Changes in intra-axonal calcium distribution following nerve crush

We used the oxalate-pyroantimonate method to demonstrate the ultrastructural distribution of calcium within rat sciatic nerve 4 h after a crush injury. In normal nerve there are discrete gradients of axoplasmic calcium precipitate with the amount of precipitate decreasing in the axoplasm beneath the Schmidt Lantermann clefts and in the paranodal regions at the node of Ranvier. Near the crush site a marked increase in endoneurial and intra-axonal calcium precipitate correlated with morphologic evidence of axonal degeneration. More distant from the crush site, both in the distal segment destined to degenerate and in the proximal segment destined to regenerate, the most prominent finding was a loss of the normal gradient of precipitate beneath the Schmidt Lantermann clefts. The calcium influx at the crush site corresponds to the known role of calcium in triggering degeneration. The alterations in the distal axon may be an early stage leading to degeneration. Alteration in calcium distribution in the proximal nerve stump may play a role in the regulation of the response to injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50077/1/480170508_ftp.pd

Cytochemical localization of Ca2+-ATPase activity in peripheral nerve

We used an electron microscopic cytochemical method to determine the localization of Ca2+-ATPase in rat peripheral nerve. We found that reaction product occurred along most cytoplasmic membranes in the dorsal root ganglia (DRG). Unmyelinated axons demonstrated reaction product on the axolemma diffusely along their length. Myelinated fibers, in contrast, had reaction product limited to the axolema in the paranodal region. Internodal axolemma never showed reaction product and nodal axolemma was only occasionally stained, usually in sections reacted for the maximum times. Schwann cell plasma membranes uniformly showed reaction product. The restricted localization of Ca2+-ATPase to the paranodal region of myelinated fibers suggests that calcium efflux may occur principally at those sites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27368/1/0000394.pd

Long-term effects of innervation at the neuromuscular junction: Induction of localized transcription of adult-type nAChRS.

The mechanisms which define the molecular components, and thus the functional characteristics, of synapses are not known but, the interaction of pre- and post-synaptic elements can induce morphological and functional changes. At the developing neuromuscular junction (NMJ), innervation causes the nicotinic acetylcholine receptor (nAChR) to become concentrated at the NMJ. This ligand-gated ion channel is composed of five protein subunits $(\alpha2\beta\gamma\delta)$ in developing muscle. During development, and coincident with innervation, the $\gamma$-subunit is replaced by an $\varepsilon$-subunit, causing a change in nAChR ion channel properties. Denervation results in the reappearance of $\gamma$-subunit-containing nAChRs in extrajunctional regions of the muscle. Thus, innervation affects synaptic properties by altering the levels, localization and subunit composition of the nAChR. The general aim of the experiments presented in this thesis was to elucidate mechanisms by which the molecular components, and thus the functional characteristics, of a model synapse, the neuromuscular junction, are regulated. I have characterized the role muscle innervation plays in nAChR RNA expression. The main results of this work are: (1) Innervation has a long-lasting effect on localization of nAChR transcripts in skeletal muscle; (2) Epsilon RNA is expressed at times which coincide with innervation and localized at the NMJ following innervation, yet, does not require the nerve to maintain this localized expression; and (3) Increased cAMP does not mediate this local endplate-specific induction but may participate in decreased epsilon gene expression in extrajunctional regions of developing muscle fibers. These results, taken together, show that innervation causes long-term changes in localization of specific transcripts at the NMJ, of which the $\varepsilon$-subunit mRNA is an example, and that this effect is not mediated by cAMP in the rat as had been proposed in previous studies.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105030/1/9624734.pdfDescription of 9624734.pdf : Restricted to UM users only

Morphological and molecular heterogeneity in release sites of single neurons

We have previously shown that labelling intensities for synaptic proteins vary strongly among synaptic boutons. Here we addressed the questions as to whether there are heterogeneous levels of integral membrane synaptic vesicle proteins at distinct active release sites of single neurons and if these sites possess the ultrastructural features of synapses. By double-immunostaining with specific antibodies against synaptophysin, synaptotagmin I, VAMP1 and VAMP2, we identified different relative levels of these integral membrane proteins of synaptic vesicles in comparison to boutons of the same rat cortical neuron. This heterogeneity could also be observed between the two isoforms VAMP1 and VAMP2. By studying pairs of these proteins implicated in neurotransmitter release, including both VAMP isoforms, we also show that the sites that contained predominantly one protein were nevertheless functional, as they internalized and released FM1-43 upon potassium stimulation. Using electron microscopy, we show that these active sites could have either synaptic specializations, or the features of vesicle-containing varicosities without a postsynaptic target. Different varicosities of the same neuron showed different intensities for synaptic vesicle proteins; some varicosities were capable of internalizing and releasing FM1-43, while others were silent. These results show that integral membrane synaptic vesicle proteins are differentially distributed among functional release sites of the same neuron

RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP)

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, making it difficult to understand how RNA structure governs function. Here we describe SHAPE mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2’-hydroxyl acylation by SHAPE are encoded as non-complementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure and was used to define a second-generation model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary structure models, enables analysis of low abundance RNAs, disentangles sequence polymorphisms in single experiments, and will ultimately democratize RNA structure analysis