8 cyclopentyl 1 3 dipropylxanthine dpcpx

The migration of unstimulated neutrophils or monocyte and chemotaxis of cells were measured as previously reported (5 (link)). Briefly, advanced RPMI (Thermo Fisher, Cat#12633–012) with the chemoattractants CXCL1 (BioLegend Cat#573702), CCL2 (R&D Systems, Cat#479-JE), or N-Formylmethionyl-leucyl-phenylalanine (fMLP; Sigma-Aldrich Cat#F3506) was placed in the bottom of the 3-μm-pore Transwell system (Neuro Probe Cat#101–3). Neutrophils were incubated with PSB12379 (Tocris) or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (Sigma-Aldrich, Cat#C101) in advanced RPMI for 10 min before being plated on top of the Transwell (0.1×10⁶ cells) and were incubated at 37°C, 5% CO₂ for 1 h. 10μl medium was taken from the bottom wells and cells were counted by a hemocytometer. The quantification of migrated neutrophils was done with the formula N=n×10⁴×0.029 (reflective of 29μl medium in the bottom well).

All components of the phosphate buffered saline (PBS) solution (in mM: 3.0 KCl, 10.0 NaH₂PO₄, 2.0 Na₂SO₄, 1.2 MgCl₂, 131.25 NaCl and 1.2 CaCl₂, with pH adjusted to 7.4) were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and used for electrode calibration. Adenosine was purchased from Sigma-Aldrich (Milwaukee, WI, USA). A 10.0 mM stock solution of Adenosine was prepared in 0.1 M perchloric acid (HClO₄) and stored in the refrigerator. A 1.0 μM Adenosine solution was made in PBS on the day of use. All aqueous solutions were prepared using deionized water (Milli-Q Biocel; Millipore, Billerica, MA, USA).
8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX, 6 mg/kg, Sigma-Aldrich) and 2-(2- Furanyl)-7-[3-(4-methoxyphenyl) propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5- amine (SCH442416, 3 mg/kg, Tocris, UK) were dissolved in dimethyl sulfoxide (DMSO, Amresco, Solon, OH, USA), whereas Nω-Nitro-L-arginine methyl ester hydrochloride ( L-NAME, 100 mg/kg, Sigma-Aldrich) was dissolved in 1 mL heated saline and all drugs were administered intraperitoneally (i.p). These doses were selected based on previous experiments that were used in the literature (Orrú et al.; Venton et al., 2003 (link); Nguyen et al., 2014 ).

All chemicals were from Fisher Scientific (Fair Lawn, NJ, USA) unless otherwise stated. Adenosine and dopamine were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in 0.1 M HClO₄ for 10 mM stock solutions. Stock solutions were diluted daily in artificial cerebral spinal fluid (aCSF) to 1 µM for electrode calibrations for brain slice experiments. The aCSF consisted of 126 mM NaCl, 2.5 mMKCl, 1.2 mM NaH₂PO₄, 2.4 mM CaCl₂ dehydrate, 1.2 mM MgCl₂ hexahydrate, 25 mM NaHCO₃, 11 mM glucose, and 15 mM tris (hydroxymethyl) aminomethane and was adjusted to pH 7.4 every day. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) (Sigma) and SCH 442416 (Tocris Biosciences, Ellisville, MO) stock solutions were made in dimethylsulfoxide (DMSO) and diluted in aCSF on the day of the experiment.

The adenosine A₁ receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A_2A receptor antagonist 3,7-Dihydro-8-[(1E)-2-(3-Methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)propyl-1-(2-propynyl)-1H-purine-2,6-dione disodium salt hydrate (MSX-3) were both obtained from Sigma Aldrich (St. Louis, MO). Vehicle for DPCPX was a maximal concentration of 30% (v/v) 0.1 M NaOH in sterile physiological saline for the highest administered dose (6 mg/kg). For MSX-3, the maximal concentration of NaOH was 16% (v/v) for the highest dose administered (4 mg/kg). The vehicle control groups had the respective maximal concentration of NaOH in saline to control for any effects the highest concentration of NaOH may have had on drinking or locomotor activity. DPCPX is 1,000-fold more selective for the A₁ receptor over the A_2A receptor (Fredholm and Lindström, 1999 (link)). MSX-3 is a prodrug for MSX-2, which has demonstrated 100-fold selectivity for A_2A over A₁ in both rat and human tissue, with no activity at A_2B or A₃ subtypes (Sauer et al., 2000 (link), Solinas et al., 2005 (link)). For drinking solutions, ethanol (190 proof; Pharmco Inc., Brookfield, CT) was diluted to 20% (v/v) in tap water and sucrose (Sigma-Aldrich, St. Louis, MO) was dissolved in tap water to 2% (w/v).