Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca(2+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location.
Among the pathologic hallmarks of Alzheimer's disease (AD) neurodegeneration, only synaptic loss in the brains of AD patients closely correlates with the degree of dementia in vivo. Here, we describe a molecular basis for this AD loss of synapses: pathological reduction of synaptogenic PKC isozymes and their downstream synaptogenic substrates, such as brain-derived neurotrophic factor. This reduction, particularly of PKC ␣ and , occurs in association with elevation of soluble  amyloid protein (A), but before the appearance of the amyloid plaques or neuronal loss in the Tg2576 AD transgenic mouse strain. Conversely, treatment of the Tg2576 mouse brain with the PKC activator, bryostatin-1, restores normal or supranormal levels of PKC ␣ and , reduces the level of soluble A, prevents and/or reverses the loss of hippocampal synapses, and prevents the memory impairment observed at 5 months postpartum. Similarly, the PKC -specific activator, DCP-LA, effectively prevents synaptic loss, amyloid plaques, and cognitive deficits (also prevented by bryostatin-1) in the much more rapidly progressing 5XFAD transgenic strain. These results suggest that synaptic loss and the resulting cognitive deficits depend on the balance between the lowering effects of A on PKC ␣ and versus the lowering effects of PKC on A in AD transgenic mice.
Using both scanning confocal and electron microscopic morphometric measurements, we analyzed single dendritic spines of CA1 pyramidal cells in the hippocampi of water maze-trained rats vs. controls. Two days after completion of all training, we observed a memory-specific increase in the number of mushroom spines-all of which make synaptic contacts-but not in the numbers of filopodia or stubby or thin spines, as quantified with double-blind protocols in both scanning confocal and electron microscopic images. This memory-specific increase of mushroom spine number was enhanced by the PKC activator and candidate Alzheimer's disease therapeutic bryostatin, blocked by the PKC␣-isozyme blocker Ro 31-8220, and accompanied by increases in the number of ''perforated'' postsynaptic densities, increased numbers of presynaptic vesicles, and the increased occurrence of double-synapse presynaptic boutons associated with the mushroom spines. These and other confocally imaged immunohistochemical results described here involving PKC substrates indicate that individual mushroom spines provide structural storage sites for long-term associative memory and sites for memory-specific synaptogenesis that involve PKC-regulated changes of spine shape, as well as PKC-regulated changes of pre-and postsynaptic ultrastructure.learning and memory ͉ synaptogenesis ͉ Alzheimer's disease ͉ mushroom spines ͉ bryostatin A s evidence has accumulated to support the hypothesis that synapses are critical storage sites for memory in the brain (1, 2), increasing attention has been given to dendritic spines (3). During development, dendritic spines appear to begin as thin extensions called filopodia that then mature with an expanded mushroom-shaped ''head'' linked by a neck to the dendrites (4). A recent two-photon laser analysis showed that single-spine enlargement is induced by long-term potentiation and glutamate release. This enlargement, however, only persists in small, nonmushroom spines (5, 6). Here, quantitative morphometric analyses in the CA1 dendritic areas were conducted with hippocampal slices taken from rats trained in the Morris water maze or exposed to control paradigms. Using double-blind protocols, a scanning confocal microscope was used to visualize different shapes of dendritic spines of CA1 apical dendrites by 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate (DiI) staining. A second set of measurements, using doubleblind protocols, involved electron microscopic visualization of pre-and postsynaptic ultrastructure associated with memoryspecific changes of the mushroom spines. Confocal immunohistochemistry was also used to assess the total number of dendritic spines and presynaptic endings. ResultsA PKC-Activating Drug Enhances Learning and Memory. For water maze training of brown Norway rats (4-5 months old), there was an overall significant difference in learning among the experimental groups (P Ͻ 0.001; ANOVA). When treated with bryostatin, rats reached a hidden platform in a water maze more quickly than controls tr...
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