Synaptosome/synaptosomal

Preparation of synaptosomes. Synaptosomes were prepared from the brains of male mice (20-30 g Blue Spruce Farms, Altamont, NY) either by a modification of the method of Hajos (8) or by the method of Dodd et al. (9). Both preparations gave qualitatively similar results, but the magnitude of all sodium fluxes per mg of synaptosomal protein was much greater with the latter preparation. A preparation enriched in synaptosomes was prepared from the brains of juvenile rainbow trout (Salmo gairdneri obtained from the New York State Fish Hatchery, Bath, NY) by homogenization in 14 volumes of 0.7 M sucrose, and centrifugation first at 2000 for 10 min and then at 31000 for 30 min. The pellet from the second centrifugation was resuspended in sodium-free buffer identical to that used in previous studies (7) except that it also contained 370 mM sucrose. 

Figure 2. Effect of phenytoin concentration on K- and veratridine-stimulated Ca uptake in rat brain synaptosomes. Synaptosomal uptake of 45Ca was stimulated by exposure of the tissue to 64 mM K (O) for 15 s or 5 /uM veratridine (%) for 5 min. In the absence of phenytoin control, stimulation was 4.7 0.3 and 6.1 0.8 fxmol Ca/g prot for K+ and veratridine, respectively. Each symbol and vertical line represents the mean and SEM, respectively, of 6 samples.

Synaptosomes. Synaptosomes are formed after homogenization of brain tissue and can be isolated by centrifugation. They are composed of the terminal parts of axons containing the synapses and form closed compartments containing either the presynaptic machinery, the postsynaptic machinery or both. 

Lonart G, Johnson KM Inhibitory effects of nitric oxide on the uptake of [3H]dopamine and [3H]glutamate by striatal synaptosomes. J Neurochem 63 2108—2117, 1994 Lovinger DM, White G Ethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in neuroblastoma cells and isolated adult mammalian neurons. Mol Pharmacol 40 263—270, 1991... 

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

Brevetoxins bind with high specificity to synaptosomes of fish (TUapia sp.)y turtles (P. scripta), and rats (Table I). In all cases, the K j was in the nanomolar... 

Figure 3. Concentration dependence of the stimulation of Na influx by PbTx-3 (4). Synaptosomes were pre-incubated for 30 min with indicated concentrations of PbTx-3 in the presence of aconitine. Influx is plotted as specific influx, points representing means of triplicate determinations.

Figure 4. Binding was measured in rat brain synaptosomes using a rapid centrifugation technique. Total ( ), and nonspecific ( ) binding of tritiated PbTx-3 were measured, their difference representing specific binding (A). Rosenthal analysis yields a of 2.6 nM and a B of 6.0 pmol toxin bound/mg protein.

Figure 5. Comparison of specific displacement of 10 nM tritiated saxitoxin (A) or 10 nM tritiated PbTx-3 ( ) by unlabeled competitor saxitoxin or brevetoxin, respectively, in rat brain synaptosomes. IC q in each case is 5-10 nM.

Several brevetoxins have been examined for their respective abilities to competitively displace tritiated brevetoxin PbTx-3 from its specific site of action in brain synaptosomes. Analysis of IC q values revealed no marked differences in the displacing abilities between any of the type-1 toxins, and similarly there was no apparent difference between displacing abilities of PbTx-1 or -7, both type-2 toxins. Although some specific details require correlation, a gross comparison indicates that sodium channels in brain are similar in the systems examined. In the system studied most extensively, the rat brain synaptosome, t-test analysis revealed no significant differences between PbTx-2 and PbTx-3 IC q, or between PbTx-1 and PbTx-7 IC q, but statistically significant differences were found between the two classes (P<0.01) (5). If the Cheng-Prusoff equation (15) is applied ... 

Figure 6. Effect of brevetoxins on tritiated PbTx-3 binding to rat brain synaptosomes. Incubations, in the presence of 50 fig synaptosomal protein and 16 nM tritiated PbTx-3 with increasing amounts of unlabeled PbTx-1 ( ), PbTx-2 ( ), PbTx-3 ( ), PbTx-5 (A), PbTx-6 ( ), or PbTx-7 (o) were for 1 hr at 4 C. Each point represents the mean of three triplicate determinations.

Table II. Speciflc Displacement of [hi] PbTx-3 Dx)m Synaptosome Binding by Unlal led Brevetoxins, Comparison with LD ...

