Synaptosomes, synaptic vesicle preparation

If there is any problem with protein detection, enriched synaptic protein preparations (synaptosomes, presynaptic plasma membrane of synaptic vesicle preparations, depending of the toxin and the substrate) can be better starting material than brain homogenate to check the proteolytic activity of clostridial neurotoxins L chains. 

The —50 kDa band (48-53 kDa) is identified as dysbindin-1 A in our WESTERNS, because it runs close to the molecular mass of our histidine-tagged recombinant mouse dysbindin-1 A. Its identity is confirmed by the fact that it is recognized by antibodies we have recently generated to amino acid sequences in the CTR of human dysbindin-lA, but not found in dysbindin-lB, -2, or -3. The —50 kDa band is the most consistently observed dysbindin-1 band across tissues. We find it in all tissues examined to date the adrenal gland, heart, kidney, liver, lung, spleen, skeletal muscle, testes, spinal cord, cerebellum, striatum, hippocampus, and cerebral cortex (e.g., Figure 2.2-12a and c). In mouse and human synaptosomes, the —50 kDa isoform is heavily concentrated in the PSD fraction with a much lesser amount in the presynaptic membrane and no detectable amount in the synaptic vesicle fraction (Talbot et al., in preparation). 

The —33 kDa band (32-34 kDa) is close in molecular mass to dysbindin-1C, which lacks the NTR of dysbindin-1 A but is otherwise identical to dysbindin-1 A (see Section 2.2.2.2.1). It is consequently difficult to determine if the —33 kDa band represents (1) dysbindin-lC itself, (2) a degradative product of dysbindin-1 A missing the NTR, or (c) some combination of the first two possibilities. The first possibility seems most likely, however, because the —33 kDa band is absent in some tissues (e.g., cerebellum, kidney, and liver) where the —50 kDa band (i.e., dysbindin-1 A) is present and hence subject to degradation ( Figure 2.2-12c). Moreover, the —33 kDa band is often stronger than the 50 kDa band even in fresh tissue. We thus believe that the —33 kDa band does represent dysbindin-1C. It is seen in the heart, lung, skeletal muscle, striatum, hippocampal formation, and cerebral cortex (e.g., O Figure 2.2-12a and c). In synaptosomes of the mouse and human brain, the —33 kDa isoform is concentrated in synaptic vesicle and PSD fractions with very little, if any, present in the presynaptic membrane fraction (Talbot et al., in preparation). 

The procedure for the preparation of synaptosomes and small synaptic vesicles (SSV) from rat brain cortex follows established methods with minor modifications (Schiavo and Montecucco, 1995). 

Subsequently it was shown that if a synaptosomal preparation was lysed, pelleted and resuspended, the uptake of - Ca by the intrasynaptosomal particles was again inhibited by prior acute in vivo or in vitro treatment (60). Similarly, an increased uptake again followed tolerance development. These drug effects were seen when ATP (3 mM) was included in the media but not in its absence, suggesting that an active process was being affected. It is unlikely that mitochondrial Ca fluxes were altered by morphine, since the effects were observed in the presence of mitochondrial inhibitors. It was considered most likely that the site of action was the synaptic vesicles. 

If ACh is synthesized extravesicularly as all experiments seem to indicate at present, there must exist a mechanism for the uptake of ACh into the synaptic vesicles. This mechanism has not yet been convincingly established. Experiments in vivo with infusion of labelled choline give labelling of ACh in synaptic vesicles (Chakrin and Whittaker, 1969 Barker et al., 1970) whereas the results from isolated synaptosome preparations are controversal. 

A 10 min period of electrical stimulation in the presence of Pi led to increased labelling of synaptosomal phosphatidate (Bleasdale Hawthorne, 1975) but no consistent, increase in phosphatidylinositol specific radioactivity. The labelling of ATP was unaffected. When sub-synaptosomal membrane fractions were prepared, the increased phosphatidate labelling was seen to be associated with the synaptic vesicle fraction, as in the acetylcholine experiments. Further work (Hawthorne Bleasdale, 1975) showed that electrical stimulation only increased the labelling of phosphatidate in the vesicle fraction when the medium contained calcium ions. This and the time course of labelling (changes seen 2 min after the onset of stimulation) suggest that the metabolism of phosphatidate is closely associated with the process of transmitter release. 

Preparation of sub-synaptosomal fractions showed that after labelling vivo, phosphatidate of Fraction E which contains marker enzymes for endoplasmic reticulum and plasma membrane (but not in higher concentration than some other fractions) had the highest specific radioactivity (Table 1). The most active phosphatidylinositol was in the synaptic vesicle fraction. Electrical stimulation provoked loss of these particular pools of phospholipid, while there was little effect on either phospholipid in the remaining fractions, as can be seen from the Table. 

---