Preparation of the mouse spinal column for single imaging using two-photon laser-scanning microscopy.

Two-photon imaging of the nervous system is now used extensively for visualizing brain dynamics and signal activities. To date, scientists have focused on the analysis either of gray matter forebrain structures, such as the cortex and cerebellum, or they have investigated muscle innervation of peripheral nerves. The spinal cord is an ideal structure to use for imaging central nervous system white matter. The dorsal columns formed by myelinated sensory axons are located directly at the surface of the spinal cord underneath the pia mater. Neuronal fibers and neighboring glial cells can be imaged in transgenic mice using cell type-specific fluorescent protein expression. This protocol describes the anesthesia and surgical procedures necessary to prepare the mouse spinal column so that neurons and glia in the spinal cord can be imaged using two-photon laser-scanning microscopy (2pLSM). These procedures are ideal for single-imaging experiments in which the spinal cord needs to be imaged at optimal spatial resolution with minimal motion artifacts.

Preparation of the mouse spinal column for repetitive imaging using two-photon laser-scanning microscopy.

Two-photon imaging of the nervous system is now used extensively for visualizing brain dynamics and signal activities. To date, scientists have focused on the analysis either of gray matter forebrain structures, such as the cortex and cerebellum, or they have investigated muscle innervation of peripheral nerves. The spinal cord is an ideal structure to use for imaging central nervous system white matter. The dorsal columns formed by myelinated sensory axons are located directly at the surface of the spinal cord underneath the pia mater. Neuronal fibers and neighboring glial cells can be imaged in transgenic mice using cell type-specific fluorescent protein expression. This protocol describes the anesthesia and surgical procedures necessary to prepare the mouse spinal column so that neurons and glia in the spinal cord can be imaged using two-photon laser-scanning microscopy (2pLSM). These procedures are ideal for repetitive-imaging experiments in which the spinal cord is to be imaged repetitively over multiple days, with time for the mouse to recover between imaging sessions.

In vivo two-photon imaging of neurons and glia in the mouse spinal cord.

Two-photon imaging of the nervous system is now used extensively for visualizing brain dynamics and signal activities. To date, scientists have focused on the analysis either of gray matter forebrain structures, such as the cortex and cerebellum, or they have investigated muscle innervation of peripheral nerves. The spinal cord is an ideal structure to use for imaging central nervous system white matter. The dorsal columns formed by myelinated sensory axons are located directly at the surface of the spinal cord underneath the pia mater. This protocol describes a method for imaging neuronal fibers and neighboring glial cells in transgenic mice using cell type-specific fluorescent protein expression and two-photon laser-scanning microscopy (2pLSM). Depending on how the mice are prepared, single imaging can be performed, or the spinal cord can be imaged repetitively over multiple days, with time for the mouse to recover between imaging sessions.

Two-color STED microscopy of living synapses using a single laser-beam pair.

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.

In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice.

The vesicular glutamate transporter VGLUT1 loads synaptic vesicles with the neurotransmitter glutamate and thereby determines glutamate release at many synapses in the mammalian brain. Due to its function and selective localization, VGLUT1 is one of the most specific markers for glutamatergic synaptic vesicles. It has been used widely to identify glutamatergic synapses, and its expression levels are tightly correlated with changes in quantal size, modulations of synaptic plasticity, and corresponding behaviors. We generated a fluorescent VGLUT1(Venus) knock-in mouse for the analysis of VGLUT1 and glutamatergic synaptic vesicle trafficking. The mutation does not affect glutamatergic synapse function, and thus the new mouse model represents a universal tool for the analysis of glutamatergic transmitter systems in the forebrain. Previous studies demonstrated synaptic vesicle exchange between terminals in vitro. Using the VGLUT1(Venus) knock-in, we show that synaptic vesicles are dynamically shared among boutons in the cortex of mice in vivo. We provide a detailed analysis of synaptic vesicle sharing in vitro, and show that network homeostasis leads to dynamic scaling of synaptic VGLUT1 levels.

In Vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS.

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1(G93A) mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ∼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.

Purinergic activation of dorsal root ganglion neurones in vivo.

Functional relevance of non-synaptic purinergic receptors on dorsal root ganglion cells was tested in vivo by the influence of ATP using 2P-LSM and Ca imaging. Within a few seconds after local application of ATP, neurones in dorsal root ganglion were activated indicated by an increase of their calcium signal. The signal reached its maximum within a few seconds and declined to control values after about 30 s. Purinergic action seems to include non-synaptic cell-to-cell communication within dorsal root ganglia.

Long-lasting post-mortem activity of spinal microglia in situ in mice.

As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion-directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser-induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post-mortem, in the progressively degrading nervous tissue. For this purpose, we used time-lapse two-photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5-10 hr post-mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post- mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue.

NO mediates microglial response to acute spinal cord injury under ATP control in vivo.

To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two-photon laser-scanning microscopy (2P-LSM), we identified the messenger nitric oxide (NO) as a modulator of injury-activated microglia. Local tissue damages evoked by high-power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO-donor spermine NONOate (SPNO) or the NO-dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High-tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.

Chronically CNS-injured adult sensory neurons gain regenerative competence upon a lesion of their peripheral axon.

Several experimental manipulations result in axonal regeneration in the central nervous system (CNS) when applied before or at the time of injury but not when initiated after a delay, which would be clinically more relevant. As centrally injured neurons show signs of atrophy and degeneration, it raises the question whether chronically injured neurons are able to regenerate. To address this question, we used adult rodent primary sensory neurons that regenerate their central axon when their peripheral axon is cut (called conditioning) beforehand but not afterwards. We found that primary sensory neurons express regeneration-associated genes and efficiently regrow their axon in cell culture two months after a central lesion upon conditioning. Moreover, conditioning enables central axons to regenerate through a fresh lesion independent of a previous central lesion. Using in vivo imaging we demonstrated that conditioned neurons rapidly regrow their axons through a fresh central lesion. Finally, when single sensory axons were cut with a two-photon laser, they robustly regenerate within days after attaining growth competence through conditioning. We conclude that sensory neurons can acquire the intrinsic potential to regenerate their axons months after a CNS lesion, which they implement in the absence of traumatic tissue.

Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles.

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing.

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.