Roles of adenosine A1 receptors in the regulation of SFK activity in the rat forebrain

Abstract Adenosine A1 receptors are widely expressed in the mammalian brain. Through interacting with Gαi/o‐coupled A1 receptors, the neuromodulator adenosine modulates a variety of cellular and synaptic activities. To determine the linkage from A1 receptors to a key intracellular signaling pathway, we investigated the impact of blocking A1 receptors on a subfamily of nonreceptor tyrosine kinases, that is, the Src family kinase (SFK), in different rat brain regions in vivo. We found that pharmacological blockade of A1 receptors by a single systemic injection of the A1 selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) induced an increase in autophosphorylation of SFKs at a consensus activation site, tyrosine 416 (Y416), in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. DPCPX also increased SFK Y416 phosphorylation in the medial prefrontal cortex (mPFC) but not the hippocampus. The DPCPX‐induced Y416 phosphorylation was time dependent and reversible. In immunopurified Fyn and Src proteins from the striatum, DPCPX elevated SFK Y416 phosphorylation and tyrosine kinase activity in Fyn but not in Src proteins. In the mPFC, DPCPX enhanced Y416 phosphorylation and tyrosine kinase activity in both Fyn and Src immunoprecipitates. DPCPX had no effect on expression of total Fyn and Src proteins in the striatum, mPFC, and hippocampus. These results demonstrate a tonic inhibitory linkage from A1 receptors to SFKs in the striatum and mPFC. Blocking this inhibitory tone could significantly enhance constitutive SFK Y416 phosphorylation in the rat brain in a region‐ and time‐dependent manner.

intracellular signaling pathway in the rat forebrain in vivo under normal conditions. Adenosine receptors are a class of purinergic G protein-coupled receptors and are activated by an endogenous neuromodulator ligand, that is, a ubiquitous purine nucleoside adenosine (Fredholm et al., 2001;Sheth et al., 2014). Four subtypes of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been identified to date (Fredholm, 2011). Each of these receptors is encoded by a separate gene and plays either different or some overlapping roles. At the G protein level, adenosine A 1 and A 3 receptors are coupled to G αi/o proteins, whereas adenosine A 2A and A 2B receptors are coupled to G αs/olf proteins. As a result, activation of A 1 and A 3 receptors inhibits adenylyl cyclase and thereby reduces cytoplasmic cAMP levels and protein kinase A (PKA) activity, whereas activation of A 2A and A 2B receptors produces the opposite effect (Fredholm et al., 2001;Kull et al., 2000;Sheth et al., 2014). In addition to the canonical cAMP pathway, adenosine receptors may trigger other signaling pathways to achieve their regulatory roles in a variety of cellular and synaptic activities Fredholm et al., 2001;Schulte & Fredholm, 2003;Sheth et al., 2014).
Among four subtypes of adenosine receptors, A 1 and A 2A receptors are principally expressed in the brain (Fredholm et al., 2005). Especially, A 1 receptors, the most conserved subtype among species , are distributed throughout the body with the highest level in the brain. A wide range of brain regions express A 1 receptors, including the cortex, hippocampus, cerebellum and striatum (Alexander & Reddington, 1989; J. F. Chen et al., 2013;Mahan et al., 1991), indicating their roles in the large range of physiological events (Kashfi et al., 2017;Sawynok, 2016;Varani et al., 2017. Within the striatum, A 1 receptors are expressed in a specific subpopulation of medium spiny projection neurons, that is, striatonigral neurons  in which dopamine D 1 receptors are co-expressed (Aubert et al., 2000;Bertran-Gonzalez et al., 2010;Gerfen et al., 1990). In fact, behavioral and biochemical studies revealed a postsynaptic A 1 -D 1 receptorreceptor interaction in the rat striatum (Ferre et al., 1994). The two receptors (A 1 and D 1 ) coimmunoprecipitated in co-transfected fibroblast cells (Gines et al., 2000) and nucleus accumbens (NAc) neurons (Toda et al., 2003). Given that D 1 receptors are coupled to G αs/olf proteins and thus stimulate the cAMP pathway when activated (Neve et al., 2004), A 1 receptors are thought to antagonistically modulate striatal D 1 signaling and fine-tune the outflow of the D 1 -mediated direct pathway in the basal ganglia (reviewed in Fuxe et al., 2007Fuxe et al., , 2010. The Src family kinase (SFK) is a family of nonreceptor tyrosine kinases (Neet & Hunter, 1996). Five out of nine SFK members (Src, Fyn, Lyn, Yes, and Lck) are expressed in the brain (S. Chen et al., 1996;Omri et al., 1996;Kalia et al., 2004). Among them, Src and Fyn are key members of SFKs and are enriched at synaptic sites (Ohnishi et al., 2011). Accumulating evidence consistently shows that Src and Fyn, by phosphorylating specific tyrosine sites on their substrates, regulate various neural and synaptic activities (Kalia et al., 2004;Ohnishi et al., 2011;Schenone et al., 2011). Meanwhile, Src and Fyn are among postreceptor kinases that are sensitive to changing cellular and synaptic input. By upregulating phosphorylation of Src and Fyn at an autophosphorylation site, tyrosine 416 (Y416), Src and Fyn kinase activity is enhanced drastically (Roskoski, 2005;Okada, 2012). However, whether Src and/or Fyn in striatal neurons are sensitive to A 1 receptor signaling remains elusive to date.
We thus initiated the present study to investigate the possible linkage between A 1 receptors and SFKs. To this end, we examined and characterized the effect of pharmacological blockade of A 1 receptors by a systemic injection of an A 1 selective antagonist on SFK Y416 phosphorylation, that is, activation of SFKs, in the two subdivisions of the rat striatum, the caudate putamen (CPu) and NAc. Other brain regions were also surveyed, which include the medial prefrontal cortex (mPFC) and hippocampus.

