Stephen G. Turney

Laminin stimulates and guides axonal outgrowth via growth cone myosin II activity

Guidance cues and signal transduction mechanisms acting at the nerve growth cone are fairly well understood, but the intracellular mechanisms operating to change the direction of axon outgrowth remain unknown. We now show that growth cones integrate myosin II–dependent contraction for rapid, coordinated turning at borders of laminin stripes in response to signals from laminin-activated integrin receptors; in the absence of myosin II activity, outgrowth continues across the borders.

Reversing the Outcome of Synapse Elimination at Developing Neuromuscular Junctions In Vivo: Evidence for Synaptic Competition and Its Mechanism

Competition between neurons for the same synaptic sites at the developing neuromuscular junction drives synaptic rearrangements.

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.

A quantitative fluorescence-imaging technique for studying acetylcholine receptor turnover at neuromuscular junctions in living animals

We have developed a technique to measure changes in the amount of fluorescently labeled acetylcholine receptors in living muscles over long time periods. The measurements of fluorescence are made relative to a novel, photolytically stable fluorescence standard (Spectralon) which allows changes in fluorescence to be followed over days, even months. The method compensates for spatial and temporal variations in image brightness due to the light source, microscope, and camera. We use this approach to study the turnover of fluorescently labeled acetylcholine receptors at a single neuromuscular junction in a living mouse by re-imaging the same junction in situ over a period of 3 weeks. In addition we show that the SIT video camera, which is generally considered inadequate for quantitative imaging (in comparison to CCD cameras), is actually a very good quantitative device, especially in situations requiring both fast acquisition and high resolution.

Axo-axonic synapses formed by somatostatin-expressing GABAergic neurons in rat and monkey visual cortex.

In cerebral cortex of rat and monkey, the neuropeptide somatostatin (SOM) marks a population of nonpyramidal cells (McDonald et al. [1982] J. Neurocytol. 11:809–824; Hendry et al. [1984] J. Neurosci. 4:2497:2517; Laemle and Feldman [1985] J. Comp. Neurol. 233:452–462; Meineke and Peters [1986] J. Neurocytol. 15:121–136; DeLima and Morrison [1989] J. Comp. Neurol. 283:212–227) that represent a distinct type of γ‐aminobutyric acid (GABA) ‐ergic neuron (Gonchar and Burkhalter [1997] Cereb. Cortex 7:347–358; Kawaguchi and Kubota [1997] Cereb. Cortex 7:476–486) whose synaptic connections are incompletely understood. The organization of inhibitory inputs to the axon initial segment are of particular interest because of their role in the suppression of action potentials (Miles et al. [1996] Neuron 16:815:823). Synapses on axon initial segments are morphologically heterogeneous (Peters and Harriman [1990] J. Neurocytol. 19:154–174), and some terminals lack parvalbumin (PV) and contain calbindin (Del Rio and DeFelipe [1997] J. Comp. Neurol. 342:389–408), that is also expressed by many SOM‐immunoreactive neurons (Kubota et al. [1994] Brain Res. 649:159–173; Gonchar and Burkhalter [1997] Cereb. Cortex 7:347–358). We studied the innervation of pyramidal neurons by SOM neurons in rat and monkey visual cortex and examined putative contacts by confocal microscopy and determined synaptic connections in the electron microscope. Through the confocal microscope, SOM‐positive boutons were observed to form close appositions with somata, dendrites, and spines of intracortically projecting pyramidal neurons of rat area 17 and pyramidal cells in monkey striate cortex. In addition, in rat and monkey, SOM boutons were found to be associated with axon initial segments of pyramidal neurons. SOM axon terminals that were apposed to axon initial segments of pyramidal neurons lacked PV, which was shown previously to label axo‐axonic terminals provided by chandelier cells (DeFelipe et al. [1989] Proc. Natl. Acad. Sci. USA 86:2093–2097; Gonchar and Burkhalter [1999a] J. Comp. Neurol. 406:346:360). Electron microscopic examination directly demonstrated that SOM axon terminals form symmetric synapses with the initial segments of pyramidal cells in supragranular layers of rat and monkey primary visual cortex. These SOM synapses differed ultrastructurally from the more numerous unlabeled symmetric synapses found on initial segments. Postembedding immunostaining revealed that all SOM axon terminals contained GABA. Unlike PV‐expressing chandelier cell axons that innervate exclusively initial segments of pyramidal cell axons, SOM‐immunoreactive neurons innervate somata, dendrites, spines, and initial segments, that are just one of their targets. Thus, SOM neurons may influence synaptic excitation of pyramidal neurons at the level of synaptic inputs to dendrites as well as at the initiation site of action potential output. J. Comp. Neurol. 443:1–14, 2002.

