Derron L. Bishop

Near-infrared branding efficiently correlates light and electron microscopy

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

Lysosomal activity associated with developmental axon pruning.

Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning.

An assay to image neuronal microtubule dynamics in mice

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

In Vivo Imaging of Dorsal Root Regeneration: Rapid Immobilization and Presynaptic Differentiation at the CNS/PNS Border

Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.

We describe a method for in vivo confocal fluorescence imaging of synaptic terminals and subsequent electron microscopic reconstructions of the same terminals. By iontophoretically applying lipophilic dye to nerve terminals at a single neuromuscular junction with a sharp microelectrode in living neonatal mice, we were able to quickly label other synaptic terminals of the same motor unit. This vital labeling technique allows the same synapses to be imaged in living animals for several days. By using two dyes applied to separate junctions we could visualize competing axons converging at the same site. We also show that similar approaches can be used to study synaptic inputs to neurons. Following photoconversion, the dye labeled axons and synapses were easily identified and distinguished from unlabeled synapses of other axons ultrastructurally. This new labeling technique thus provides a useful means to study reorganization of synaptic structure at high temporal and spatial resolution.

Simvastatin Inhibits Staphylococcus aureus Host Cell Invasion through Modulation of Isoprenoid Intermediates

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.

The Effects of Denervation Location on Fiber Type Mix in Self-Reinnervated Mouse Soleus Muscles

Mouse soleus muscles were denervated by crushing the soleus nerve where it enters the muscle to determine if denervation followed by self-reinnervation can permanently alter the mix of fiber types in a muscle. Reinnervated and contralateral control muscles were sectioned at 2 and 7 months postdenervation and histochemically stained for myosin ATPase to determine the percentages of fast and slow twitch fibers in the muscles. It was found that, at both 2 and 7 months postdenervation, reinnervated muscles had a significantly higher percentage of slow twitch fibers than did contralateral control muscles (86.7 versus 67.8% at 2 months and 90.0 versus 69.3% at 7 months). Soleus muscles were also denervated by crushing the soleus nerve where it exists the gastrocnemius muscle (approximately 4 mm proximal to where the nerve enters the soleus muscle) to ascertain if the location of the nerve lesions plays a role in the ultimate outcome of the process of self-reinnervation. Reinnervated muscles and their contralateral muscles were sectioned at 2 months postdenervation and histochemically stained for myosin ATPase as before. It was found that, in contrast to muscles denervated at the point of nerve entry, muscles denervated 4 mm more proximal exhibited only a small increase in their percentage of slow twitch fibers which was not statistically significant (71.4 versus 68.4%). These results suggest that denervation followed by self-reinnervation can cause a permanent change in a muscles fibers type mix and that the location of the nerve lesion strongly influences the final outcome of the reinnervation process.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination

Summary Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Nimodipine Suppresses Preferential Reinnervation of Mouse Soleus Muscles by Slow α-Motoneurons

Denervation of mouse soleus muscle followed by self-reinnervation causes a significant increase in slow twitch (type I) muscle fiber content, suggesting preferential reinnervation by slow α-motoneurons. Since intracellular Ca2+influences both axonal elongation rate and branching, we examined the process of self-reinnervation in mouse soleus muscles in the presence of the L-type Ca2+channel blocker nimodipine. Soleus muscles in both control and experimental animals were denervated by crushing the soleus nerve where it enters the muscle. Experimental animals received a daily i.p. injection of a 0.1% nimodipine solution beginning 4 days prior to denervation and ending 2 weeks postdenervation. At 2 months postdenervation reinnervated and contralateral muscles from both control and experimental animals were sectioned and histochemically stained for myosin ATPase to determine the percentage of slow twitch fibers in the muscles. It was found that, in agreement with previous experiments, untreated reinnervated muscles had a significantly higher percentage of slow twitch fibers than did their contralateral controls (91.3 versus 74.6%). However, in nimodipine-treated animals only a small, but not statistically significant, difference between reinnervated and contralateral control muscles was observed (76.5 versus 72.8%). These results suggest that Ca2+influx through L-type calcium channels in growing neurites may play a role in the outcome of the reinnervation process.

Glial imaging during synapse remodeling at the neuromuscular junction.

Glia are an indispensable structural and functional component of the synapse. They modulate synaptic transmission and also play important roles in synapse formation and maintenance. The vertebrate neuromuscular junction (NMJ) is a classic model synapse. Due to its large size, simplicity and accessibility, the NMJ has contributed greatly to our understanding of synapse development and organization. In the past decade, the NMJ has also emerged as an effective model for studying glia-synapse interactions, in part due to the development of various labeling techniques that permit NMJs and associated Schwann cells (the glia at NMJs) to be visualized in vitro and in vivo. These approaches have demonstrated that Schwann cells are actively involved in synapse remodeling both during early development and in post-injury reinnervation. In vivo imaging has also recently been combined with serial section transmission electron microscopic (ssTEM) reconstruction to directly examine the ultrastructural organization of remodeling NMJs. In this review, we focus on the anatomical studies of Schwann cell dynamics and their roles in formation, maturation and remodeling of vertebrate NMJs using the highest temporal and spatial resolution methods currently available.