Animals
The study was not pre-registered.

Experimental arrangements
We randomly divided rats into different groups using a computergenerated randomization Two groups of rats were used for each time point (n = 4-5 per group).
Changes in Y416 phosphorylation were analyzed in the CPu, NAc, and mPFC. Third, we attempted to identify the members of SFKs that were regulated by A 1 receptor signaling in two groups of rats (n = 4 per group). To this end, we analyzed changes in Y416 phosphorylation in immunopurified Fyn and Src proteins from the striatum and mPFC after vehicle or DPCPX administration (2.5 mg/kg, i.p.; 20-30 min).
Finally, tyrosine kinase activity of immunopurified Fyn and Src was assayed in two groups of rats (n = 5 per group). Changes in Fyn and Src kinase activity in response to vehicle or DPCPX (2.5 mg/kg, i.p.; 20-30 min) were monitored in the rat striatum and mPFC. Doses of DPCPX (0.25 and 2.5 mg/kg) were chosen based on previous findings that an i.p. injection of DPCPX at 3 mg/kg blocked the anxiolytic-like effect induced by the A 1 selective agonist and positive allosteric modulator (Prediger et al., 2006;Vincenzi et al., 2016).

Western blot
Rats were anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and decapitated. Brains were removed and coronal slices were cut. The brain regions of interest (CPu, NAc, and hippocampus) were dissected.
In addition, the mPFC was dissected, which includes the anterior cingulate, prelimbic, and infralimbic cortex (Heidbreder & Groenewegen, 2003 were used for immunoblots. Western blot was performed as described previously (Mao et al., 2013;Mao & Wang, 2015). Briefly, proteins were separated on NuPAGE Novex 4%-12% gels (Invitrogen, Carlsbad, CA). Separated proteins were transferred to membranes (polyvinylidene fluoride) which were blocked and incubated with a primary antibody overnight at 4 • C. After membranes were incubated with a secondary antibody linked to horseradish peroxidase, immunoblots were visualized using an enhanced chemiluminescence reagent.
Optical density of immunoblots was measured using analysis software (NIH ImageJ). Values were normalized to β-actin.

