioned (20 μm) for histological
370
verification of electrode location within the striatum.
371 372
Isolation and mRNA expression analysis. Adult mice were sacrificed by decapitation and
373
brains rapidly removed. Midbrain from PICK1 wildtype and KO mice was rapidly dissected in
374
the presence of RNAse away (Molecular Bioproducts, Fischer scientific). Samples were
375
collected on dry ice and placed at -80˚C until RNA extraction. Samples were lyzed using a
376
Qiagen microRNA column (Qiagen) according to the manufacturer’s instructions, including
377
DNase treatment. Conversion from RNA to cDNA was done with SuperScript III
378
(Thermofisher). Reaction 1 (70oC 5 min): max 1 µg RNA, 50 ng random primer, 0.2 µmol
13
379
DTT, H2O to a final volume 15 µl. Reaction 2 (10 min 25oC, 50 min 50oC, 5 min 80oC); 10X
380
RT buffer, 10 nmol dNTP mix, 40U RNaseOUT, 200U Superscript, H2O to a final volume 5
381
µl, thereafter added to Reaction 1. Before running qPCR on the samples, cDNA was diluted
382
10X with sterile water. The Agilent Mx3000p (Agilent Technologies) real-time thermocycler
383
was used to perform the qPCR using SYBR green (PrecisionPLus 2x qPCR Mastermix
384
Primer design Ltd.) as probe. Data collection was performed by MxPro software and
385
analyzed in Microsoft Excel as well as GraphPad Prism version 6. All samples were run in
386
duplicates and beta-actin was used as reference/housekeeping gene. Relative expression
387
was calculated through the ΔΔCq method (Pfaffl, 2001). Primers; beta-actin: forward
388
(TTCTACAATGAGCTGCGTGTG)
389
hydroxylase:
390
(CCTGGGAGAACTGGGCAAATG).
forward
reverse
(GGGGTGTTGAAGGTCTCAAA).
(CCGTCATGCCTCCTCACCTATG)
Tyrosine reverse
391 392
Protein expression, purification and fluorescence polarization assay. E. Coli
393
transformed with a PICK1 encoding plasmid (pET41) (Madsen et al., 2005) were inoculated
394
overnight in 50 mL of LB media (+kanamycin), diluted into 1L LB media (+kanamycin) and
395
grown at 37oC to OD 0.6. Protein expression was induced with 0.5 mM IPTG and grown
396
overnight at 20oC. Cells were harvested and resuspended in lysis buffer containing 50 mM
397
Tris, 125 mM NaCl, 2 mM DTT (Sigma), 1% TritonX-100 (Sigma), 20 µg/mL DNAse 1 and
398
cOmplete protease inhibitor cocktail (Roche). Lysate was frozen at -80oC to induce cell lysis.
399
The bacterial suspension was thawed and cleared by centrifugation (F20 rotor, 36.000 x g
400
for 30 min at 4oC). Supernatant was incubated with Glutathione-Sepharose 4B beads
401
(GEHealthcare) for 2 hours at 4oC under gentle rotation. The beads were pelleted at 4.000 x
402
g for 5 min, and washed twice in 50 mM Tris, 125 mM NaCl, 2 mM DTT and 0.01% Triton-
403
X100. Beads were transferred to a PD10 gravity column and were washed 3 times. Protein
404
was separated from GST by Thrombin (Millipore) incubation overnight at 4oC with gentle
405
rotation. PICK1 was eluted on ice and absorption at 280 nm was measured on a TECAN
406
plate reader, followed by protein concentration calculations using lambert beers law (A =
14
407
εcl), εA280PICK1 = 32320(cm*mol/L)-1. A peptide of the 11 most C-terminal residues of
408
mouse TH (HTLTQALSAIS) was ordered from TAGcopenhagen A/S with > 95% purity. The
409
peptide was dissolved in 10% DMSO and wash buffer to a concentration of 2 mM, and a
410
final assay max concentration of 1 mM was used. The fluorescence polarization assay was
411
performed as previously described (Madsen et al., 2005; Erlendsson et al., 2014) with a
412
fixed concentration of PICK1 (1 µM, non-saturated) and fluorescent tracer (Oregon-Green
413
DATC13, 20 nM), pre-incubated for 15 min after which increasing concentrations of
414
unlabeled TH-C11 peptide were added and incubated for 20 min on ice in a black 190 µL
415
non-binding surface 96-well plate (Corning). Fluorescence polarization was measured on an
416
Omega POLARstar plate reader, 488 nm excitation and 535 nm emission.
