Controlling FIB-SBEM slice thickness by monitoring the transmitted ion beam.

Serial block-face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack-acquisition time to several days. Here, we describe a method for controlling the ion-beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted-beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion-mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in-column detector is explored.

Automated in-chamber specimen coating for serial block-face electron microscopy.

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally ('sample charging'). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block-face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1-2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal-to-noise ratio (SNR) caused by the film is smaller than that caused by the widely used low-vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non-conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.

In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness.

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly along the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatiotemporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 mum) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4- to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5- to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.

Fly motion vision is based on Reichardt detectors regardless of the signal-to-noise ratio.

The computational structure of an optimal motion detector was proposed to depend on the signal-to-noise ratio (SNR) of the stimulus: At low SNR, the optimal motion detector should be a correlation or "Reichardt" type, whereas at high SNR, the detector would employ a gradient scheme [Potters, M. & Bialek, W. (1994) J. Physiol. (Paris) 4, 1755-1775]. Although a large body of experiments supports the Reichardt detector as the processing scheme leading to direction selectivity in fly motion vision, in most of these studies the SNR was rather low. We therefore reinvestigated the question over a much larger SNR range. Using 2-photon microscopy, we found that local dendritic [Ca(2+)] modulations, which are characteristic of Reichardt detectors, occur in response to drifting gratings over a wide range of luminance levels and contrasts. We also explored, as another fingerprint of Reichardt detectors, the dependence of the velocity optimum on the pattern wavelength. Again, we found Reichardt-typical behavior throughout the whole luminance and contrast range tested. Our results, therefore, provide strong evidence that only a single elementary processing scheme is used in fly motion vision.

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.

Dendritic processing.

Dendritic processing multiplies the computational power of a single neuron by enabling the processing of inputs in a spatio-temporally differentiated manner. Recently, the development of new and refined optical, electrophysiological and molecular-biological techniques has led to new insights into dendritic function and revealed an astonishing plethora of computational mechanisms.

Odour-evoked [Ca2+] transients in mitral cell dendrites of frog olfactory glomeruli.

We measured Ca2+ concentration, [Ca2+], transients in mitral cell distal apical dendritic tufts produced by physiological odour stimulation of the olfactory epithelium and electrical stimulation of the olfactory nerve (ON) using two-photon scanning and conventional wide-field microscopy of Ca2+-Green-1 dextran in an in vitro frog nose-brain preparation. Weak or strong ON shock-evoked fluorescence transients always had short latency with an onset 0-10 ms after the onset of the bulb local field potential, rapidly increasing to a peak of up to 25% fractional fluorescence change (DeltaF/F) in 10-30 ms, were blocked by 10 microM CNQX, decaying with a time constant of about 1 s. With stronger ON shocks that activated many receptor axons, an additional, delayed, sustained AP5-sensitive component (peak at approximately 0.5 s, up to 40% DeltaF/F maximum) could usually be produced. Odour-evoked [Ca2+] transients sometimes displayed a rapid onset phase that peaked within 50 ms but always had a sustained phase that peaked 0.5-1.5 s after onset, regardless of the strength of the odour or the amplitude of the response. These were considerably larger (up to 150% DeltaF/F) than those evoked by ON shock. Odour-evoked [Ca2+] transients were also distinguished from ON shock-evoked transients by tufts in different glomeruli responding with different delays (time to onset differed by up to 1.5 s between different tufts for the same odour). Odour-evoked [Ca2+] transients were increased by AMPA-kainate receptor blockade, but substantially blocked by AP5. Electrical stimulation of the lateral olfactory tract (5-6 stimuli at 10 Hz) that evoked granule cell feedback inhibition, blocked 60-100% of the odour-evoked [Ca2+] transient in tufts when delivered within about 0.5 s of the odour. LOT-mediated inhibition was blocked by 10 microM bicuculline.

Initiation and propagation of calcium-dependent action potentials in a coupled network of olfactory interneurons.