Immunoprecipitation
Immunoprecipitation was performed as described previously (Mao & Wang, 2016). Briefly, brain tissue was lysed in a RIPA buffer. Lysates were centrifuged at 800 g (10 min, 4 • C). We then collected supernatants for immunoprecipitation. Supernatant proteins (300-500 μg) were incubated in a solution containing a mouse antibody against Fyn or Src (2-3 μg). Immunocomplexes were precipitated with 50% protein A/G agarose/sepharose bead slurry (GE Healthcare). Precipitated proteins were analyzed by immunoblots with a rabbit antibody indicated.

SFK kinase activity assay
A Takara Universal Tyrosine Kinase Assay Kit (Clontech Laboratory, Inc., Mountain View, CA) was used to assess SFK kinase activity as described previously (D. Z. Liu et al., 2014;Mao et al., 2018). Briefly, we used a mouse antibody against Fyn or Src to immunoprecipitate a protein of interest from striatal or mPFC homogenates. Immunopurified Fyn or Src proteins and ATP were added to microplate wells that were covered with an immobilized tyrosine kinase substrate poly (Glu-Tyr) at 37 • C for 30 min. Wells were washed and blocked.
A horseradish peroxidase-conjugated anti-phosphotyrosine antibody was added into wells, followed by measurement of phosphorylated substrates in absorbance at 450 nm.

Antibodies and pharmacological agents
Several primary antibodies were used in this study (Table 1) hydroxide (Uzbay et al., 2007). DPCPX was freshly prepared at the day of experiments.

Statistics
After the normality of data was tested, we utilized GraphPad Prism 6 (RRID:SCR_002798; GraphPad software, La Jolla, CA) to analyze data.
We used a two-tailed unpaired Student's t-test or one-or two-way analysis of variance (ANOVA) with a multiple comparison post hoc test.
The level of statistical significance was determined by a P value less than 0.05. Test for outliers was not performed. No rats were excluded from the analysis.

Effects of DPCPX on SFK Y416 phosphorylation
We first examined whether blocking A 1 receptors with DPCPX has any impact on SFK Y416 phosphorylation. To this end, we subjected rats to a single i.p. injection of vehicle or DPCPX at either 0.25 or 2.5 mg/kg. Rats were then sacrificed 20 min after DPCPX injection.
Changes in Y416 phosphorylation were analyzed in different brain regions using western blots. We found that DPCPX at 0.25 mg/kg did not alter pY416 levels in the CPu ( Figure 1A). Remarkably, DPCPX at 2.5 mg/kg markedly increased pY416 levels in the region. Similarly,
DPCPX altered Y416 phosphorylation in the NAc. While DPCPX at 0.25 mg/kg had no effect, DPCPX at 2.5 mg/kg elevated pY416 levels in the NAc ( Figure 1B). At either dose, DPCPX did not affect cellular levels of total Fyn and Src proteins in the CPu and NAc. These data indicate that pharmacological blockade of A 1 receptors with a selective antagonist upregulates SFK Y416 phosphorylation in the CPu and NAc.
In addition to the CPu and NAc, we assessed responses of SFK Y416 to DPCPX in the mPFC and hippocampus. As shown in Figure 2A,

Time-dependent effects of DPCPX on SFK Y416 phosphorylation
We next attempted to determine the temporal profile of the effect of DPCPX on SFK Y416 phosphorylation. Rats received an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed at three different time points (20 min, 1 h, and 3 h) after drug injection. Changes in pY416 levels in three brain regions (CPu, NAc, and mPFC) were assayed using western blots. In the CPu, DPCPX induced a significant increase in pY416 levels at an early time point (20 min, Figure 3A Figure 3D for Src). These results indicate that DPCPX induced a rapid and reversible increase in SFK Y416 phosphorylation in the CPu.
Similar results were observed in the NAc. An increase in pY416 levels was seen in this region at 20 min and 1 h after DPCPX injection ( Figure 4A and B). The increase declined to a level insignificantly different from the vehicle control at 3 h. No change in expression of total Fyn and Src in the NAc was seen at three time points ( Figure 4C for Fyn and Figure 4D for Src). In the mPFC, we observed a marked increase in pY416 levels 20 min after DPCPX injection ( Figure 5A and B). The increase persisted at 1 h in DPCPX-treated rats compared to vehicle-treated rats. At 3 h, pY416 levels were not significantly different between two groups. Fyn and Src expression was not altered following DPCPX administration ( Figure 5C and D). Thus, like the CPu, the NAc and mPFC exhibit a similar dynamic pattern of SKF Y416 phosphorylation in response to DPCPX.

