Fritjof Helmchen

Deep tissue two-photon microscopy

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional—including confocal—fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

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.

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

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.

Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat.

1. Simultaneous whole‐cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0.4 ms at 36 degrees C and 0.9 ms at 23‐24 degrees C. The amplitude of the L‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor‐mediated component of the excitatory postsynaptic currents (EPSCs) was 2‐13 nA (at ‐80 mV). 3. Current‐voltage relations showed that presynaptic Ca2+ channels were of the high voltage‐activated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0.3‐0.7 s, depending on the concentration (60‐200 microM) of the Ca2+ indicator Calcium Green‐5N (CG‐5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole‐cell configuration in the presynaptic terminal when the pipette contained 50 microM BAPTA. In contrast, with 1 mM BAPTA, peak amplitudes of EPSCs were reduced to one‐third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50 microM BAPTA, whereas depression was reduced with 1 mM BAPTA. 7. In low (1 mM) [Ca2+]o, facilitation instead of depression of EPSCs was observed. 8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2+ buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.

In vivo dendritic calcium dynamics in deep layer cortical pyramidal neurons

Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.

Calcium dynamics associated with a single action potential in a CNS presynaptic terminal

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.

Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance

UNLABELLED An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity. VIDEO ABSTRACT

Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones.

1. Simultaneous measurements of intracellular free calcium concentration ([Ca2+]i) and intrasomatic and intradendritic membrane potential (Vm) were performed using fura‐2 fluorimetry and whole‐cell recording in neocortical layer V pyramidal neurones in rat brain slices. 2. Back‐propagating action potentials (APs) evoked [Ca2+]i transients in the entire neurone including the soma, the axon initial segment, the apical dendrite up to the distal tuft branches, and the oblique and basal dendrites, indicating that following suprathreshold activation the entire dendritic tree is depolarized sufficiently to open voltage‐dependent calcium channels (VDCCs). 3. The [Ca2+]i transient peak evoked by APs showed large differences between various compartments of the neurone. Following a single AP, up to 6‐fold differences were measured, ranging from 43 +/‐ 14 nM in the soma to 267 +/‐ 109 nM in the basal dendrites. 4. Along the main apical dendrite, the [Ca2+]i transients evoked by single APs or trains of APs had the largest amplitude and the fastest decay in the proximal region; the [Ca2+]i transient peak and decay time constant following a single AP were 128 +/‐ 25 nM and 420 +/‐ 150 ms, respectively, and following a train of five APs (at 10‐12 Hz), 710 +/‐ 214 nM and 390 +/‐ 150 ms, respectively. The [Ca2+]i transients gradually decreased in amplitude and broadened in more distal portions of the apical dendrite up to the main bifurcation. 5. In the apical tuft branches, the profile of the [Ca2+]i transients was dependent on AP frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo

In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two–photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action–potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learning.