Bio-sample environment manipulation using advanced microscopy techniques

2010/2011Under physiological conditions in the brain, molecules are released with high spatial and temporal resolution. A lot of efforts have been done in the last years in order to develop techniques that mimic this situation. Among them, we mention the use of micropipettes for the ejection of fluids, the use of AFM (Atomic Force Microscopy), microfluidic devices and optical manipulation. The latter approach exploits light to manipulate the samples, e.g. to create transient pores in the cell membrane or to move small objects carrying a stimulus. This Thesis concerns with the development of new techniques for the local delivery of molecules based on optical manipulation technologies, and in particular on optical tweezers. Sub-micrometer particles in a compact trap, such as the single-beam gradient or optical tweezers, can be localized within a small fraction of a wavelength of light or moved over long distances of many centimeters without any mechanical contact. A three-dimensional trap is simply created by focusing a laser beam through a microscope objective with high numerical aperture. We studied three types of vectors for local delivery of molecules, which can be optically manipulated: microbeads, micron-sized liposomes and Quantum dots (Qdots). Silica microbeads can be covalently functionalized on their surface with the protein of interest and placed in contact with the desired part of a cell. In order to validate the technique, we functionalized beads with a secretory molecule, the neurotrophin Brain-derived neurotrophic factor (BDNF). BDNF is a key regulator of neuronal development and plasticity. We showed that single BDNF-coated microbeads can be extracted with optical tweezers from small reservoirs and positioned with submicrometric precision to specific sites on the dendrites of cultured hippocampal neurons. Localized contact of microbeads functionalized with BDNF induced focal increase of Calcium signaling in the stimulated dendrite, specific activation of the TrkB receptor pathway and influenced the development of growth cones. Remarkably, a single BDNF-coated bead positioned on a dendrite was found to be enough for TrkB phosphorylation, an efficient and long-lasting activation of Calcium signaling in the soma, and c-Fos signaling in the nucleus, comparable to bath stimulation conditions. Moreover, since BDNF is covalently cross-linked to the bead surface we could demonstrate that activation of some of the TrkB receptor pathway does not necessarily require BDNF endocytosis. In the case of liposomes, the molecules of interest were encapsulated within their lumen. Single liposomes were trapped and transported by means of optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses that broke the liposome membrane. In order to test the effect of the UV-induced release, liposomes with a diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca2+ dye, were monitored by imaging intracellular Ca2+. An efficient release from the liposomes was demonstrated by detectable Calcium signals, indicating induced depolarization of the neuronal cells by KCl. Afterwards, this technique was used to address a biological issue, that is the effect of two proteins (Semaphorin 3A and Netrin-1) on growth cones. The growth cone is an intracellular apparatus located at the tip of the neurite of developing neurons. Its motility governs axonal path-finding and the construction of neuronal networks. Growth cones are highly dynamic structures that respond to external stimuli turning towards or away from the chemical gradient. We were able to demonstrate an attractive effect of Netrin-1 on the growth cones of primary hippocampal neurons. On the contrary, Semaphorin 3A showed a repellant behavior. To correlate the high resolution of vector manipulation with high resolution of imaging we used STimulated Emission Depletion (STED) to investigate the intimate organization of two main cytoskeleton components: actin and tubulin filaments. STED microscopy allowed imaging of actin bundles in the filopodia and organized network in lamellipodia with un-precedent resolution, beyond the diffraction barrier. Lastly, we used liposomes to encapsulate Quantum dots. Qdots are bright and photostable nanocrystals. Due to their small size, similar to that of proteins, Qdots may be endocyted along the receptor-mediated endocytosis pathway, when they are functionalized with the appropriate ligand. As case study we considered the BDNF-TrkB endocytotic pathway. We optimized the protocol for the direct binding of BDNF to Qdots and we demonstrated the possibility of encapsulating and releasing them from liposomes. Concluding, two different approaches for local stimulation of neurons, based on optical manipulation of microvectors, were presented and validated in this thesis. Indirect optical manipulation of nanovectors (Qdots) encapsulated in liposomes has been demonstrated as well. The techniques were then successfully applied to address some biological issues, that in turn required the optimization of other imaging tools (super resolution microscopy and Qdots).XXIV Ciclo198

Optical delivery of liposome encapsulated chemical stimuli to neuronal cells

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca(2+) dye, were monitored by imaging intracellular Ca(2+). An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds

Use of optical tweezers technology for long-term, focal stimulation of specific subcellular neuronal compartments

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Actin waves do not boost neurite outgrowth in the early stages of neuron maturation

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration

Neuron-oligodendrocyte potassium shuttling at nodes of Ranvier protects against

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient (OL-Kir4.1-deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy

Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR-Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging

Fluorogenic probes for live-cell imaging of the cytoskeleton

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells