A structural basis for enhancement of long-term associative memory in single dendritic spines regulated by PKC

Abstract

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...

Postischemic PKC activation rescues retrograde and anterograde long-term memory

“…Our present EM data show that the bryostatin-1 treatment prevented the ischemic loss of the preexisting ultra-structures for long-term associative memory, that is, perforated PSD mush- room spines that form synapses with an axonal bouton containing a high volume of presynaptic vesicles (10). Double-blind electron microscopy was used to investigate the effects of ischemia/hypoxia and/or bryostatin-1 on the number of mushroom spines and the number of axonal boutons with high concentration of presynaptic vesicles (n ϭ 48-62 electron micrographs; see Fig.…”

“…Data were analyzed by double-blind quantification (unknown treatment and animal subject) from electron micrographs (10 m ϫ 10 m). Morphology of mushroom-shape dendritic spines, presynaptic axonal boutons, and synapses were quantitatively analyzed according to Hongpaisan and Alkon (10). The criteria of mushroom spines are dendritic spines with (cross-sectionally visualized) diameter Ͼ600 nm.…”

“…3A) (16). Because the number of presynaptic boutons was not altered by memory, the memory-increased mushroom spines form multiple synapses with preexisting axonal boutons that already synapse with other dendritic spines (10,17). The increase in the number of multiple synapse boutons results in an increase in presynaptic vesicle concentration (10).…”

Poststroke neuronal rescue and synaptogenesis mediated in vivo by protein kinase C in adult brains

“…Experimental cerebral ischemia is usually induced focally through, for example, occlusion of the middle cerebral artery, or globally, through permanent occlusion of two arteries (bilateral vertebral arteries in a 4-vessel occlusion model or carotid arteries) associated with a controlled period of occlusion of the other two arteries (bilateral carotid arteries), hypotension, or hypoxia (6, 15). In young adult animals, the combined procedure is essential to induce ischemic pathophysiologic consequences (6, 16-19).Using confocal and electron microscopy, the potent PKC activator bryostatin was recently shown to enhance precisely those structural and ultrastructural changes in hippocampal synapses specifically affecting rat spatial maze learning and memory (20). Based on that study and other studies (21-24), as well as on previous observations of ischemia/hypoxia-induced damage to CA1 hippocampal neurons (14), we investigated the possibility that bryostatin also may reduce or prevent the aforementioned neuronal and synaptic damage in the postischemic brain.…”

“…Bryostatin-1, which crosses the blood-brain barrier when administered peripherally (28), produces a relatively selective activation of the PKC isozyme, which is neuroprotective (29-31) and has a lower median effective dose than PKC␦ for bryostatin-1 in its translocation (thus activation), whereas PKC␦ is most likely involved in ischemic injury during ischemia-reperfusion (32-34). We recently showed that PKC activation with bryostatin-1 enhanced exactly those synaptogenesis, presynaptic/postsynaptic ultrastructural specialization, and protein synthesis functions involved in rat maze learning and memory (20,35). PKC activation has at least two actions that may be relevant to PKC signal processing: activation of PKC isozymes at low concentrations and protection of active, membrane-bound PKC from degradation under stress.…”