Phosphorylation of GluA1-Ser831 by CaMKII Activation in the Caudate and Putamen Is Required for Behavioral Sensitization After Challenge Nicotine in Rats

Abstract Background Phosphorylation of the glutamate receptor (GluA1) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor plays a crucial role in behavioral sensitization after exposure to psychostimulants. The present study determined the potential role of serine 831 (Ser831) phosphorylation in the GluA1 subunit of the caudate and putamen (CPu) in behavioral sensitization after challenge nicotine. Methods Challenge nicotine (0.4 mg/kg) was administered subcutaneously (s.c.) after 7 days of repeated exposure to nicotine (0.4 mg/kg, s.c.) followed by 3 days of withdrawal in rats. Bilateral intra-CPu infusions of drugs were mainly performed to test this hypothesis. Results Challenge nicotine increased both phosphorylated (p)Ser831 immunoreactivity (IR) and pCa2+/calmodulin-dependentprotein kinases II (pCaMKII)-IR in the medium spiny neurons (MSNs) of the CPu. These increases were prevented by bilateral intra-CPu infusion of the metabotropic glutamate receptor 5 (mGluR5) antagonist MPEP (0.5 nmol/side) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 (2 nmol/side). However, the dopamine D1 receptor (D1R) antagonist SCH23390 (7.5 nmol/side) prevented only pSer831-IR alone. Bilateral intra-CPu infusion of the Tat-GluA1D peptide (25 pmol/side), which interferes with the binding of pCaMKII to GluA1-Ser831, decreased the challenge nicotine–induced increase in locomotor activity. Conclusions These findings suggest that the GluA1-Ser831 phosphorylation in the MSNs of the CPu is required for the challenge nicotine–induced behavioral sensitization in rats. CaMKII activation linked to mGluR5 and NMDA receptors, but not to D1R, is essential for inducing the CaMKII-Ser831 interaction.


Introduction
The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is an ionotropic glutamate receptor consisting of 4 subunits (GluA1-4) (Hollmann and Heinemann, 1994). Stimulation of the AMPA receptor in response to drug exposure upregulates fast ionic conductance. This alters the electrochemical environment of neurons, leading to psychomotor sensitization (Wolf, 1998;Cornish and Kalivas, 2000;Pierce and Wolf, 2013). Intracellular Ca 2+ mobilization This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. elevated by the stimulation of other glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptor and group I metabotropic glutamate receptor (mGluR1/5), after drug exposure further regulates the function of the AMPA receptor (Wang et al., 2005;Oh et al., 2013).
Activation of Ca 2+ -dependent kinases, such as Ca 2+ / calmodulin-dependent protein kinases II (CaMKII) and/or protein kinase C (PKC), which are coupled to glutamate receptors, phosphorylates serine residue at the position of 831 (Ser831) in the C-terminus of GluA1 subunit after cocaine exposure (Roche et al., 1996;Barria et al., 1997;Wang et al., 2006). However, activation of cAMP and/or protein kinase A (PKA), which are coupled to dopamine D1 receptor (D1R), phosphorylates Ser845 in the GluA1 subunit after cocaine exposure (Snyder et al., 2000;Surmeier et al., 2007;Oh et al., 2013). These findings suggest that the psychomotor function of the AMPA receptor is regulated by the phosphorylation of serine residues via activation of protein kinases coupled to glutamate and dopamine receptors in the brain after drug exposure.
Nicotine, a major psychoactive component of tobacco, produces physical and psychological dependence in mammals and human beings (Stolerman and Jarvis, 1995;Le Foll and Goldberg, 2006). Nicotine upregulates glutamate release by stimulating nicotinic acetylcholine receptors (nAChRs) in the terminals of the neurons in the caudate and putamen (CPu), a component of the basal ganglia involving habit learning such as cigarette smoking (Yin and Knowlton, 2006;Changeux, 2010;Ryu et al., 2017). It has been documented that stimulation of α7 nAChR, which is expressed mainly in the glutamate terminals of the CPu, enhances glutamate release after nicotine exposure (Engelman and MacDermott, 2004;Ryu et al., 2017). Challenge exposure to nicotine after withdrawal increases locomotor activity by stimulating α7 nAChR in the CPu of rats (Ryu et al., 2017(Ryu et al., , 2020, suggesting that elevation of glutamate release leads to behavioral sensitization. Previous studies have demonstrated that increased phosphorylation of Ser831 in the CPu after cocaine exposure causes the elevation of locomotor activity in rats (Martínez-Rivera et al., 2017;Wang et al., 2017;Yang et al., 2018). Taken together, Ser831 in the GluA1 of the AMPA receptor in the neurons of the CPu can be phosphorylated as a result of enhanced glutamate release in response to psychostimulants. However, it is not known how electrochemical changes in the neurons of the CPu lead to behavioral sensitization in response to challenge nicotine. Therefore, we investigated whether the interaction between CaMKII and Ser831 phosphorylation in the GluA1 of AMPA receptor in the CPu drives behavioral sensitization after challenge nicotine in rats. This study shows a novel finding that the binding of activated CaMKII to GluA1-Ser831 in the γ-aminobutyric acid (GABA) neurons of the CPu is responsible for behavioral sensitization in rats after challenge nicotine.