Coherent oscillatory electrical activity and apical-basal wave propagation have been described previously in the procerebral (PC) lobe, an olfactory center of the terrestrial slug Limax maximus. In this study, we investigate the physiological basis of oscillatory activity and wave propagation in the PC lobe. Calcium green dextran was locally deposited in the PC lobe; this led to cellular uptake and transport of dye by bursting and nonbursting neurons of the PC lobe. The change of intracellular calcium concentration was measured at several different positions in neurites of individual bursting neurons in the PC lobe with a two-photon laser-scanning microscope. Fluorescence measurements were also made from neurons intracellularly injected with calcium green-1. Two different morphological classes of bursting neurons were found, varicose (VB) and smooth (SB). Our results from concurrent optical and intracellular recordings suggest that Ca2+ is the major carrier for the inward current during action potentials of bursting neurons. Intracellular recordings from bursting neurons with nystatin perforated-patch electrodes made while simultaneously recording the local field potential (LFP) with extracellular electrodes indicate that the burster spikes are precisely phase-locked to the periodic LFP events. By referencing successive calcium measurements to the common LFP signal, we could therefore accurately determine the relative timing of calcium transients at different points along a neurite. Measuring the relation of temporal to spatial differences allowed us to estimate the velocity of action potential propagation, which was 4.3 +/- 0.2 (SE) mm/s in VBs, and 1.3 +/- 0.2 mm/s in SB.

Coincidence detection in single dendritic spines mediated by calcium release.

Cerebellar long-term depression (LTD) is a calcium-dependent process in which coincident activity of parallel fiber (PF) and climbing fiber (CF) synapses causes a long-lasting decrease in PF synaptic strength onto Purkinje cells. Here we show that pairing CF activation with bursts of PF activity triggers large (>10 microM) calcium signals in Purkinje cell dendrites. When PFs are densely activated, signals span whole dendritic branchlets and are mediated by voltage-dependent calcium entry. When PFs are sparsely activated, however, signals are restricted to single spines and blocked by metabotropic glutamate receptor antagonists. Single-spine signals and sparse-stimulation LTD are also blocked by thapsigargin, indicating that calcium must be released from stores. Single-spine signals and sparse-stimulation LTD are greatest when PF activation precedes the CF activation within 50-200 ms. This timing rule matches the properties of several forms of motor learning, providing a link between behavior and functional properties of cerebellar synaptic plasticity.

Action potentials reliably invade axonal arbors of rat neocortical neurons.

Neocortical pyramidal neurons have extensive axonal arborizations that make thousands of synapses. Action potentials can invade these arbors and cause calcium influx that is required for neurotransmitter release and excitation of postsynaptic targets. Thus, the regulation of action potential invasion in axonal branches might shape the spread of excitation in cortical neural networks. To measure the reliability and extent of action potential invasion into axonal arbors, we have used two-photon excitation laser scanning microscopy to directly image action-potential-mediated calcium influx in single varicosities of layer 2/3 pyramidal neurons in acute brain slices. Our data show that single action potentials or bursts of action potentials reliably invade axonal arbors over a range of developmental ages (postnatal 10-24 days) and temperatures (24 degrees C-30 degrees C). Hyperpolarizing current steps preceding action potential initiation, protocols that had previously been observed to produce failures of action potential propagation in cultured preparations, were ineffective in modulating the spread of action potentials in acute slices. Our data show that action potentials reliably invade the axonal arbors of neocortical pyramidal neurons. Failures in synaptic transmission must therefore originate downstream of action potential invasion. We also explored the function of modulators that inhibit presynaptic calcium influx. Consistent with previous studies, we find that adenosine reduces action-potential-mediated calcium influx in presynaptic terminals. This reduction was observed in all terminals tested, suggesting that some modulatory systems are expressed homogeneously in most terminals of the same neuron.

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 [Ca 2+] 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.

Longitudinal spread of second messenger signals in isolated rod outer segments of lizards.

1. In vertebrate rods activation of the phototransduction cascade by light triggers changes in the concentrations of at least two diffusible intracellular second messengers (cGMP and Ca2+) whose actions depend on how far they spread from their site of production or entry. To address questions about their spatial spread, cell-attached patch current recording and fluorescence imaging of Calcium Green-dextran were used to measure the longitudinal spread of cGMP and Ca2+, respectively, in functionally intact isolated Gecko gecko lizard rod outer segments under whole-cell voltage clamp. 2. The light-evoked changes in cGMP and Ca2+ concentrations decayed with distance from a site of steady focal activation by two-photon absorption of 1064 nm light with similar decay lengths of approximately 3.5 microm. 3. These results can be understood on the basis of a quantitative model of coupled diffusible intracellular messengers, which is likely to have broad relevance for second messenger signalling pathways in general. 4. The decay length for the spread of adaptation from a site of steady local illumination was about 8 microm, i.e. substantially longer than the decay lengths measured for the spread of cGMP and Ca2+. There are a number of factors, however, that could broaden the apparent relationship between functional changes in the light response and the concentration of a diffusible messenger. For these reasons the measured decay length is an upper limit estimate of the spread of adaptation and does not rule out the possibility that Ca2+ and/or cGMP carry the adaptation signal.

