Labelling of synaptosomes and axoplasmic flow in the brain stem of the rat

EVIDENCE for axoplasmic flow in the peripheral nervous system is well documented but fewer attempts have been made to demonstrate the phenomenon in the CNS (see FOULKES and ROBINSON, 1969~). In both, it is possible to demonstrate that protein-incorporated radioactive precursors are Iocaliz+d in nerve soma after a short period but that with increasing time after injection the radioactive tracers become distributed along nerve axons by migration from the cell body. Earlier work on the distribution of labelled protein, following injection of [14C]leucine into the rat brain reticular formation, revealed no si@cant migration of the protein over a period of 21 days. This was explained by postulating that the brain stem reticular formation was mainly populated by short synapsing neurones (FOWLKES and ROBINSON, 1968). However, BARONDES (1964, 1966, 1968) demonstrated axoplasmic transport within whole mouse brain by showing that the specific activity of labelled protein from synaptosomes, and from fractions derived from them by lysis in water, exhibited a substantial increase over a period from 2 h to 10 days, while the specific activities of proteins from other subcellular fractions steadily decreased. This alternative method, demonstrating significant axonal flow in whole mouse brain, has been applied to the brain stem reticular formation previously investigated (FOULKES and ROBINSON, 1968). Ten PI of [14C]leucine (311 mCi/mmol, 62.5 PCi/ml) made isotonic with NaCl were injected into the brain stem of male Lister rats of approximately 250 g secured in a stereotaxic apparatus, at a precise position as previously described (FOULKES and ROBINSON, 1968). At times varying from 4 h to 10 days the animals were sacrificed, the brain stem quickly dissected out and homogenized in 5 vol. 0.32 M-sucrose (containing 0.1 hi-tns, pH 7.6; 0025 M-KCI and 0-005 M-MgCI,) at 0°C (HOAGLAND, SCORNIK and PFEPFERKON, 1964). For the small amount of tissue investigated (approximately 0.25 g wet weight), this medium was found to give a better yield of synaptosomes than 0-32 M-SUCrOSe alone. Following dilution of the homogenate with sucrose solution to a tissue concentration of 10% (w/v), aliquots were taken for spmlic activity determinations before the remainder was differentially centrifuged by the method of GRAY and W ~ A K E R (1962), as modified by BARONDES (1966). The crude mitochondria1 fraction was washed twice with the medium, suspended in 6 ml of the 0.32 M S U ~ ~ O S ~ solution and layered onto a discontinuous density gradient of 10 rnl each of 0.8 Mand 1.2 Msucrose (GRAY and W ~ A K E R , 1962). Centrifugation at 53,OOOg for 2 h in a Spinco 25 swing-out head produced a ‘myelin’ layer at the 0.32/08 M interface (discarded), a synaptosomal layer at the 08/1.2 M interface, and a mitochondria1 sediment. The mitochondria were washed twice with 0.32 M-sucrose solution before collection (BARONDES, 1966). The absence of aggregates within this fraction was verified by electron microscopy. Synaptosomes were removed from the gradient with a Pasteur pipette, diluted with 3 vol. of the 0.32 M S U ~ ~ O S ~ solution and spun at 100,OOOg for 1 h in the Spinco 40 angled head centrifuge to produce a compact pellet . Microsomes were separated from the supernatant by centrifugation at 100,OOOg for 1 h, and their purity checked by electron microscopy. hotein was extracted from the total homogenate aliquots and the subcellular fractions by the method of VRBA, GAITONDE and R I C H ~ R (1962) and dissolved in 0.5 M-NaOH. The protein solutions were assayed according to WADDELL (1956) and radioactivities determined in a Packard Tri-Carb liquid scintillation spectrometer, the activities being corrected for background radiation and to 100 per cent efficiency. The results are expressed as dis. /min/mg protein (Table 1) and are the means of the stated numbers of animals investigated. The standard errors calculated for each mean are included in brackets: the high values obtained have been discussed in a previous communication on the reproducibility of the results in a similar experiment (FOULKES and ROBINSON, 1969b). All proteins were heavily labelled by 4 h, specific activities showing a general decline during the subsequent period of investigation; this is attributable to protein turnover. It is noteworthy that the synaptosomal fraction was heavily labelled at 4 h. If nerve endings receive protein from the cell body by axoplasmic transport, they might be expected to exhibit a low protein specific activity immediately following the introduction of a labelled precursor, with a subsequent increase as protein is transported to the nerve endings. The results indicate that nerve endings are either capable of local synthesis of protein, as suggested for rat cerebral cortex ( A u m and MORGAN, 1967) and mouse cerebrum (DROZ