Animals
Sprague-Dawley male rats (6 weeks, n = 146) were obtained from Hyo-Chang Science Co. (Daegu, Korea). Rats were housed in pairs in a controlled environment with food and water available ad libitum. They were housed in a 12-hour-light/-dark cycle room (light on at 7 am) at a temperature and humidity of 21°C-23°C and 45%-55%, respectively. The rats were allowed to acclimate for a week, and the experiment started when they weighed 250-310 g. Experiments were performed within the light cycle period. On the day of the experiment, injections were given in home cages to minimize stress. All animal procedures were approved by the Institutional Animal Care and Use Committee of Pusan National University and conducted in accordance with the provisions of the NIH Guide for the Care and Use of Laboratory Animals.

Drugs and Peptides
Nicotine hydrogen tartrate salt was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in sterile 0.9% physiological saline (NaCl), and adjusted to pH 7.2-7.4 with sodium hydroxide. Nicotine was administered subcutaneously (s.c.) in a volume of 1 mL, and the concentration of nicotine (0.4 mg/kg/d) was determined in previous studies (Ryu et al., 2017. Rats were randomly divided into 2 groups and administered saline or nicotine for 7 consecutive days. After repeated injections followed by a 3-day withdrawal period, the rats received a challenge saline or nicotine injection the day after the withdrawal period. All drugs, except nicotine, were purchased from Tocris Bioscience (Bristol, UK), and solutions were freshly prepared before use. KN62 (CaMKII inhibitor), MPEP (mGluR5 antagonist), MK801 (NMDA receptor antagonist), and SCH23390 (D1R antagonist) were dissolved in the minimum concentration of dimethylsulfoxide and then diluted in artificial cerebrospinal fluid containing (mM) 123 NaCl, 0.86 CaCl 2 , 3.0 KCl, 0.89 MgCl 2 , 0.50 NaH 2 PO 4 , and 0.25 Na 2 HPO 4 aerated with 95% O 2 /5% CO 2 (pH 7.2-7.4) or NaCl. The same dimethylsulfoxide-artificial cerebrospinal fluid solution was used as a vehicle control. The concentrations of pharmacological drugs used in this study were determined from previous studies Lee et al., 2010;Oh et al., 2013). Two Tat-GluA1-derived peptides (Peptron, Daejeon, South Korea), Tat-GluA1 D and Tat-GluA1 A , contained the Ser831 site of GluA1 in which the Ser831 residue is modified to aspartate (Tat-GluA1 D , D-peptide) or alanine (Tat-GluA1 A , A-peptide). D-peptide was synthesized as a competitive inhibitor to compete with phosphorylated (p)Ser831 in the GluA1 subunit (Lee et al.,

