Semi-automated reconstruction of neural processes from large numbers of fluorescence images.

We introduce a method for large scale reconstruction of complex bundles of neural processes from fluorescent image stacks. We imaged yellow fluorescent protein labeled axons that innervated a whole muscle, as well as dendrites in cerebral cortex, in transgenic mice, at the diffraction limit with a confocal microscope. Each image stack was digitally re-sampled along an orientation such that the majority of axons appeared in cross-section. A region growing algorithm was implemented in the open-source Reconstruct software and applied to the semi-automatic tracing of individual axons in three dimensions. The progression of region growing is constrained by user-specified criteria based on pixel values and object sizes, and the user has full control over the segmentation process. A full montage of reconstructed axons was assembled from the approximately 200 individually reconstructed stacks. Average reconstruction speed is approximately 0.5 mm per hour. We found an error rate in the automatic tracing mode of approximately 1 error per 250 um of axonal length. We demonstrated the capacity of the program by reconstructing the connectome of motor axons in a small mouse muscle.

Mechanisms of amyloid plaque pathogenesis.

The first ultrastructural investigations of Alzheimer's disease noted the prominence of degenerating mitochondria in the dystrophic neurites of amyloid plaques, and speculated that this degeneration might be a major contributor to plaque pathogenesis. However, the fate of these organelles has received scant consideration in the intervening decades. A number of hypotheses for the formation and progression of amyloid plaques have since been suggested, including glial secretion of amyloid, somal and synaptic secretion of amyloid-beta protein from neurons, and endosomal-lysosomal aggregation of amyloid-beta protein in the cell bodies of neurons, but none of these hypotheses fully account for the focal accumulation of amyloid in plaques. In addition to Alzheimer's disease, amyloid plaques occur in a variety of conditions, and these conditions are all accompanied by dystrophic neurites characteristic of disrupted axonal transport. The disruption of axonal transport results in the autophagocytosis of mitochondria without normal lysosomal degradation, and recent evidence from aging, traumatic injury, Alzheimer's disease and transgenic mice models of Alzheimer's disease, suggests that the degeneration of these autophagosomes may lead to amyloid production within dystrophic neurites. The theory of amyloid plaque pathogenesis has thus come full circle, back to the intuitions of the very first researchers in the field.

Mitochondrial degeneration in dystrophic neurites of senile plaques may lead to extracellular deposition of fine filaments.

Recent data show that amyloid precursor protein accumulates inside axons after disruption of fast axonal transport, but how this leads to mature plaques with extracellular amyloid remains unclear. To investigate this issue, primitive plaques in prefrontal cortex of aged rhesus monkeys were reconstructed using serial section electron microscopy. The swollen profiles of dystrophic neurites were found to be diverticula from the main axis of otherwise normal neurites. Microtubules extended from the main neurite axis into the diverticulum to form circular loops or coils, providing a transport pathway for trapping organelles. The quantity and morphology of organelles contained within diverticula suggested a progression of degeneration. Primitive diverticula contained microtubules and normal mitochondria, while larger, presumably older, diverticula contained large numbers of degenerating mitochondria. In advanced stages of degeneration, apparent autophagosomes derived from mitochondria exhibited a loose lamellar to filamentous internal structure. Similar filamentous material and remnants of mitochondria were visible in the extracellular spaces of plaques. This progression of degeneration suggests that extracellular filaments originate inside degenerating mitochondria of neuritic diverticula, which may be a common process in diverse diseases.

Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro.

Hippocampal slices often have more synapses than perfusion-fixed hippocampus, but the cause of this synaptogenesis is unclear. Ultrastructural evidence for synaptogenic triggers during slice preparation was investigated in 21-day-old rats. Slices chopped under warm or chilled conditions and fixed after 0, 5, 25, 60, or 180 minutes of incubation in an interface chamber were compared with hippocampi fixed by perfusion or by immersion of the whole hippocampus. There was no significant synaptogenesis in these slices compared with perfusion-fixed hippocampus, but there were other structural changes during slice preparation and recovery in vitro. Whole hippocampus and slices prepared under warm conditions exhibited an increase in axonal coated vesicles, suggesting widespread neurotransmitter release. Glycogen granules were depleted from astrocytes and neurons in 0-min slices, began to reappear by 1 hour, and had fully recovered by 3 hours. Dendritic microtubules were initially disassembled in slices, but reassembled into normal axial arrays after 5 minutes. Microtubules were short at 5 minutes (12.3 +/- 1.1 microm) but had recovered normal lengths by 3 hours (84.6 +/- 20.0 microm) compared with perfusion-fixed hippocampus (91 +/- 22 microm). Microtubules appeared transiently in 15 +/- 3% and 9 +/- 4% of dendritic spines 5 and 25 minutes after incubation, respectively. Spine microtubules were absent from perfusion-fixed hippocampus and 3-hour slices. Ice-cold dissection and vibratomy in media that blocked activity initially produced less glycogen loss, coated vesicles, and microtubule disassembly. Submersing these slices in normal oxygenated media at 34 degrees C led to glycogen depletion, as well as increased coated vesicles and microtubule disassembly within 1 minute.