417 418
Confocal microscopy. Confocal microscopy was performed using a Zeiss LSM 510
419
confocal laser-scanning microscope with an oil immersion 63×1.4 numerical aperture
420
objective (Carl Zeiss, Germany). Alexa Fluor 488 dye was excited with a 488 nm laserline
421
from an argon-krypton laser, and emitted light detected using a 505-530 nm bandpass filter.
422
Alexa Fluor 568 dye was excited with a 543nm helium-neon laser and fluorescence was
423
recorded using a 560 nm long-pass filter. Images were analysed with ImageJ (FIJI)
424
software.
425 426
Immunohistochemistry of perfused brain sections for confocal microscopy. Adult mice
427
were anaesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M PBS.
428
Coronal midbrain sections (40 m) were used for fluorescence immunohistochemistry.
429
Initially, antigen retrieval was performed by treatment with 10 mM sodium citrate buffer (pH
430
6.0) for 30 min at 80°C. Brain sections were then rinsed in PBS and preincubated in PBS
431
containing 5% goat serum, 1% bovine serum albumin and 0.3% Triton X-100 for 1 hour.
432
Sections were subsequently incubated with a custom generated mouse monoclonal PICK1
433
antibody (2G10; described in (Jansen et al., 2009), 1:1000) and rabbit polyclonal TH
15
434
(1:1000, OPA-04050, Affinity Bioreagents) at 4˚C overnight. Sections were rinsed in washing
435
buffer (0.25% BSA, 0.1% Triton X-100 in PBS) and then incubated with biotinylated goat
436
anti-mouse (1:400, E0433, DAKO Cytomation A/S) or Alexa-568 goat anti-rabbit IgG
437
(1:1000, A11036, Molecular Probes). Following rinsing, slices were incubated with avidin-
438
biotin-peroxidase complex (Vector laboratories, USA), rinsed and incubated with biotinyl
439
tyramide amplification reagent (Perkin Elmer) and incubated with streptavidin-conjugated
440
Alexa-488 (1:400, S11223, Thermo Fisher Scientific) to detect PICK1. Additional rinsing was
441
followed by mounting on SuperFrost slides (Menzel-Gläser, Braunschweig, Germany) and
442
cover-slipping using Prolong Gold antifade reagent (Molecular Probes, USA).
443 444
Culturing, transduction and immunocytochemistry of dopaminergic neurons for
445
confocal microscopy. Dopaminergic neurons were isolated from ventral midbrain tissue
446
dissected from 1-3 days old rat pups and plated on a monolayer of glia cells on coverslips
447
using a protocol modified from (Rayport et al., 1992) as described in (Eriksen et al., 2009).
448
For lentiviral transduction of the neurons, vectors were driven by the dual promoter FUGW
449
with the H1 promoter driving the expression of the small hairpin (ShPICK1) targeting the
450
PICK1 sequence (CTATGAGTACCGCCTTATCCT) and the ubiquitin promoter driving the
451
expression of GFP (PICK1 knockdown (KD)) (Citri et al., 2010). In the control vector (GFP),
452
the sequence of the ShPICK1 was removed before the PCR product of the deleted ShPICK1
453
fragment and the PICK1 KD vector was digested and ligated together using AfeI and BamHI.