Significance Statement
Challenge nicotine leads to behavioral sensitization by increasing glutamate release via stimulation of α7 nicotinic acetylcholine receptor (nAChR) in the caudate and putamen (CPu) of rats. However, it is not known how electrochemical changes in the neurons of the CPu result in behavioral sensitization after challenge nicotine. Phosphorylation of α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptor by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), which is linked to the stimulation of other glutamate receptors such as the N-methyl-D-aspartate (NMDA) receptor and metabotropic glutamate receptor 5 (mGluR5) in the medium spiny neurons of the CPu, is crucial for regulating behavioral sensitization after challenge nicotine. The present study unraveled that GluA1-Ser831 phosphorylation of AMPA receptor by activated CaMKII is required for behavioral sensitization after challenge nicotine. This finding enhances the understanding of neuroadaptation caused by interaction between CaMKII and AMPA receptor on challenge nicotine-induced behavioral sensitization of rats. 2013). A-peptide was used as a negative control as described previously (Lee et al., 2013). Both peptides containing the HIV-1 Tat N-terminus domain (YGRKKRRORRR) enhance cell permeability (Schwarze et al., 1999).

Surgery and Intra-CPu Drug Infusion
The rats were anesthetized with a mixture of Zoletil 50 (tiletamine and zolazepam, 75 μL/kg; Virbac Korea, Seoul) and Rompun (xylazine, 50 µL/kg; Bayer Korea, Seoul), then placed in a stereotaxic apparatus. Under aseptic conditions, a 23-gauge, stainless-steel, double-guide cannula (0.29-mm inner diameter, 13 mm long) was implanted 1 mm anterior to the bregma, 2.5 mm left/right of the midline, and 5 mm below the surface of the skull. The guide cannula was sealed with a stainless-steel wire of the same length. The rats were then allowed 7 days to recover from surgery prior to the experiment. On the day of the experiment, the inner steel wire was replaced with a 30-gauge stainless-steel injection cannula (0.15-mm inner diameter, 13.5 mm long) that protruded 0.5 mm from the guide cannula. Throughout the experiments, all drugs and peptides were infused bilaterally into the central part of the CPu 5 minutes prior to the final injection of saline or nicotine in a volume of 1 μL at a rate of 0.2 μL/min in freely moving rats. The progress of the injection was monitored by observing the movement of a small air bubble along the length of precalibrated PE-10 tubing inserted between the injection cannula and a 2.5-μL Hamilton microsyringe. After the infusion, the injector was left in place for an additional 5 minutes to reduce any possible backflow along the injection tract. The physical accuracy of the injection was verified by the reconstruction of microinjection placements.

Behavioral Assessments
Behavioral assessments were conducted as described previously (Ryu et al., 2017. Rats received a bilateral intra-CPu infusion of D-or A-peptide prior to the final injection of saline or nicotine. After the peptide infusion, locomotor activity was measured in an open field using an infrared photocell-based Opto-Varimex-4 Auto-Track (Columbus Instruments, Columbus, OH, USA) under sound-attenuated and illuminated conditions. Rats were placed in a standard transparent rectangular chamber (44.5 cm × 44.5 cm × 24 cm) and habituated for 30 minutes prior to the experiment to avoid environmental variations. Three pairs of sensors were positioned on orthogonal axes in the cage to provide the coordinates of rat movements in the locomotor testing chamber. Each pair of sensors produced 16 infrared light beams intersecting the animal cage (beam scan rate = 10 Hz). This Auto-Track system sensed the presence of animals by using data generated by infrared beam blocking. Locomotor activities were recorded for over 60 minutes after the final injection of saline or nicotine. The measurements were transferred to a computer using Opto-Varimex-4 Auto-Track Rapid Release software (v4.99B software, Columbus Instruments). All rats were anesthetized with a mixture of Zoletil 50 and Rompun and then brains were removed to identify the placements of guide cannula. One of 48 rats was excluded from the experiment due to health problems after surgery.

