Notes: Summary GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.

Notes: Summary The growth-associated protein GAP-43 (B-50, F1, pp46), has been found in elongating axons during development and regeneration, and has also been associated with synaptic plasticity in mature neurons. We have examined the loss of GAP-43 labelling from cerebellar granule cells with immunocytochemical localization of a polyclonal antibody to GAP-43. One day after plating, the plasma membrane of cell bodies, neurites and growth cones were all labelled with anti-GAP-43. By 10 days, most of the cell body labelling was lost, and by 20 days the neuritic and growth cone labelling was greatly reduced. Beginning at six days, anti-GAP-43 labelling of growth cones, which was initially uniform, became clustered. When growth cones were double-labelled with antibodies to GAP-43 and the synaptic vesicle protein, p65, inverse changes in the distribution of label was observed. While growth cone labelling with anti-p65 increased from three to 20 days in culture, GAP-43 label began to be lost from some growth cones by six days and showed continuing decline through 20 days. For individual growth cones, the loss of GAP-43 appeared to parallel the accumulation of p65, and first growth cones to lose GAP-43 appeared to be the first to accumulate p65 label. When cultures were grown on a substrate of basement membrane material, the time frames of neuritic outgrowth, loss of GAP-43 labelling, and increase in p65 labelling were all accelerated. At five days, labelling for GAP-43 was weak and labelling for p65 was strong, in a pattern comparable to that seen in older cultures on a polylysine substrate. These results suggest several conclusions concerning the expression and loss of GAP-43 in cultured cerebellar granule neurons. First, GAP-43 label is initially distributed in all parts of these cells. With increasing time in culture the label is first lost from cell bodies and later from neurites and growth cones. Second, the loss of GAP-43 label from growth cones is correlated with the appearance of the synaptic vesicle protein p65. Finally,in vitro developmental changes in the loss of GAP-43 can be altered by changing the growth substrate.

Notes: Abstract Despite substantial increases in atmospheric pollution and significant correlations between specific inputs and red spruce (Picea rubens Sarg.) decline at certain locations in eastern North America, it has not been possible to demonstrate a consistent relationship between severity of decline and either pollution inputs or climatic factors throughout the region of decline. These results indicate that different pollutants or environmental features may be associated with red spruce deterioration in different regions and raise the question of why red spruce, and not other sympatric species, might be predisposed to injury from a diversity of inputs and over a large geographic area. In this paper we present data to indicate red spruce is a relatively uniform species genetically, and we discuss the possibility that it may be deteriorating because it lacks the genetic flexibility to cope with and respond to abrupt environmental modification. Estimates of genetic variability in red spruce were determined from electrophoretic assessments of 30 trees from each of 10 populations distributed throughout the region of red spruce decline. An examination of genetic variation at 36 loci revealed an average observed heterozygosity for red spruce of 6.9% with only 21.3% polymorphic loci and 1.43 alleles per locus. These genetic variability estimates are considerably lower than those reported for other north-temperate woody plant species, including conifer species that are sympatric with red spruce and are presumably exposed to similar selection pressures. In one population, individual trees that were high in observed heterozygosity were generally more vigorous (as estimated from sapwood area/basal area ratios) than less genetically variable individuals. As a result, it appears highly heterozygous individuals may be more resilient and capable of surviving and reconstituting viable populations in the future. We discuss five additional lines of evidence that are consistent with a hypothesis of insufficient genetic variability and limited adaptability in red spruce.