454
Removal of ShPICK1 was achieved by synthesis of a 500 bp PCR fragment using the
455
primers
456
TCGCCGAGAAGGGACTACTTTTCCTCGCCTG and the original PICK1 KD construct as
457
template. Consequently, the control GFP vector only drives the expression of GFP under the
458
ubiquitin promoter. Verification of both constructs was performed by dideoxynucleotide
459
sequencing (Eurofins Genomics, Ebersberg, Germany). Transduction of dopaminergic
460
neurons was initiated the first day post plating dopaminergic neurons on the monolayer of
461
glia cells. Before transduction, half the medium was removed from each well and kept in
AGTAACGGATCCTTTTTCTAGCCCCAAGGGCG
and
16
462
incubator. Equal amounts of virus inducing expression of either PICK1 KD or GFP were
463
added below the medium surface in 2-3 spots. After 6-8 hours, the media was transferred
464
back to the dopaminergic neurons and 5-fluorodeoxyuridine was added to the medium to
465
inhibit cell growth. For immunocytochemistry, dopaminergic neurons were washed followed
466
by 15 min fixation in 4% paraformaldehyde. Cells were washed followed by 20 min in
467
permeabilization+blocking buffer (0.2% saponin + 5% goat serum in PBS). Neurons were
468
stained with a rabbit anti-TH antibody (1:1000, OPA1-04050, Thermo Fischer Scientific) and
469
a mouse anti-PICK1 antibody (2G10; described in (Jansen et al., 2009), 1:1000) for 1 hour
470
at RT. Cells were washed in blocking buffer and incubated for 45 min with Alexa Fluor 568
471
goat anti-rabbit and Alexa Fluor 647 goat anti-mouse (both 1:500, Life Technologies).
472 473
Statistical analysis. The dataset was examined for normality (Shapiro-Wilk and KS
474
normality test), outliers were removed by Grubbs outlier analysis, and all data are presented
475
as mean ± SEM. Significance level was set to p < 0.05. GraphPad Prism was used for data
476
analyses except for three-way ANOVAs, which were analyzed in SPSS. All statistical
477
analysis run, are reported in detail in “table of statistics”.
478
Data were compared between groups using unpaired t-tests when a Gaussian distribution
479
was observed. Otherwise, the unparametric Mann-Whitney test was used. When comparing
480
two normalized groups, with wildtype set to 1 (western blot), one-sample t test was used.
481
For cocaine administration without habituation of animals, as well as the self-administration
482
paradigm, we used a two-way ANOVA with genotype and dose as factors. F values are
483
reported as F (degrees of freedom between groups, degrees of freedom within groups).
484
Pairwise multiple comparison procedures were made using the Holm–Sidak method. For
485
drug-induced locomotion with habituation of animals as well as the sensitization paradigm,
486
we instead used a three-way ANOVA with genotype, injection (pre vs post drug) and dose
487
as factors.
488
17
489
Results
490
Attenuated locomotor response to cocaine in mice with deletion of PICK1
491
To investigate a possible role of PICK1 in mediating the behavioral actions of cocaine, we
492
assessed cocaine-induced hyperactivity in PICK1 KO mice and littermate wildtype controls
493
(Fig. 1). Mice were injected with 0, 5, 10 or 30 mg/kg cocaine and placed directly in activity
494
boxes for an hour. Analysis of their locomotion revealed an overall treatment effect of
495
cocaine with a significant difference between genotypes (FGENOTYPE(1,87) = 14.91, p = 0.0002,
496
FTREATMENT(3,87) = 17.5, p < 0.0001, FINTERACTION(3,87) = 1.88, p = 0.32 Fig. 1A). Further
497
statistical assessment of the genotype effect revealed a significantly attenuated cocaine-
498
induced behavioral response in the PICK1 KO mice after injection of both 10 and 30 mg/kg
499
cocaine (saline; t87 = 0.76, p = 0.45, 5mg/kg; t87 = 1.32, p = 0.342, 10mg/kg; t87 = 2.59, p =
500
0.033, 30mg/kg; t87 = 2.97, p = 0.015, Fig. 1A). Wildtype mice responded in a dose-
501
dependent manner to 5, 10 and 30 mg/kg cocaine i.p. by increased locomotor activity, while
502
KO mice showed a significantly attenuated locomotor response with treatment effect only at
503
the highest dose of cocaine (F(3,87) = 17.5, p < 0.0001, wildtype: 5 mg/kg; t87 = 0.93, p = 0.35,
504
10 mg/kg; t87 = 3.41, p = 0.002, 30 mg/kg; t87 = 6.15, p < 0.0001, KO: 5 mg/kg; t87 = 0.24, p =
505
0.81, 10 mg/kg; t87 = 1.28, p = 0.36, 30 mg/kg; t87 = 3.45, p = 0.003, Fig. 1A).
