Journal of Neuroscience Methods 203 (2012) 335–337 Contents lists available at SciVerse ScienceDirect Journal of Neuroscience Methods jou rnal h om epa ge: www.elsevier.com/locate/jneumeth Basic Neuroscience Synaptosomal protein synthesis in P2 and Ficoll purified fractions Maria Eyman, Carolina Cefaliello, Annapaola Bruno 1 , Marianna Crispino, Antonio Giuditta ∗ Department of Biological Sciences, University of Naples ‘Federico II’, Via Mezzocannone 8, Naples 80134, Italy a r t i c l e i n f o Article history: Received 29 September 2011 Received in revised form 10 October 2011 Accepted 11 October 2011 Keywords: Synaptosomes Protein synthesis Brain plasticity Learning a b s t r a c t Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Eukaryotic cells are well known to contain two separate systems of protein synthesis, which include the high capacity cytoplasmic system, and the mitochondrial system responsible for the synthesis of few intrinsic protein species. Either system is selectively inhib- ited by different sets of inhibitors. Cytoplasmic and mitochondrial systems of proteins synthesis are present in brain in the perinuclear cytoplasm of nerve and glial cells as well as in neuronal processes such as axons, nerve terminals and dendrites (for reviews, see Alvarez et al., 2000; Giuditta et al., 2002, 2008; Martin and Zukin, 2006; Crispino et al., 2009). The local synthesis of synaptic proteins is readily determined in subcellular brain fractions enriched in particles enclosed by a plasma membrane (synaptosomes) which are derived from presynaptic terminals and fragments of dendritic and glial pro- cesses (Whittaker, 1993; Giuditta et al., 2008). The activity of the cytoplasmic system of brain synaptosomes is selectively assayed in an incubation medium containing chloramphenicol, a spe- cific inhibitor of the mitochondrial system, but lacking added energy sources and soluble factors which are essential for the activity of the cytoplasmic system. Their absence in the assay medium is irrelevant for the activity of cytoplasmic protein ∗ Corresponding author at: Department of Biological Sciences, Via Mezzocannone 8, Naples 80134, Italy. Tel.: +39 081 2535089; fax: +39 081 2535090. E-mail address: giuditta@unina.it (A. Giuditta). 1 Present address: Department of Pharmaceutical Sciences, University of Salerno, via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy. synthesis in synaptosomes which natively contain soluble fac- tors and energy sources (mitochondria), but it strongly hinders the activity of extrasynaptosomal cytoplasmic protein synthe- sis. First identified in the late sixties (Austin and Morgan, 1967; for review, see Giuditta et al., 2008), the cytoplasmic system of pro- tein synthesis of brain synaptosomes has recently been shown to be involved in brain plastic responses elicited by learning (Eyman et al., 2007) and by brain ischemia (Mariucci et al., 2007). 2. Materials and methods Male Wistar rats 2–3 month old (Charles River Co., Calco, Italy) were kept in the animal room at a temperature of 23 ◦ and a light:dark regime of 12:12 (light on at 6.00 am) with free access to food and water. Rats trained for a two-way active avoidance task were taken at random and exposed to a session starting at about 9.00 am which consisted of 3 training periods of 30 min separated by rest intervals of the same duration (Eyman et al., 2007). Soon after training, rats were anestesized with CO 2 and decapitated with a guillotine. Cerebral hemispheres were quickly dissected and homogenized in a Dounce homogenizer with 9 vol- umes of cold isotonic medium (HM) containing 0.32 M sucrose and 10 mM Tris–Cl pH 7.4. Subcellular fractions were prepared accord- ing to a routine method (Eyman et al., 2007) slightly modified from Rao and Steward (1991). Briefly, following centrifugation of the homogenate in rotor JA17 of a Beckman JA21 centrifuge (4200 rpm, 1 min, 4 ◦ C), the sediment was resuspended in the same volume of HM and centrifuged under the same conditions to yield a sed- iment containing nuclei, cell debris and large synaptosomes (P1). 0165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2011.10.007