Effect of collagenase on the release of dopamine and acetylcholine from slices of rat corpus striatum

SGARAGLI & COOPER (1974) recently reported that collagenase-treated slices of bovine superior cervical ganglion released significantly less acetylcholine (ACh) in response to potassium-induced depolarization than did normal tissue slices. This effect appeared to be due only to interference with transmitter release mechanisms since the total radioactivity of ACh in the slices was the same whether or not collagenase was present in the incubation medium. This same effect was noted in synaptosomes prepared from collagenase-treated slices as compared to synaptosomes isolated from control slices. In this paper we report the effect of collagenase on the release of two transmitters, dopamine (DA) and ACh, from striatal slices prepared from rat brain. The corpora striata of male rats (250-300 g) were dissected out rapidly after decapitation. Tissue slices of 0.175 mm thickness were prepared with a Sorvall tissue chopper. The tissue was incubated for 25 min a t 37°C in 2 ml of Krebs-Ringer phosphate buffer (KRP), pH 7.4, which contained NaCl, 1 2 8 m ~ ; KCl, 48mM; CaCl,, 075mM; MgSO,, 1.2 mM; glucose, 16 mM; Na,HPO,, 16 mM; and EDTA, 0.027 mM. The incubation was continuously oxygenated by a mixture of 95% 02-5% CO,. The incubation medium also contained a labelled neurotransmitter or its precursor: C3H]DA (specific activity 9.3 Ci/mmol, final concentration 5.3 x lo-'' M, New England Nuclear), ['Hltyrosine (specific activity 55.5 Ci/mmol, final concentration 1.8 x lo-' M, New England Nuclear), or ['H]methylcholine (specific activity 16.5 Ci/mmol, final concentration 5.3 x lo-* M. New England Nuclear), with or without purified collagenase (EC 3.4.24.3, 400 units, Worthington). In experiments involving ACh, lo-, M-diisopropyl phosphorofluoridate (DFP) was included in the incubation medium. At the end of 25 min the incubation mixture was centrifuged for 5 min at 3000 g and 4"C, and the supernatant was discarded. The slices were transferred to a superfusion apparatus which has been described previously (BLJSTOS & ROTH, 1972). The striatal slices were washed rapidly with about 20 ml of KRP, poured through the perfusion chamber. KRP, prewarmed to 37T, was superfused through the tissue slices at the rate of 4 ml/min for 9-10 min. This procedure washed away loosely associated radioactive material, producing a low steady background of spontaneously released labelled compounds. A system of 3-way taps allowed a change of perfusing solutions to be made without disturbing the flow. The slices were stimulated by superfusion of KRP containing high K + concentrations (53 mM) for 1 min or KRP containing lo-, amphetamine or 6.8 x lo-, M carbachol for 3 min. Samples of the superfusate were collected every minute in test tubes containing either 0.2 ml of 0.5 M-HCI and 0 2 mg cold carrier DA for studies on dopamine release