Atomoxetine Increases Cortical Catecholamine Levels
MATERIALS AND METHODSTransporter Binding Studies
Membranes from HEK 293, MDCK, and HEK293 celllines transfected with human 5-HT, NE and DA trans-porters, respectively, were obtained from Receptor Biol-ogy, Inc. (Beltsville, MD). All assays were performed intriplicate in a final volume of 0.8 ml containing either a buffer consisting of 50 mM Tris Cl, pH 7.4, 150 mMNaCl, and 5 mM KCl for 5-HT and NE transporters or 50mM Tris Cl, pH 7.4, and 100 mM NaCl for the DA trans-porter. The radioligands for 5-HT, NE and DA humantransporters were [
H]-paroxetine (0.2 nM, 25 Ci/mmol,Dupont NEN products, Boston, MA), [
H]-nisoxetine(1.0 nM, 86 Ci/mmol, New England Nuclear) and[
H]-WIN35,428 (1.0 nM, 86 Ci/mmol, New EnglandNuclear), respectively. Membranes equivalent to proteinin amounts of 10.3
g or 6.2
g, respectively,were used in the assays. After incubation at 37
C for 40min for the 5-HT transporter and 25
C for 30 min for NEand DA transporters, the binding was terminated byrapid vacuum filtration over Whatman GF/B filters (pre-soaked in 0.5% polyethylenimine) and the filters werewashed four times with cold 50 mM Tris Cl buffer, pH7.4. The filters were then placed in vials containing liquidscintillation fluid and radioactivity was measured by liq-uid scintillation spectrometry. Non-specific binding wasdetermined in separate samples with 1
M desipramine or 10
M nomifensine, for 5-HT, NEand DA transporters, respectively.
Determination of Binding to Neuronal Receptors
Inhibition of binding to neuronal receptors was pro-vided by NovaScreen (Hanover, MD) using proprietaryreceptor binding assays.
Blockade of p-Chloramphetamine and DSP-4 Effects
The 5-HT selective neurotoxin p-chloramphetamine hy-drochloride (p-CA) was dissolved in sterile water for in-traperitoneal (i.p.) administration. Male Sprague-Daw-ley rats (Harlan Industries, Indianapolis, IN) in groupsof five weighing 140–170 grams were injected withp-CA (10 mg/kg i.p.) 2 h before cervical dislocation. Ve-hicle or test drugs were dissolved in sterile water andinjected 1 h prior to p-CA. The brains were quicklyremoved, frozen on dry ice and stored at
C untilassayed. Whole brain 5-HT concentrations were mea-sured using high pressure liquid chromatography withelectrochemical detection (HPLC-EC) as previously de-scribed (Fuller and Perry 1989).DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyla-mine hydrochloride), a noradrenergic neurotoxin (Jonssonet al. 1981; Grzanna et al. 1989), was dissolved in 0.01 NHCl and injected at 30 mg/kg i.p. one hour after adminis-tration of drugs. Cortical NE concentrations were mea-sured 6 h after administration of DSP-4 according to amodification of the method of Fuller and Perry (1977).Cortical tissue was homogenized in 2 ml 0.1 N trichloro-acetic acid with 3,4-dihydrobenzylamine hydrobromideadded as an internal standard. After centrifugation, 1 mlof the supernatant was added to 150 mg alumina, and thepH adjusted to 8.6 with 0.5 M Tris containing 0.1 M EDTA.Samples were mixed for 10 min, centrifuged and the su-pernatant decanted. The alumina was washed with 1ml 50 mM Tris/10 mM EDTA. One ml of 0.1 N formicacid was added to the alumina, and the samples weremixed for 10 min and centrifuged. Cortical NE wasmeasured using a HPLC-EC technique by injecting a 20
l aliquot of the supernatant onto a BDS Hypersil C-18analytical column (Keystone Scientific, Inc.). The elu-tion buffer contained 75 mM sodium phosphate, 0.5mM EDTA, 350 mg/l 1-octanesulfonate sodium, 4% ac-etonitrile (v/v) and 0.8% tetrahydrofuran, pH 3.0 andthe flow rate was 1.2 ml/minute. Dihydrobenzylamineand NE peaks were measured at 600 mV with 10 nAsensitivity using an electrochemical method and com-pared with samples containing known amounts of NE.
Two hours after administration of atomoxetine (3 mg/kg, i.p.), the rats were deeply anesthetized with sodiumpentobarbital (60 mg/kg, i.p.) and transcardially per-fused with 100 ml of phosphate buffered saline (PBS)followed by 100 ml of 4% paraformaldehyde in PBS.The brain was rapidly removed and postfixed for 90min in 4% paraformaldehyde, and then was transferredto 30% sucrose at 4
C until saturated. After quick freez-ing, serial 30
m sections were cut and placed in PBSuntil processed for immunohistochemistry. In brief,sections were incubated in PBS containing blocking se-rum and 0.5% Triton-X 100 for 1 h. Sections were thenincubated with anti-Fos antibody (Santa Cruz Biotech-nology, Inc.) at 4
C overnight. Visualization of the Fos-like immunoreactivity was performed with a VectastainABC Elite Kit (Vector Labs, Burlingame, CA) using thestandard protocol supplied with the kit. Nickel-intensi-fied diaminobenzidine (DAB) was used as the chroma-gen to yield a gray-black precipitation product. Follow-ing visualization of the Fos immunoreactivity, thesections were mounted on gelatin-coated glass slidesand allowed to dry. The sections were then dehydratedand cover slipped. Fos expressing cells were quanti-tated using the MCID M2 imaging system (Imaging Re-search, St. Catherines, Ontario).
Male Sprague-Dawley rats from Harlan Industries (Indi-anapolis, IN) were used for all studies. For the microdial-