Effect of the AMPA/kainate receptor antagonist, NBQX, on odor responses of mitral cells.

<p>(A) Whole-cell recording of a mitral cell response to odor stimulation (food extract; bar) before (black) and during (red) bath-application of NBQX and after washout (gray). (B1–B5) Five examples illustrating effects of NBQX on odor responses. Ticks denote individual action potentials. Each row shows one trial. Black: control; red: during NBQX application; gray: after wash-out of NBQX. Continuous lines are peri-stimulus time histograms, averaged over all trials under each condition. Thick portions depict time bins where peri-stimulus time histograms were significantly different (Student's t-test; P<0.05) from the corresponding time bin in the control peri-stimulus time histogram (black). Bar indicates odor stimulation. Responses are from different cells and were recorded in the whole-cell, cell-attached or loose-patch configuration.</p

Ionotropic glutamate receptors are essential for odor responses of mitral cells.

<p>(A) Whole-cell recording from a mitral cell during odor stimulation (food extract; bar) before (black) and during (red) application of NBQX and AP5. (B) Local field potential recording during odor stimulation (food extract; bar) before (black) and during (red) application of NBQX and AP5 and after washout (gray). Traces are band-pass filtered between 8–43 Hz. (C) Power spectrum of local field potential traces (average of 6 trials; from unfiltered data) for the examples shown in (B). (D) Average local field potential power (15–30 Hz) in the presence of NBQX and AP5, normalized to control (n = 4 olfactory bulbs). ***, P<0.001.</p

Effect of NBQX on mitral cell responses measured by 2-photon Ca²⁺ imaging.

<p>(A) Odor-evoked Ca²⁺ signals in mitral cells before, during and after application of NBQX (stimulus: Trp, 10 µM). Arrows depict somata of neurons identified as mitral cells by expression of the genetically encoded fluorescence marker HuC-YC. (B) Average somatic Ca²⁺ signals before (control) and during application of NBQX, normalized to control. Error bars show standard deviation. **, P = 0.002 (sign test). (C) Cumulative distribution of Ca²⁺ signal amplitudes before (black) and during application of NBQX (red) and after washout (gray). (D) Comparison of Ca²⁺ signal amplitudes evoked by the same odors in the same mitral cells before and during application of NBQX. Data were pooled over all cells, odors and animals (n = 190 responses). r, Pearson correlation coefficient. Inset shows the density of data points in the boxed region. Lines are diagonals with slope one. (E) Left: mitral cell odor responses ranked according to the Ca²⁺ signal before application of NBQX. Inset shows an enlargement of a subregion. Right: Responses of the same mitral cells to the same odors in the presence of NBQX, ranked in the same order as in the control.</p

Effect of the NMDA receptor antagonist, AP5, on odor responses of mitral cells.

<p>(A) Whole-cell recording of a mitral cell response to odor stimulation (Lys, 10 µM; bar) before (black) and during (red) application of AP5. (B1–B5) Five examples illustrating effects of AP5 on odor responses. Conventions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone-0001416-g003" target="_blank">Fig. 3</a>. Responses are from different cells and were recorded in the whole-cell, cell-attached or loose-patch configuration.</p

Simplified architecture of synaptic pathways in the olfactory bulb.

<p>Within glomeruli, glutamatergic olfactory sensory neurons provide excitatory synaptic input to mitral cells and a subpopulation of periglomerular cells via AMPA/kainate and NMDA receptors. Periglomerular cells also receive glutamatergic input from mitral cell dendrites and provide GABAergic output to mitral cells of the same and neighbouring glomeruli. In addition, GABA (green arrow) and dopamine (not shown) released from periglomerular cells reduces glutamate release from olfactory sensory neuron axon terminals by acting on GABA_B and D₂ receptors, respectively, in the same glomerulus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Murphy1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Wachowiak3" target="_blank">[49]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Hsia1" target="_blank">[53]</a>. In subglomerular layers, glutamate release from mitral cell dendrites and axon collaterals stimulates granule cells via AMPA/kainate and NMDA receptors. Granule cells release GABA back onto GABA_A receptors on the same and other mitral cells. Glutamate release from a mitral cell can therefore cause recurrent inhibition of the same mitral cell and lateral inhibition of other mitral cells via periglomerular and granule cells. These interactions, here collectively referred to as the mitral cell→interneuron→mitral cell pathway, can extend over distances corresponding to multiple glomeruli. An additional pathway mediating lateral inhibition that is not detailed in this scheme is the short axon cell (SAC)→periglomerular→mitral cell pathway identified in rodents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Wachowiak1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Aungst1" target="_blank">[54]</a>. Centrifugal inputs from higher brain areas are also not shown in detail. Many of these inputs terminate on interneurons and are glutamatergic. Not included in the scheme are metabotropic glutamate receptors, interactions among interneurons in the granule cell layer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Pressler1" target="_blank">[55]</a>, glutamate spillover <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Isaacson3" target="_blank">[56]</a>, and a small glutamatergic subpopulation of granule cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Didier1" target="_blank">[57]</a>. Strong excitatory interactions across glomeruli, as revealed in the antennal lobe of Drosophila <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Olsen1" target="_blank">[58]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001416#pone.0001416-Root1" target="_blank">[60]</a>, have not been found in the vertebrate olfactory bulb. Abbreviations: OSN: olfactory sensory neuron, PGC: periglomerular cell, MC: mitral cell, GC: granule cell, SAC: short axon cell.</p

Effect of AP5 on interneuron responses measured by 2-photon Ca²⁺ imaging.

