Long-term storage of functional, isolated nerve endings by slow freezing and rapid thawing

Nerve endings (synaptosomes) were isolated from homogenized rat brain corpora striata following centrifugation on discontinuous sucrose gradients. The synaptosomes (in 0.8 M sucrose) were (i) slowly frozen by placing the tube containing the suspension in a freezer at -10 degrees C for 1 h followed by (ii) swirling in a mixture of acetone and dry ice for 15 min and (iii) were stored in liquid nitrogen for up to 6 weeks. Freshly isolated synaptosomes and synaptosomes from the same preparation that were frozen for 2, 4, or 6 weeks and rapidly thawed in a water bath at 37 degrees C were re-equilibrated with a physiological salt solution and assayed for their ability to accumulate Ca and to release transmitter (dopamine) upon depolarization in high K medium. K-dependent Ca uptake gradually declined to approximately 1/3 the value observed with freshly isolated synaptosomes after 6 weeks of storage. K-stimulated dopamine release (only from intact synaptosomes) was normal over the entire period of storage. It is concluded that synaptosomes retain their physiological properties when stored frozen for a few weeks and that cold storage may be a useful technique for experiments requiring lengthy or repeated assays or accumulation of material.