Richard Hammerschlag

Nerve impulse-enhanced release of amino acids from non-synaptic regions of peripheral and central nerve trunks of bullfrog

The release of glutamate from vertebrate central nervous tissue following electrical stimulation has been demonstrated in a variety of preparations: spinal cord slices 11,13, spinal hemicord 19, brain slices 17, exposed cerebral cortex 14,~8, exposed hippocampus 4, retina 2~, and cerebral cortical synaptosomes 3. These findings are frequently discussed in the context of a possible synaptic transmitter role for glutamate in the vertebrate central nervous systemS,10, ~5. A cautionary note to such discussions is that an efflux of this amino acid from non-synaptic regions of neurons could contribute to the release observed in many of the above studies. Such a nonsynaptic release of glutamate has been described from desheathed sciatic nerves 7,~3, and more importantly, this release appears enhanced during neuronal activity. In the present study, isolated dorsal and ventral spinal roots of bullfrog were chosen to further characterize amino acid efflux from nerve trunks during impulse conduction, and to compare parameters of this release with those established for the release of substances from synaptic regions. Since spinal roots lack the thick perineurium--epineurium that surrounds peripheral nerve trunks, diffusion rates of small molecules are similar to those found in desheathed sciatic nerves6,2°; in addition, problems of tissue swelling and metabolic alterations subsequent to desheathing 2° are avoided. Both types of roots were examined to determine if the glutamate release previously observed from sciatic nervesT, 23 was solely from afferent fibers where it may serve a transmitter role 10, or was equally released from the cholinergic efferent fibers. Impulse-enhanced release of amino acids was also examined in isolated optic nerves of bullfrog to study the phenomenon in the central nervous system. A preliminary report of these findings has appeared 22. Dorsal and ventral spinal roots and optic nerves were excised from 4-6 in. bullfrogs, Rana catesbeiana, during the months of March through early July. The

Membrane delivery by fast axonal transport

Abstract Newly-synthesized proteins destined for fast axonal transport initially reach the axon by a pathway similar to that of proteins destined for membrane insertion or secretion in non-neural systems. The analogy can be pursued to suggest that fast-transported materials are moved through the cell body and axon in the form of preassembled membrane and that a major function of these materials is to maintain functionally diverse regions of axon and synaptic terminal membrane.

DIVALENT CATION SPECIFICITY OF THE CALCIUM REQUIREMENT FOR FAST TRANSPORT OF PROTEINS IN AXONS OF DESHEATHED NERVES

The presence of a requirement for calcium during the fast transport of [3H]protein in axons was assessed in desheathed spinal nerves of bullfrog. The nerves were desheathed locally along 4 mm of their length, and desheathing was judged effective on the basis of an enhanced uptake of [3H]leucine into that region of nerve trunk. Desheathing per se had a slight inhibitory effect on transport. Incubation of desheathed nerve trunks in calcium‐free medium reduced transport by 60‐80% relative to that in desheathed nerves incubated in normal medium. Addition of Mg2+ or Sr2+ to the calcium‐free medium allowed transport to proceed normally. Addition of Co2+ or Mn2+ to normal medium did not affect transport in desheathed isolated nerve trunks. When ganglia and nerve trunks were both incubated in medium containing 0.18 mM‐CoCl2, transport was depressed to a similar extent proximal and distal to the desheathed region. This again indicates that Co2+ does not inhibit transport in desheathed nerves, whereas it does inhibit transport in the ganglia. Additive inhibitory effects were observed when ganglia were incubated in medium containing 0.018 mM‐CoCl2, and desheathed nerves were incubated in calcium‐free medium. Differences in the divalent cation specificities of the axonal and ganglionic calcium requirements suggest that calcium supports transport in nerves in a manner distinct from its role in maintaining transport in spinal ganglia. It is concluded that the ganglionic calcium requirement involves initiation of axonal transport in the soma rather than translocation in the intraganglionic region of axon.

Relation of somal lipid synthesis to the fast axonal transport of protein and lipid

The role of somal lipid synthesis in the fast axonal transport of protein and lipid was examined in vitro utilizing spinal/sciatic nerve preparations of bullfrog. Inhibition of phospholipid synthesis in dorsal root ganglia by the amphiphilic cation, fenfluramine (0.1-2.0 mM) was monitored as decreased incorporation of [3H]choline into phosphatidyl choline. This inhibition was directly proportional to a decrease in the amount of [3H]protein undergoing fast axonal transport, the two variables being related by a slope close to unity. [3H]Choline-labeled lipid undergoing fast transport in the axon was unaffected by inhibition of somal phospholipid synthesis. Levels of fenfluramine up to 1.0 mM had no effect on uptake or incorporation of [3H]leucine. Selective exposure of desheathed nerve trunks to 1.0 mM fenfluramine had no effect on [3H]protein translocation, indicating that local phospholipid synthesis is not required to maintain ongoing transport in the axon. Inhibition of cholesterol synthesis in the ganglia with the analog 20,25-diazacholesterol also resulted in depression of [3H]protein transport. Since synthesis of both phospholipid and cholesterol are required at the level of the ganglion, it is suggested that the initiation of fast axonal transport of protein is dependent on the assembly of lipoprotein structures in the soma.

Differential effects of cobalt on the initiation of fast axonal transport.

