Axel Nimmerjahn

Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.

The Role of Microglia in the Healthy Brain

Microglia were recently shown to play unexpected roles in normal brain development and adult physiology. This has begun to dramatically change our view of these resident “immune” cells. Here, we briefly review topics covered in our 2011 Society for Neuroscience minisymposium “The Role of Microglia in the Healthy Brain.” This summary is not meant to be a comprehensive review of microglia physiology, but rather to share new results and stimulate further research into the cellular and molecular mechanisms by which microglia influence postnatal development, adult neuronal plasticity, and circuit function.

Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo

It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from α-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

Miniaturized integration of a fluorescence microscope

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

High-speed, miniaturized fluorescence microscopy in freely moving mice

A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca2+ dynamics.

Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective

We present a miniature, flexible two-photon microscope consisting of a fused coherent optical fiber bundle with 30,000 cores and a gradient-index lens objective. The laser focus of a standard two-photon laser-scanning microscope was scanned over the entrance surface of the fiber bundle, resulting in sequential coupling into individual cores. Fluorescent light was detected through the fiber bundle. Micrometer-sized fluorescent beads and pollen grains were readily resolved. In addition, fluorescently labeled blood vessels were imaged through the fiber bundle in rat brain in vivo.

Motor Behavior Activates Bergmann Glial Networks

Although it is firmly established that neuronal activity is a prime determinant of animal behavior, relationships between astrocytic excitation and animal behavior have remained opaque. Cerebellar Bergmann glia are radial astrocytes that are implicated in motor behavior and exhibit Ca(2+) excitation. However, Ca(2+) excitation in these cells has not previously been studied in behaving animals. Using two-photon microscopy we found that Bergmann glia exhibit three forms of Ca(2+) excitation in awake, behaving mice. Two of these are ongoing within the cerebellar vermis. During locomotor performance concerted Ca(2+) excitation arises in networks of at least hundreds of Bergmann glia extending across several hundred microns or more. Concerted Ca(2+) excitation was abolished by anesthesia or blockade of either neural activity or glutamatergic transmission. Thus, large networks of Bergmann glia can be activated by specific animal behaviors and undergo excitation of sufficient magnitude to potentially initiate macroscopic changes in brain dynamics or blood flow.

In Vivo Calcium Imaging of Circuit Activity in Cerebellar Cortex

In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-d-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.

Stepwise Recruitment of Transcellular and Paracellular Pathways Underlies Blood-Brain Barrier Breakdown in Stroke

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.

Distortion-free delivery of nanojoule femtosecond pulses from a Ti:sapphire laser through a hollow-core photonic crystal fiber

We demonstrate propagation of femtosecond pulses in the 800-nm range through a hollow-core photonic crystal fiber with preserved temporal and spectral profiles for pulse energies up to 4.6 nJ. Without the use of a prechirping unit, 170-fs pulses were transmitted essentially undistorted at 812 nm, near the zero-dispersion wavelength. Because of the air guidance of pulses, intensity-dependent nonlinear effects were minimal, with only 15% pulse broadening occurring at 350-mW average output power. This fiber thus is excellently suited for applications that require single-mode delivery of high-energy ultrashort pulses to the fiber output face such as, for example, miniaturized multiphoton microscopes.