506
We then used an open field test to investigate cocaine-induced hyperactivity following a
507
120-min habituation period (Fig. 1B-D). Three-way ANOVA comparing injection (activity 60
508
min pre vs post drug injection), genotype (wildtype vs knockout) and treatment (0 vs 10 vs
509
30 mg/kg cocaine), revealed significant effects of genotype, treatment and injection (pre vs
510
post) (FGENOTYPE = 4.16, df = 1, p = 0.048, FTREATMENT = 14.24, p< 0.0001, df = 2, FINJECTION =
511
131.69, p < 0.0001, df = 1, FGENOTYPE*TREATMENT = 1.62, p = 0.21, df = 2, Fig. 1B) with post-hoc
512
analysis revealing a significant difference at 10 mg/kg cocaine (saline; t 8 = 0.36, p = 0.73,
513
10mg/kg; t17 = 2.87, p = 0.01, 30mg/kg; t16 = 0.82, p = 0.44, Fig. 1C) supporting the
514
observation that the attenuated cocaine-induced locomotor response in PICK1 KO mice is
515
present both with and without habituation (Fig. 1A).
18
516 517
Decreased number of self-administered cocaine reinforcements in PICK1 KO mice
518
We next assessed whether the lack of PICK1 affected long-term behavioral effects of
519
cocaine by testing PICK1 KO mice in a sensitization paradigm (Fig. 2). Mice were given
520
daily injections of 10 mg/kg cocaine for 6 days followed by cocaine challenges on day 12
521
and day 20 (Fig. 2A). Control groups were given saline on the 6 induction days and cocaine
522
on challenge days. Cocaine instigated increased cocaine-induced locomotor activity with
523
progressively increasing response to cocaine in both wildtype and PICK1 KO mice with
524
significant sensitization and no genotype difference (FDAY1vs6 = 4.61, p = 0.04, df = 1,
525
FDAY*GENOTYPE = 0.13, p = 0.72, df = 1, FDAY*TREATMENT = 28.73, p < 0.0001, df = 1,
526
FDAY*GENOTYPE*TREATMENT = 0.07, p = 0.79, df = 1, Fig. 2B). In addition, there was no genotype
527
difference in the maintenance of sensitization (FDAY6vs12vs20 = 5.13, p = 0.01, df = 2,
528
FDAY*GENOTYPE = 5.13, p = 0.46, df = 2, FDAY*TREATMENT = 5.32, p = 0.01, df = 2,
529
FDAY*GENOTYPE*TREATMENT = 0.35, p = 0.71, df = 2, Fig. 2B).
530
We further explored whether lack of PICK1 affected cocaine’s reinforcing properties by
531
testing PICK1 KO mice in an intravenous cocaine self-administration paradigm. PICK1 KO
532
mice learned to self-administer cocaine as fast as their wildtype counterparts (Table 1), i.e.
533
both genotypes acquired the task with similar number of reinforcers earned as well as days
534
spent to reach criteria for baseline, extinction, and re-baseline (Table 1). However, when
535
subsequently examining the dose-effect relationship (saline, 0.03, 0.1, 0.3, 1.0
536
mg/kg/infusion of cocaine), KO mice obtained significantly fewer cocaine reinforcements
537
(FINTERACTION(4,70) = 0.68, p = 0.61, FCOCAINE DOSE(4,70) = 5.28, p = 0.0009, FGENOTYPE(1,70) = 4.46,
538
p = 0.04, Fig. 2C) indicating an effect of lacking PICK1 on long-term effects of cocaine. A
539
group of naïve mice was used for the control experiment with liquid food self-administration.
540
While PICK1 KO mice were slightly slower to learn this task and earned fewer
541
reinforcements during this phase of training, no differences were seen between genotypes
542
during extinction and re-baseline criteria (Table 1), or during assessment of the dose-effect
543
relationship (FINTERACTION(4,85) = 0.30, p = 0.87, FFOOD
DOSE(4,85)
= 18.82, p