Effects of DPCPX on Y416 phosphorylation in immunopurified SFK members
To identify the member(s) of SFKs that respond to the A 1 receptor antagonist, we combined western blots with immunoprecipitation.
After an i.p. injection of DPCPX (2.5 mg/kg), rats were sacrificed 20-30 min after drug injection. Fyn and Src proteins were immunoprecipitated from striatal and mPFC lysates. Phosphorylation at Y416 was then analyzed in Fyn and Src immunoprecipitates using western blots.
In the striatum containing the CPu and NAc, a significant increase in pY416 levels was shown in immunopurified Fyn proteins in DPCPXtreated rats compared to vehicle-treated rats ( Figure 6A). The pY416 signal in Src immunoprecipitates showed a tendency of an increase after DPCPX administration, although it did not reach a statistically significant level ( Figure 6B). In the mPFC, we observed a significant increase in pY416 levels in both Fyn and Src precipitates after DPCPX administration ( Figure 6C and D). These results indicate that DPCPX upregulates Y416 phosphorylation of Fyn rather than Src proteins in

Effects of DPCPX on Fyn and Src tyrosine kinase activity
Given an elevated response of Y416 phosphorylation to DPCPX, SFK tyrosine kinase activity is reasoned to be increased by the antagonist.
To determine this, rats were treated with DPCPX (2.5 mg/kg, i.p.; 20-30 min). They were then sacrificed and striatal and mPFC lysates were prepared. Fyn and Src proteins were immunoprecipitated from these lysates. Kinase activity levels of immunopurified Fyn and Src were assayed. As shown in Figure 7A, DPCPX elevated Fyn kinase activity in the striatum, while DPCPX did not significantly alter Src kinase activity in the region ( Figure 7B). In the mPFC, DPCPX increased kinase activity of Fyn and Src precipitates in this region ( Figure 7C and D).
Thus, DPCPX possesses the ability to upregulate Fyn although not Src kinase activity in the striatum and both Fyn and Src kinase activity in the mPFC.

