Journal: JARO: Journal of the Association for Research in Otolaryngology

Article Title: BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau

doi: 10.1007/s10162-016-0585-5

Figure Lengend Snippet: Afferent nerve fibers and bouton terminals were partially enveloped by a BX-GAG glycocalyx (green). A BODIPY fluorescence was present in hair cell bodies (hc) and appearing to surround afferent (a) nerve fibers (scale bar 50 μm). B Higher magnification of BX-GAGs present in the basement membrane (bm) of crista and at heminodes (n) (scale bar 30 μm). C FM1-43 (magenta), injected into endolymph over the same time scale as that of BX (B), labeled hair cells and neurons; scale bar: 15 μm. D Maximum intensity of 20-μm two-photon projection following whole nerve intracellular labeling with NBN350 (blue) revealing colocalization of BX-GAGs (green) with bouton (b) nerve terminals. Unmylenated (u) dendrites branching to bouton terminals showed little evidence of BX-GAGs. The initial heminode (n) within the basement membrane (bm) of the crista ampullaris was enveloped by a xyloside primed glycocalyx (g) that continued to envelop the afferent (a) nerve fibers projecting centrally (scale bar 50 μm)

Article Snippet: Neurobiotin350 (NBN350; Vector Labs, Burlingame, CA) was introduced to the ganglion for retrograde bulk labeling of neuronal projections in the crista ampullaris.

Techniques: Fluorescence, Injection, Labeling

Journal: JARO: Journal of the Association for Research in Otolaryngology

Article Title: BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau

doi: 10.1007/s10162-016-0585-5

Figure Lengend Snippet: Hair cells from saccule-staining positive for NBN350 (blue) following administration into the endolymph of the living oyster toadfish for 2 h, either without gentamicin (A) or with 1 h pretreatment of gentamicin (1 mM) (B). C–F Two-photon images of functional (1) vs. blocked METs (2) in the semicircular canal crista (C) and in the utricular macula (D–F). NBN350 (blue) labeled cilia of hair bundles (C, inset). G Semicircular canal microphonics recorded in response to mechanical stimulation show MET currents were reduced to 20 % 2 h after BX administration and recovered to 53 % 18 h after administration (n = 5). The inset shows reduction of the microphonic in three example animals (onset time constant ∼25 min). Twenty-four percent of hair cells in the utricular macula labeled for functional METs 2 h after BX administration, confirming that ∼75 % of hair cell MET channels were blocked by the BX compound (n = 5). H Single-unit afferent discharge rates were reduced (n = 47) following BX treatment. The average afferent discharge rate (spk s−1) decreased 59 %. H Sensitivity to sinusoidal mechanical stimulation at 5 Hz (spks s−1 μm−1) decreased 91 % re: controls, while sensitivity to infrared heat pulse stimulation (spks s−1 °C−1) of the crista ampullaris decreased 71 % re: controls. Error bars show standard error of the mean (A, F), with all results normalized to the animal-specific mean in the control condition prior to BX administration (scale bars: 15 μm, Fig. 5 A–B; 50 μm, Fig. 5 C–F)

Article Snippet: Neurobiotin350 (NBN350; Vector Labs, Burlingame, CA) was introduced to the ganglion for retrograde bulk labeling of neuronal projections in the crista ampullaris.

Techniques: Staining, Functional Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Dopaminergic Neurons Exhibit an Age-Dependent Decline in Electrophysiological Parameters in the MitoPark Mouse Model of Parkinson's Disease

doi: 10.1523/JNEUROSCI.1395-15.2016

Figure Lengend Snippet: Verified tyrosine hydroxylase-positive neurons from MitoPark mice exhibit altered electrophysiological parameters. In a separate experiment, neurons were filled with 0.2% neurobiotin and recorded with an emphasis on obtaining multiple dependent electrophysiological measures from each cell. Cells were later stained (A) for neurobiotin (left) and TH (middle), and cells that definitively co-stained for both markers (right) were analyzed for their electrophysiological parameters. Consistent with previous results, recordings in neurons from MitoPark mice exhibited decrease cell capacitance (B), increased input resistance (C), and decreased Ih (D), spike width (E), and peak current observed in response to iontophoresis of dopamine (F). Two-tailed unpaired t tests. **p < 0.01, ***p < 0.001.

Article Snippet: Spontaneous firing was measured with either the loose cell attached method using pipettes of 9–10 MΩ resistance filled with Na HEPES plus 20 m m NaCl (290 mOsm/L, pH 7.40), or with pipettes of 2.5–4 MΩ resistance filled with internal solution containing the following (in m m ): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl 2 , 10 HEPES, 10 BAPTA, 2 ATP, and 0.4 GTP, pH 7.35–7.40, 267–275 mOsm/L, plus 0.2% neurobiotin (Vector Laboratories) immediately before gaining access to the interior compartment of the cell.