<p>(A) Odor-evoked Ca²⁺ signals in interneurons before, during and after application of AP5 (stimulus: food odor). (B) Average somatic Ca²⁺ signals before (control) and during application of AP5, normalized to control. Error bars show standard deviation. (C) Cumulative distribution of Ca²⁺ signal amplitudes before (black) and during (red) application of AP5. (D) Comparison of Ca²⁺ signal amplitudes evoked by the same odors in the same interneurons before and during application of AP5. r, Pearson correlation coefficient. Inset shows the density of data points in the boxed region. Lines are diagonals with slope one. (E) Left: interneuron odor responses ranked according to the Ca²⁺ signal before application of AP5. Data were pooled over all cells, odors and anminals (n = 14884 responses). Right: Responses of the same interneurons to the same odors in the presence of AP5, ranked in the same order as in the control. Inset shows an enlargement of a subregion to demonstrate that low-amplitude values are interspersed between high amplitude values. The visual impression in the full diagram that many amplitudes are increased during AP5 treatment is therefore an artifact caused by crowding of bars in the graph.</p

Effect of NBQX on odor responses of mitral cells: quantitative analysis.

<p>(A) Mean firing rate change evoked by odor stimulation before (control) and during NBQX treatment in the time window between 0.25 and 0.75 s after response onset. Error bars show standard deviation. *, P = 0.02 (sign test). (B) Cumulative distribution of odor-evoked firing rate changes in mitral cells before (control) and during NBQX application. (C) Left: mitral cell odor responses ranked according to the firing rate change measured before NBQX application. Right: Responses of the same mitral cells to the same odors in the presence of NBQX (same rank order as control). Asterisks denote responses that were significantly changed in the presence of NBQX (Student's t-test; P<0.05). (D) Top (continuous lines): average peri-stimulus time histogram of mitral cell odor responses before (control) and during NBQX treatment. Thick portions depict time bins where the peri-stimulus time histogram in the presence of NBQX was significantly different from the control peri-stimulus time histogram in the corresponding time bin (sign test; P<0.05). Dashed lines show standard deviation. (E) Differences of peri-stimulus time histograms (NBQX–control) for all mitral cell odor responses.</p

Effect of AP5 on mitral cell responses measured by 2-photon Ca²⁺ imaging.

<p>(A) Odor-evoked Ca²⁺ signals in mitral cells before, during and after application of AP5 (stimulus: food extract). Arrows depict somata of neurons identified as mitral cells by expression of the genetically encoded fluorescence marker HuC-YC. Black and white arrows show mitral cells whose response was increased and decreased, respectively, by AP5 treatment. (B) Average somatic Ca²⁺ signals before (control) and during application of AP5, normalized to control. Error bars show standard deviation. (C) Cumulative distribution of Ca²⁺ signal amplitudes before (black) and during application (red) of AP5 and after washout (gray). (D) Comparison of Ca²⁺ signal amplitudes evoked by the same odors in the same mitral cells before and during application of AP5. Data were pooled over all cells, odors and anminals (n = 742 responses). r, Pearson correlation coefficient. Inset shows the density of data points in the boxed region. Lines are diagonals with slope one. (E) Left: mitral cell odor responses ranked according to the Ca²⁺ signal before application of AP5. Inset shows an enlargement of a subregion. Right: Responses of the same mitral cells to the same odors in the presence of AP5, ranked in the same order as in the control.</p

Effect of NBQX on interneuron responses measured by 2-photon Ca²⁺ imaging.

<p>(A) Odor-evoked Ca²⁺ signals in interneurons before, during and after application of NBQX (stimulus: food odor). (B) Average somatic Ca²⁺ signals before (control) and during application of NBQX, normalized to control. Error bars show standard deviation. ***, P<0.001 (sign test). (C) Cumulative distribution of Ca²⁺ signal amplitudes before (black) and during (red) application of NBQX. (D) Comparison of Ca²⁺ signal amplitudes evoked by the same odors in the same interneurons before and during application of NBQX. Data were pooled over all cells, odors and anminals (n = 5878 responses). r, Pearson correlation coefficient. Inset shows the density of data points in the boxed region. Lines are diagonals with slope one. (E) Left: interneuron odor responses ranked according to the Ca²⁺ signal before application of NBQX. Right: Responses of the same interneurons to the same odors in the presence of NBQX, ranked in the same order as in the control. Inset shows an enlargement of a subregion to demonstrate that low-amplitude values are interspersed between high amplitude values. The visual impression in the full diagram that many amplitudes are increased during NBQX treatment is therefore an artifact caused by crowding of bars in the graph.</p

Image_1_SBEMimage: Versatile Acquisition Control Software for Serial Block-Face Electron Microscopy.pdf

<p>We present SBEMimage, an open-source Python-based application to operate serial block-face electron microscopy (SBEM) systems. SBEMimage is designed for complex, challenging acquisition tasks, such as large-scale volume imaging of neuronal tissue or other biological ultrastructure. Advanced monitoring, process control, and error handling capabilities improve reliability, speed, and quality of acquisitions. Debris detection, autofocus, real-time image inspection, and various other quality control features minimize the risk of data loss during long-term acquisitions. Adaptive tile selection allows for efficient imaging of large tissue volumes of arbitrary shape. The software’s graphical user interface is optimized for remote operation. In its user-friendly viewport, tile grids covering the region of interest to be acquired are overlaid on previously acquired overview images of the sample surface. Images from other sources, e.g., light microscopes, can be imported and superimposed. SBEMimage complements existing DigitalMicrograph (Gatan Microscopy Suite) installations on 3View systems but permits higher acquisition rates by interacting directly with the microscope’s control software. Its modular architecture and the use of Python/PyQt make SBEMimage highly customizable and extensible, which allows for fast prototyping and will permit adaptation to a wide range of SBEM systems and applications.</p