Optical recording of light-evoked calcium signals in the functionally intact retina.

Using two-photon excitation of fluorescent indicator dyes, we measured calcium concentration transients in retinal ganglion and amacrine cells without destroying the light sensitivity of the retina by maximally activating or bleaching the photoreceptors. This allowed an immediate assessment of the cellular morphology and study of the calcium signals evoked by visual stimuli. Calcium dynamics in individual dendritic processes could be examined for extensive periods without deterioration and with little apparent phototoxicity at excitation wavelengths of from 930 to 990 nm. Light-evoked increases in calcium were resolved in ganglion- and amacrine-cell neurites, making it possible to use optical recording to study the relationship between calcium signaling and retinal function.

[Pump-gun as a weapon. Type of injuries, prohibition of weapons]. / Pumpgun--Waffe, Verletzungsbilder, Waffenverbot.

Pumpguns are shotguns with pump action whose injuries and wound mechanisms have several special features: extremely high kinetic energy of the shot (2500 to 3500 J) frequent cases of "Krönlein shots" (exenteration of the brain) punchmark/imprint immediately adjacent to the entrance wound from the front of the pipe magazine exit wounds from buckshot may be similar to pellet entrance injuries from a distant shotgun discharge the use of various shotgun cartridges (plastic ammunition, slug bullet, various lead pellets) within the same weapon. The change in the Austrian gun law and the banning of the pumpgun in 1995 is also discussed in the article.

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.

Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis.

Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for calcium influx: high-threshold voltage-sensitive calcium channels, NMDA receptors, and an APV-resistant influx consistent with calcium-permeable AMPA or kainate receptors. These pathways vary among different populations of spines and are engaged under different stimulation conditions, with peak calcium concentrations reaching >10 microM. Furthermore, as a result of the biophysical properties of the NMDA receptor, the calcium dynamics of spines are exquisitely sensitive to the temporal coincidence of the input and output of the neuron. Our results confirm that individual spines are chemical compartments that can perform coincidence detection. Finally, we demonstrate that functional studies and optical quantal analysis of single, identified synapses is feasible in mammalian CNS neurons in brain slices.

Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis.

Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 micrometers below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of approximately 70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Identification of chemical synapses in the pharynx of Caenorhabditis elegans.

The rhythmic contraction of the Caenorhabditis elegans pharynx is unique in that the network of 12 neurons, including two M3 neurons, that regulate the contraction is known. The neurotransmitters secreted by these cells, and the target cells responding to these chemical signals, are not known. Here, we describe an approach to obtain this missing information and use the M3 cells as an example. Electrical recordings (electropharyngeograms) were used in conjunction with temporally and spatially defined application of neurotransmitters via photolysis of inactive, photolabile precursors. To illustrate the technique we used pharyngeal preparations in which the two M3 neurons are intact and preparations in which they were removed by laser irradiation. Removal of M3 neurons results in the loss of the small negative peaks in the electropharyngeograms and an increase in time during which the pharynx remains contracted. We demonstrate that the application of glutamate by photolysis of caged glutamate to a pharynx from which the two M3 neurons were removed produces effects similar to those observed before removal of the M3 neurons. In control experiments, photolytic release from photolabile precursors of carbamoylcholine, a stable and well characterized analog of acetylcholine, or of gamma-aminobutyric acid, from photolabile precursors did not have this effect. The response depended on the amount of glutamate released. By reducing the size of the photolytic beam, glutamate was released at several different locations of the pharynx. Two areas of the pharynx mainly respond to the application of glutamate; one corresponds to the pm4 muscle cells in the metacorpus, and the other to the junction between muscle cells pm5 in the isthmus and pm6 in the terminal bulb.

Pulsing mercury arc lamps for uncaging and fast imaging.

It is shown that the light intensity from a mercury short arc light bulb can be boosted to about 100 times its steady-state value for a period of about 1 ms by superimposing a current pulse of up to 100 amperes in magnitude and 1-2 ms in duration on a simmer current of 3 amperes. The output spectrum changes in a remarkable way from a line to a broadband distribution. The radiance delivered during a 1-ms pulse is comparable to what can be obtained from a Xe flash bulb. The lack of a high voltage ignition pulse accounts for an extreme reduction of the electrical artifact seen for the pulsed Hg bulb as compared to a Xe flash lamp. Possible applications include the release of caged compounds, high speed imaging, and wavelength ratio-imaging without filter switching.