Statistical Analysis
Analysis was conducted using GraphPad Prism 6 software (GraphPad Software Incorporation, San Diego, CA, USA). Results are presented as means ± standard errors, and the statistical significance of differences was accepted for P < .05. Intergroup differences between numbers of immunoreactive pixels per measured area determined by western blotting and immunofluorescence were determined using the unpaired t test or 2-way ANOVA followed by Tukey's multiple comparisons test. Statistical significance of differences in total distance travelled between groups was determined by 2-way ANOVA or 2-way mixed ANOVA followed by Tukey's multiple comparisons test.

Challenge Nicotine Increased Phosphorylation of Ser831 and CaMKII in MSNs of CPu
The first set of experiments was conducted to determine whether challenge nicotine alters Ser831 phosphorylation in the GluA1 subunit of AMPA receptor in the CPu (Fig. 1A, B). Challenge nicotine increased both pSer831 and CaMKII-immunoreactivities (IR), but not pSer845, pPKC, and pPKA-IR compared with those of challenge saline groups (Fig. 1C, D: 1C, t(6) = 4.401, P = .0046, unpaired t test; 1D, t(6) = 4.182, P = .0058, unpaired t test). IR of all measured proteins from the acute nicotine exposure group and the challenge saline group did not show any difference (data not shown). In triple-immunofluorescence staining, challenge nicotine increased the number of neurons, which were co-localized with both pSer831 and pCaMKII-IR that were located mainly in the neurons of the CPu ( Fig. 2A, B: 2B, t(6) = 2.988, P = .0244, unpaired t test). Furthermore, >97% of neurons in the CPu were identified as MSNs (supplementary Figure). In addition, the co-localized puncta of pSer831 and pCaMKII-IR were increased within neuronal cell bodies and neuropils ( Fig.  2A, C: 2C, t(6) = 5.038, P = .0024, unpaired t test). Because challenge nicotine increased both pSer831 and pCaMKII-IR, the second set of experiments was conducted to determine whether pCaMKII is capable of phosphorylating Ser831 after challenge nicotine. Bilateral intra-CPu infusion of the selective CaMKII inhibitor KN62 (20 nmol/side) attenuated the challenge nicotine-induced increase in pSer831-IR ( Fig. 2D: interaction, F(1, 13) = 9.061, P = .0100; drug effect, F(1, 13) = 2.051, P = .1758; nicotine effect, F(1, 13) = 38.78, P < .0001, 2-way ANOVA). The physical accuracy of intra-CPu infusion of KN62 was confirmed by reconstructing microinjection placements (Fig. 2D). Western immunoblotting was conducted at least 3 times throughout the experiments.