Interactive Histology of Large-Scale Biomedical Image Stacks

Histology is the study of the structure of biological tissue using microscopy techniques. As digital imaging technology advances, high resolution microscopy of large tissue volumes is becoming feasible; however, new interactive tools are needed to explore and analyze the enormous datasets. In this paper we present a visualization framework that specifically targets interactive examination of arbitrarily large image stacks. Our framework is built upon two core techniques: display-aware processing and GPU-accelerated texture compression. With display-aware processing, only the currently visible image tiles are fetched and aligned on-the-fly, reducing memory bandwidth and minimizing the need for time-consuming global pre-processing. Our novel texture compression scheme for GPUs is tailored for quick browsing of image stacks. We evaluate the usability of our viewer for two histology applications: digital pathology and visualization of neural structure at nanoscale-resolution in serial electron micrographs.

Myosin II Regulates Activity Dependent Compensatory Endocytosis at Central Synapses

Recent evidence suggests that endocytosis, not exocytosis, can be rate limiting for neurotransmitter release at excitatory CNS synapses during sustained activity and therefore may be a principal determinant of synaptic fatigue. At low stimulation frequencies, the probability of synaptic release is linked to the probability of synaptic retrieval such that evoked release results in proportional retrieval even for release of single synaptic vesicles. The exact mechanism by which the retrieval rates are coupled to release rates, known as compensatory endocytosis, remains unknown. Here we show that inactivation of presynaptic myosin II (MII) decreases the probability of synaptic retrieval. To be able to differentiate between the presynaptic and postsynaptic functions of MII, we developed a live cell substrate patterning technique to create defined neural circuits composed of small numbers of embryonic mouse hippocampal neurons and physically isolated from the surrounding culture. Acute application of blebbistatin to inactivate MII in circuits strongly inhibited evoked release but not spontaneous release. In circuits incorporating both control and MIIB knock-out cells, loss of presynaptic MIIB function correlated with a large decrease in the amplitude of evoked release. Using activity-dependent markers FM1–43 and horseradish peroxidase, we found that MII inactivation greatly slowed vesicular replenishment of the recycling pool but did not impede synaptic release. These results indicate that MII-driven tension or actin dynamics regulate the major pathway for synaptic vesicle retrieval. Changes in retrieval rates determine the size of the recycling pool. The resulting effect on release rates, in turn, brings about changes in synaptic strength.

Nonmuscle myosin II is a critical regulator of clathrin-mediated endocytosis.

Variable requirements for actin during clathrin‐mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) stimulation of embryonic mouse sensory axon outgrowth is MII dependent. NGF regulates two actomyosin processes: transverse actin bundling and peripheral retrograde (radial) network actin flow. These two processes oppose microtubule advance and differentially involve MIIA and MIIB, respectively.

Chapter 11: Imaging fluorescent mice in vivo using confocal microscopy.

In vivo imaging is the most direct way to uncover the dynamic events that occur during neural development. This approach is especially challenging in developing mammals where technical hurdles related to optical resolution, animal movement, phototoxicity, and postoperative complications need to be addressed. In our work concerning the process of naturally occurring synapse elimination at developing neuromuscular junctions, these technical issues are critical because we need to resolve multiple and very fine single axons that converge on the same synaptic site. In previous studies, we used wide-field microscopy with either intensified or high quantum efficiency cameras. We now have begun to use laser scanning confocal microscopy which improves contrast and resolution but comes with its own challenges. In this chapter, we describe the approaches we have taken to permit both rapid time-lapse (minutes to hours) and long-term time-lapse (days to months) to visualize the synaptic alterations associated with the development and maturation of the neuromuscular system.