Effects of Co2+ on the fast axonal transport of individual proteins were examined in vitro in bullfrog spinal/sciatic nerves.35S-methionine-labeled proteins, fast-transported in control and Co2+-treated preparations were separated via two-dimensional gel electrophoresis. While the overall amount of protein transported was reduced, no qualitative differences could be seen when gel fluorographic patterns were compared. Quantitative analyses of the 48 most abundantly transported species revealed two significantly different populations (p < 0.01) differentially sensitive to Co2+ and distinguishable to a large extent by molecular weight. Those proteins less sensitive to Co2+ ranged from ~20,000 to 35,000 daltons while those more sensitive to Co2+ were >~35,000 daltons. The finding that all proteins are affected by Co2+ supports the proposal that fast-transported proteins are subject to a common Co2+-sensitive, Ca2+-requiring step. The observed differential effects are consistent with more than one Ca2+-dependent step occurring during the initiation phase of fast transport.

Involvement of coated vesicles in the initiation of fast axonal transport

The present study examines whether coated vesicles play a role in the intrasomal transit of newly synthesized fast-transported proteins. Coated vesicles isolated from bullfrog brain were shown to have a protein composition and ultrastructure similar to purified bovine brain coated vesicles. Bullfrog brain was then used as unlabeled carrier for the isolation of coated vesicles from dorsal root ganglia labeled with [3H]leucine. Fast-transported [35S]methionine-labeled proteins were generated in separate preparations from sciatic nerve, and co-electrophoresed on two-dimensional gels with [3H]proteins of the coated vesicle fraction. The [35S]Met fluorographic X-ray film pattern was used as a guide to remove gel regions which were tested for the presence of 3H. By this means, 45 of 67 individual fast-transported proteins examined were found to contain significant levels of 3H. The fact that these proteins have similar net charge and molecular weight characteristics to the mature fast-transported proteins with which they co-migrated, suggests that such species have already undergone post-translational modifications prior to becoming associated with coated vesicles. Since most modifications of this type occur in the Golgi apparatus, it appears that the majority of fast-transported proteins are isolated in association with a population of post-Golgi coated vesicles. The role of coated vesicles is incorporated into a model describing the pathway taken by fast-transported proteins during the initiation of fast axonal transport.

An increase in smooth endoplasmic reticulum and a decrease in golgi apparatus occur with ionic conditions that block initiation of fast axonal transport

The ultrastructure of bullfrog spinal ganglia was analyzed after incubation in media containing concentrations of calcium and cobalt known to inhibit export of proteins from the soma to the axon. Although most somal organelles were morphologically unchanged by the various incubation media, striking changes were seen in the smooth endoplasmic reticulum (SER) and the Golgi apparatus (GA). In order of effect, calcium-free medium (CFM), normal medium supplemented with cobalt (NM--Co), and CFM supplemented with cobalt (CFM--Co) produced increasing amounts of SER coupled with decreasing densities of GA stacks. In the extreme case, CFM--Co incubation resulted in a nearly 10-fold increase in SER volume as well as in a virtually complete depletion of GA stacks. Axons originating within the ganglion were also examined and showed little change after the various incubations. The rank order of the altered incubation media in producing morphological changes was the same as the relative effectiveness of the media in depressing the fast axonal transport of [3H]protein within the dorsal root ganglion neurons. The morphological and biochemical results are discussed with respect to establishing the localization of the calcium-dependent step(s) that has been proposed to occur in the neuronal soma during the initiation of axonal transport.

Glycosylation as a Criterion for Defining Subpopulations of Fast-Transported Proteins

The role carbohydrate residues may play in the sorting of newly synthesized fast‐transported proteins during the initiation of fast axonal transport has been examined by identifying individual fast‐transported glycoproteins that contain either or both fucose and galactose. [3H]Fucose or [3H]galactose was incorporated together with [35S]methionine in vitro in bullfrog dorsal root ganglia. Fast‐transported proteins that accumulated proximal to a ligature on the spinal nerve were separated via two‐dimensional gel electrophoresis, and 92 gel spots were analyzed quantitatively for the presence of 35S and 3H. Of these spots, 56 (61%) contained either or both fucose and galactose. Glycomoieties were generally associated with families of charged spots whose isoelectric points could be altered with neuraminidase treatment. Single spots tended to be unglycosylated and were unaffected by neuraminidase. The prevalence of glycoproteins was considerably greater in the higher‐molecular weight range. Of the 55 spots analyzed with molecular weight greater than approximately 35,000 daltons, 89% were glycosylated, whereas only 19% of the 37 spots with lower molecular weight contained sugar moieties. When considered in light of previous studies in which similar Subpopulations have been described, the current findings suggest that the presence or absence of glycomoieties may represent another criterion by which proteins are sorted during the initiation of fast axonal transport.

Fast-Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate

Abstract: 35SO4‐labeled fast‐transported proteins of bullfrog dorsal root ganglion neurons were separated by two‐dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine‐labeled fast‐transported proteins. The majority of these well resolved spots appeared as “families,” previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side‐chain charge that underlies microheterogeneity. The most heavily 35SO4‐labeled species, however, corresponded to fast‐transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.

Ca2+- or Mg2+-Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport

Abstract: Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1‐2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM‐Sr2+ or Ba2+, Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50‐60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal gangliodspinal nerve preparations, incubated for 20 h in medium containing 0.2 mM‐Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM‐Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+‐dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg2+ ATPase in the transport process.