Discussion
The present study shows that interaction between CaMKII and GluA1-Ser831 in the MSNs of the CPu is necessary for behavioral sensitization after challenge nicotine administration. In the present study, challenge nicotine increased the phosphorylation of Ser831 and CaMKII, but not Ser845, PKC, and PKA phosphorylation in the CPu of rats. Previous studies documented that nicotine upregulates glutamate release in the in vivo recordings of the CPu, resulting in the stimulation of glutamate receptors (Howe et al., 2016;Ryu et al., 2017). Blockade of NMDA receptor and mGluR5 decreased the challenge or chronic nicotine-induced increase in behavioral sensitization in rats (Shoaib et al., 1997;Shim et al., 2002;Paterson et al., 2003). Stimulation of NMDA receptor and mGluR5 increases intracellular Ca 2+ mobilization either by itself or by interacting with them (Kotecha et al., 2003). This increase in turn activates the Ca 2+ -dependent protein kinases, CaMKII and/or PKC, leading to phosphorylation of GluA1-Ser831 (Kim et al., 2009). Parallel to glutamate release, nicotine upregulates dopamine release, stimulating D1R in the CPu of rats (Shim et al., 2001;Janhunen et al., 2005). Activation of cAMP and/or PKA, which are coupled to D1R, preferentially phosphorylate Ser845 rather than Ser831 in the GluA1 (Snyder et al., 2000;Ahn and Choe, 2009). Taken together, these findings suggest that challenge exposure to nicotine phosphorylates GluA1-Ser831 by activating CaMKII rather than PKC, which is linked to the NMDA receptor and mGluR5 in the CPu.
The present data obtained from triple immunofluorescence staining show that phosphorylation of Ser831 and CaMKII is co-localized in the neurons of the CPu. Furthermore, the co-localized cluster of phosphorylated Ser831 and CaMKII is observed in cell bodies as well as dendrites. It is well-known that medium spiny GABA neurons, which project to the globus pallidus, constitute >90% of the CPu (Kemp and Powell, 1971;Märtin et al., 2019). Consistently, the present study also demonstrates that >97% of neurons in the CPu are overlapped with the MSN marker DARPP-32. These findings support the fact that challenge nicotine phosphorylates GluA1-Ser831 by activating CaMKII in the GABA neurons of the CPu. This notion can be supported by the present finding that inhibition of CaMKII partially reduces the challenge nicotine-induced increase in Ser831 phosphorylation in the MSNs of the CPu.
The present data show that blockade of mGluR5, a dominant group I mGluR subtype in the CPu, reduces the challenge nicotine-induced increase in the phosphorylation of Ser831 and CaMKII. This finding suggests that stimulation of mGluR5 after challenge nicotine phosphorylates Ser831 by activating CaMKII. It is well-documented that stimulation of phospholipase C (PLC) coupled to mGluR5 upregulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ) (Nishizuka, 1984;Abe et al., 1992). Activation of DAG in turn activates PKC, which leads to Ca 2+ influx by phosphorylating NMDA receptor and/or autophosphorylating CaMKII (Tingley et al., 1997;Liao et al., 2001;Bayer et al., 2001;Giese, 2021). In parallel with PKC activity, elevation of IP 3 further increases intracellular Ca 2+ release from the endoplasmic reticulum, resulting in CaMKII activation (Berridge and Irvine, 1984;Yang et al., 2021). In this study, blockade of ionotropic NMDA glutamate receptor reduced the phosphorylation of Ser831 and CaMKII, which were elevated by challenge exposure to nicotine. Taken together, these findings suggest that elevation of Ca 2+ mobilization, resulting from the stimulation of NMDA receptor and mGluR5, or possibly by an interaction between these 2 receptors, activates CaMKII and phosphorylates GluA1-Ser831 in the MSNs of the CPu. The present data also show that blockade of D1R decreased the challenge nicotine-induced increase in Ser831 phosphorylation, but not CaMKII phosphorylation. Stimulation of D1R after challenge nicotine also contributes to Ser831 phosphorylation by phosphorylating NMDA receptors (Dudman et al., 2003;Chen et al., 2004). Taken together, the present finding suggests that activation of CaMKII due to glutamate receptor stimulation after challenge nicotine is crucial for GluA1-Ser831 phosphorylation in the MSNs of the CPu. D-peptide that is designed to interfere with the binding of activated CaMKII to Ser831 attenuated the challenge nicotineinduced increase in locomotor activity in rats in this study. Previous studies have demonstrated that Ser831 phosphorylation is necessary for cocaine-induced locomotor sensitization in rats (Martínez-Rivera et al., 2017;Yang et al., 2018). Prevention of Ser831 phosphorylation by inhibiting CaMKII decreases the α7 nAChR-stimulated upregulation of AMPA currents in the rat prefrontal cortex (Tang et al., 2015). Taken together, the present findings suggest that the interaction between activated CaMKII and Ser831 is necessary for behavioral sensitization after challenge nicotine in rats.
In summary, as proposed in Figure 5, increased glutamate release after challenge nicotine stimulates mGluR5 and NMDA receptors in the CPu, which leads to CaMKII activation by enhancing intracellular Ca 2+ mobilization. Stimulation of D1R after challenge nicotine also results in the phosphorylation of Ser831 without activating CaMKII. Activated CaMKII in the MSNs of the CPu binds directly to Ser831 in the C-terminus of GluA1, leading to behavioral sensitization. Therefore, the phosphorylation of GluA1-Ser831 by activated CaMKII in the MSNs of the CPu may be necessary for behavioral sensitization in rats after challenge exposure to nicotine. Under these circumstances, stimulation of mGluR5 and NMDA receptor, but not D1R, is linked to CaMKII activation.