Structural changes at dendritic spine synapses during long-term potentiation.

Two key hypotheses about the structural basis of long-term potentiation (LTP) are evaluated in light of new findings from immature rat hippocampal slices. First, it is shown why dendritic spines do not split during LTP. Instead a small number of spine-like dendritic protrusions may emerge to enhance connectivity with potentiated axons. These 'same dendrite multiple synapse boutons' provide less than a 3% increase in connectivity and do not account for all of LTP or memory, as they do not accumulate during maturation. Second, polyribosomes in dendritic spines served to identify which of the existing synapses enlarged to sustain more than a 30% increase in synaptic strength. Thus, both enhanced connectivity and enlarged synapses result during LTP, with synapse enlargement being the greater effect.

Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices.

The presence of polyribosomes in dendritic spines suggests a potential involvement of local protein synthesis in the modification of synapses. Dendritic spine and synapse ultrastructure were compared after low-frequency control or tetanic stimulation in hippocampal slices from postnatal day (P)15 rats. The percentage of spines containing polyribosomes increased from 12% +/- 4% after control stimulation to 39% +/- 4% after tetanic stimulation, with a commensurate loss of polyribosomes from dendritic shafts at 2 hr posttetanus. Postsynaptic densities on spines containing polyribosomes were larger after tetanic stimulation. Local protein synthesis might therefore serve to stabilize stimulation-induced growth of the postsynaptic density. Furthermore, coincident polyribosomes and synapse enlargement might indicate spines that are expressing long-term potentiation induced by tetanic stimulation.

Dendritic spine pathology: cause or consequence of neurological disorders?

Altered dendritic spines are characteristic of traumatized or diseased brain. Two general categories of spine pathology can be distinguished: pathologies of distribution and pathologies of ultrastructure. Pathologies of spine distribution affect many spines along the dendrites of a neuron and include altered spine numbers, distorted spine shapes, and abnormal loci of spine origin on the neuron. Pathologies of spine ultrastructure involve distortion of subcellular organelles within dendritic spines. Spine distributions are altered on mature neurons following traumatic lesions, and in progressive neurodegeneration involving substantial neuronal loss such as in Alzheimer's disease and in Creutzfeldt-Jakob disease. Similarly, spine distributions are altered in the developing brain following malnutrition, alcohol or toxin exposure, infection, and in a large number of genetic disorders that result in mental retardation, such as Down's and fragile-X syndromes. An important question is whether altered dendritic spines are the intrinsic cause of the accompanying neurological disturbances. The data suggest that many categories of spine pathology may result not from intrinsic pathologies of the spiny neurons, but from a compensatory response of these neurons to the loss of excitatory input to dendritic spines. More detailed studies are needed to determine the cause of spine pathology in most disorders and relationship between spine pathology and cognitive deficits.

Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane.

Endosomes are essential to dendritic and synaptic function in sorting membrane proteins for degradation or recycling, yet little is known about their locations near synapses. Here, serial electron microscopy was used to ascertain the morphology and distribution of all membranous intracellular compartments in distal dendrites of hippocampal CA1 pyramidal neurons in juvenile and adult rats. First, the continuous network of smooth endoplasmic reticulum (SER) was traced throughout dendritic segments and their spines. SER occupied the cortex of the dendritic shaft and extended into 14% of spines. Several types of non-SER compartments were then identified, including clathrin-coated vesicles and pits, large uncoated vesicles, tubular compartments, multivesicular bodies (MVBs), and MVB-tubule complexes. The uptake of extracellular gold particles indicated that these compartments were endosomal in origin. Small, round vesicles and pits that did not contain gold were also identified. The tubular compartments exhibited clathrin-coated tips consistent with the genesis of these small, presumably exosomal vesicles. Approximately 70% of the non-SER compartments were located within or at the base of dendritic spines. Overall, only 29% of dendritic spines had endosomal compartments, whereas 20% contained small vesicles. Small vesicles did not colocalize in spines with endosomes or SER. Three-dimensional reconstructions revealed that up to 20 spines shared a recycling pool of plasmalemmal proteins rather than maintaining independent stores at each spine.