DISCUSSION
This study investigated the role of adenosine A 1 receptors in the regulation of SFKs in the rat brain in vivo. We found that a systemic injection of the A 1 antagonist DPCPX at a higher dose (2.5 mg/kg) but not a lower dose (0.25 mg/kg) induced a substantial increase in the autophosphorylation of SFKs at Y416 in the two subdivisions of the striatum (CPu and NAc). DPCPX also increased Y416 phosphorylation in the mPFC but not the hippocampus. The increase in Y416 phosphorylation was rapid and reversible as revealed in a time course study.
The DPCPX-elevated Y416 phosphorylation occurred in immunopuri- early study shows that a single dose of DPCPX and the nonselective adenosine receptor antagonist caffeine increased expression of the immediate early genes (c-fos, zif/268, and arc) in the striatum at both mRNA and protein levels (Dassesse et al., 1999). DPCPX after an i.p. or intrastriatal injection enhanced damage to striatal neurons induced by a mitochondrial complex II inhibitor malonate (Alfinito et al., 2003). An A 1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine significantly altered Thr34 phosphorylation of dopamine-and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) in the striatum (Yabuuchi et al., 2006), whereas A 2A receptors were also essential for the regulation of striatal DARPP-32 phosphorylation (Shen et al., 2013).
In addition, sleep deprivation decreased PKA phosphorylation in the striatum and hippocampus and impaired memory (Oliveira et al., 2019). DPCPX prevented the decrease in PKA phosphorylation in the striatum but not hippocampus and reversed the memory impairment.
In this study, we found that the A 1 antagonist DPCPX upregulated   (Fuxe et al., , 2010 Early studies have established a positive linkage from the cAMP/PKA pathway to SFK/Fyn activity (Schmitt & Stork, 2002 ;Yang et al., 2011;Yeo et al., 2011), which supports the notion that A 1 receptors regulate Fyn activity via a signaling pathway involving cAMP and PKA. Alternatively, A 1 receptors have been found to reside on incoming dopaminergic terminals within the striatum (Borycz et al., 2007). These presynaptic A 1 heteroreceptors act to inhibit dopamine release (Borycz et al., 2007;O'Neill et al., 2007;Wood et al., 1989;Yabuuchi et al., 2006).
Thus, DPCPX could block the presynaptic A 1 receptor-mediated inhibition of dopamine release and induce an increase in synaptic dopamine levels. Released dopamine could in turn activate D 1 receptors specif-ically expressed in striatonigral neurons (Aubert et al., 2000;Bertran-Gonzalez et al., 2010;Gerfen et al., 1990) to upregulate Fyn phosphorylation in these neurons. Previous work has consistently demonstrated phosphorylation of Fyn in the rat striatum in response to pharmacological activation of G αs/olf -coupled D 1 receptors (Mao & Wang, 2015 ;Mao et al., 2018). In addition to dopamine, presynaptic A 1 heteroreceptors exert a tonic inhibition of glutamate release in the rat NAc (Quarta et al., 2004). Moreover, presynaptic A 1 and A 2A receptors form heteromers in striatal glutamatergic nerve terminals (Ciruela et al., 2006;Ferré et al., 2007). This heteromerization provides a concentrationdependent switch. Namely, low (under basal conditions) and high con-  kinase activity in the mPFC. Note that DPCPX increased Fyn but not Src kinase activity in the striatum and both Fyn and Src kinase activity in the mPFC. Rats were given an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed 20-30 min after drug injection. Tyrosine kinase activity levels were measured in immunopurified Fyn and Src proteins from the striatum and mPFC. Data are presented as means ± standard error of the mean (n = 5 per group) with 'n' equal to the number of animals and were analyzed with unpaired Student's t-test: striatum/Fyn: t (8) = 3.473, n = 10, p = 0.008; striatum/Src: t (8) = 0.722, n = 10, p = .491; mPFC/Fyn: t (8) = 2.536, n = 10, p = .035; and mPFC/Src: t (8) = 3.119, n = 10, p = .014. *p < .05 versus vehicle.
A 1 receptor agent injected i.p. could affect central Fyn phosphorylation indirectly via its effect on peripheral cardiovascular activities.
The expression level of Fyn is much greater than Src in the striatum (Pascoli et al., 2011). In the striatum, we found that DPCPX selectively affected Fyn. Unlike the striatum, DPCPX also increased phosphorylation of Src in addition to Fyn in the mPFC, indicating that A 1 receptors are negatively coupled to Src in the mPFC. Of note, in contrast to this negative connection, A 1 receptors could positively modulate Src in different models. For instance, an A 1 receptor agonist activated Src to form a signaling pathway linking A 1 receptors to activation of nuclear factor kappaB in HEK293 cells (A. M. F. Liu & Wong, 2004 ). An A 1 receptor antagonist CPT reduced Src Y416 phosphorylation in acutely prepared mouse cortical slices containing somatosensory cortex (Deng et al., 2011).
Our studies on SFK phosphorylation were conducted in male rats.
While sex differences in the role of A 1 receptors in SFK phosphorylation are poorly understood at present, it is imperative to investigate the response of SFK phosphorylation to adenosine receptor agents in female animals in future studies.

ACKNOWLEDGMENTS
This work was supported by an NIH grant R01MH61469 (JQW). We thank Daozhong Jin and Nan He for their assistance.

CONFLICT OF INTEREST
The authors declare that there is no potential conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.