Figure 7. Inhibition of tritiated PbTx-3 specific binding by unlabeled brevetox-ins, PbTx-2, PbTx-1, PbTx-3, PbTx-7. Specific binding was measured in intact synaptosomes at 4 C degrees in standard binding medium using four different tritiated toxin concentrations—5.0, 7.5, 10.0, and 15.0 nM (inverse [ H] PbTx-3 abscissa values)—in the presence of unlabeled toxins at 0 (o), 5.0 ( ), 7.5 (A), 10.0 (A), 25.0 ( ), 50.0 ( ), or 100.0 (data not shown) nM. Points are means of triplicate determinations at each concentration.

A NT might be expected to be concentrated in nerve terminals and this can be ascertained since when nervous tissue is appropriately homogenised the nerve endings break off from their axons and surrounding elements and then reseal. Such elements are known as synaptosomes. They have been widely used to study NT release in vitro (Chapter 4) and some NT should always be found in them, at least if it is released from vesicles. 

Synaptosomes are pinched-ofP nerve terminals which become severed from the parent axon during gentle homogenisation of brain tissue and then subsequently reseal. They... 

The main advantage of using synaptosomes is that they are free from any influence of the parent axon. Another is that, since the volume of extracellular space (the incubation medium) is functionally infinite, transmitter will not accumulate near the synaptosomes. This means that reuptake of released transmitter is unlikely to occur and that, under drug-free conditions, transmitter release will not be modified by activation of auto- or heteroceptors (see below). 

A disadvantage of using synaptosomes is that they cannot be used to study transmitter release evoked by propagated nerve impulses, but the release, like that from intact neurons, is Ca +-dependent and K+-sensitive. Pharmacological studies using synaptosomes have also provided evidence that the amount of transmitter that is released following their depolarisation is regulated by the activation of presynaptic receptors. 

Much evidence supports this scheme. For example, neuronal depolarisation increases the amount of free synapsin in the cytosol and microinjection of CAM kinase II into the terminals of the squid giant axon or brain synaptosomes increases depolarisation-evoked transmitter release. By contrast, injection of dephosphorylated synapsin I into either the squid giant axon or goldfish Mauthner neurons inhibits transmitter release. 

Regulation of transmitter release does not rest solely on the frequency at which nerve impulses reach the terminals. Early experiments using stimulated sympathetic nerve/end-organ preparations in situ, or synaptosomes, indicated that release of [ HJnoradrenaline was attenuated by exposure to unlabelled, exogenous transmitter. This action was attributed to presynaptic adrenoceptors, designated a2-adrenoceptors, which were functionally distinct from either aj- or )S-adrenoceptors. Later experiments have confirmed that ai-adrenoceptors comprise a family of pharmacologically and structurally distinct adrenoceptor subtypes. 

Figure 6.2 Diagrammatic representation of a cholinergic synapse. Some 80% of neuronal acetylcholine (ACh) is found in the nerve terminal or synaptosome and the remainder in the cell body or axon. Within the synaptosome it is almost equally divided between two pools, as shown. ACh is synthesised from choline, which has been taken up into the nerve terminal, and to which it is broken down again, after release, by acetylcholinesterase. Postsynaptically the nicotinic receptor is directly linked to the opening of Na+ channels and can be blocked by compounds like dihydro-jS-erythroidine (DH/IE). Muscarinic receptors appear to inhibit K+ efflux to increase cell activity. For full details see text...

There is some evidence that receptors for other neurotransmitters on 5-HT nerve terminals also modify release of 5-HT. These include nicotinic receptors (increase release from striatal synaptosomes), a2A-adrenoceptors (depress cortical release) and H3-receptors (cortical depression). Because changes in 5-HT release on activation of these receptors is evident in synaptosomal preparations, it is likely that these are true heteroceptors . 

ATP certainly fulfils the criteria for a NT. It is mostly synthesised by mitochondrial oxidative phosphorylation using glucose taken up by the nerve terminal. Much of that ATP is, of course, required to help maintain Na+/K+ ATPase activity and the resting membrane potential as well as a Ca +ATPase, protein kinases and the vesicular binding and release of various NTs. But that leaves some for release as a NT. This has been shown in many peripheral tissues and organs with sympathetic and parasympathetic innervation as well as in brain slices, synaptosomes and from in vivo studies with microdialysis and the cortical cup. There is also evidence that in sympathetically innervated tissue some extracellular ATP originates from the activated postsynaptic cell. While most of the released ATP comes from vesicles containing other NTs, some... 

---