Techniques: Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The fluorescence of the induced fluorescent pseudopupil originates from the rhabdomeres. (a) Diagram of a single ommatidium of a dipteran compound eye. Dotted lines denote the plane where optical cross sections of the eye in (b–d) were taken. From top to bottom: corneal facet (C), pseudocone (PC) and distal tip of the rhabdomeres (Rh). (b,c) Eristalis tenax compound eye after application of Lucifer Yellow and scanned with a confocal microscope, with a 63× glycerol objective. Scale bar 100 µm. The fluorescence observed at the surface of the eye (b) originates from the fluorescent rhabdomere tips as no other cells or parts of the photoreceptors are fluorescent when we focus below the cornea to the plane of the rhabdom tips (c) . (d) Magnified view from a single ommatidium, showing the distinctive trapezoidal shape formed by the distal tips of 7 adjacent rhabdomeres, typical of dipteran flies. Scale bar 5 µm. (e) Maximum intensity projection of a z-stack of the left eye of a female Eristalis tenax. When the dye (in this case Neurobiotin 488) had been left in the head for more than 3 h we experienced glowing in the entire eye. Image acquired with a Leica SP8 DLS confocal microscope and a 2.5× air objective lens (see also Supplementary Information video , part 2).

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: Induced fluorescent and dark pseudopupil overlap. (a) Head of the bee Anthophora sp. These bees have clear eyes and its dark pseudopupil is evident as a black spot on the eye surface. Image acquired with a Nikon SMZ18 equipped with a bright field filter cube (P2-EFLBF) and a ¼ wave plate. The eye was stained with Neurobiotin 488 (see “ ). Scale bar: 500 µm. (b) Zoomed-in view of the area marked by the white rectangle in (a) showing more clearly the dark pseudopupil in the left eye. It is common to see multiple pseudopupils resulting from the incident light from the environment around the eye. (c) Zoomed-in view of the area marked by the white rectangle in (a) , after using a P2-EFLGFP-B filter cube—the induced fluorescent pseudopupil was visible in the same sample (in vivo). (d) Co-visualization of the dark (b) and induced fluorescent pseudopupil (c) showing a clear overlap of the two. (b–d) Scale bar: 200 µm. (e) Relative pixel values for the induced fluorescent and dark pseudopupils in the area marked in (d) by a white rectangle . Note that the centres of the two (i.e. max relative pixel value) coincide, with the induced fluorescent pseudopupil having the centre much more easily identifiable compared to the dark pseudopupil.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining, In Vivo

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: Dark and induced fluorescent pseudopupils. (a) Section of an insect apposition compound eye illustrating formation of the principal pseudopupil when illuminated by incident co-axial light ( continuous black lines ); modified after . To the experimenter, looking through the lens of the microscope, the surface of the eye ( diagram below ) will appear pale in the majority of the facets where incident light is scattered from secondary pigment cells ( black dashed lines ). However, in ommatidia aligned with the viewing axis, light is absorbed by rhabdoms and primary pigment cells, making them appear dark ( diagram below ). C corneal facet, Cc crystalline cone, Rh rhabdom. (b) Section of an apposition compound eye illustrating formation of the fluorescent pseudopupil. In this case the compound eye has been injected with a fluorescent dye that has been taken up by the rhabdomeres ( green shade in the diagram, e.g. Lucifer Yellow or Neurobiotin 488, see “ ” section and Fig. ). Short wavelength incident light ( blue shade ) will excite the fluorophore in the rhabdomeres so that only those ommatidia aligned with the optical axis of the microscope will re-emit longer wavelengthlight ( green line ). These re-emitted light rays will cause the facet lenses through which they pass to be bright compared to the surrounding facet lenses which will be dark ( diagram below ). (c) Images taken from the eye of a dragonfly, Aeshna cyanea , whose induced fluorescent pseudopupil was obtained using Lucifer Yellow (see “ ). Scale bar: 500 µm. Images from Supplementary Information video 1, part 1. A. cyanea has a frontal acute zone that is very broad ( black arrows ) as seen by the dark pseudopupil occupying a large area of the frontal part of the eye when the animal is perpendicular to the axis of the objective, i.e. at 0° longitude. However, the induced fluorescent pseudopupil ( blue arrow ) is easily identified and because it is confined to a smaller region of the eye, its centre is easy to pinpoint.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Modification, Microscopy, Injection

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The dimensions of the induced fluorescent pseudopupil depends on the numerical aperture (NA) of the objective lens. (a) Heatmap of the induced fluorescent pseudopupil in a honeybee forager eye stained with Lucifer Yellow (see “ ) and acquired with a Nikon SMZ18 microscope fitted with a P2-EFLGFP-B filter cube, a 1 × SHR PlanApo objective with two different numerical apertures (NA), 0.15 ( top ) and 0.075 ( bottom ). (b) Relative pixel values of the rectangular area in (a) showing that the two pseudopupils have the same centre despite subtending a broader area when the NA is higher ( orange line ).

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: A induced fluorescent pseudopupil in the dark-pigmented compound eye of the honeybee, Apis mellifera . (a) The pigmented compound eye of a naive honeybee has no visible pseudopupil due to the presence of pigments around the retinula cells . The red arrow shows the direction of rotation in (b–d) . (b–d) Zoomed-in view of the left eye of a honeybee forager, Apis mellifera (area of the eye as in the red inset in (a) ), acquired using a Nikon SMZ18. The eye was stained with Lucifer Yellow as described in the Methods and the bright induced fluorescent pseudopupil is thus visible ( red arrow ) and shifts its location in the eye as the head of the honeybee is turned (c, d) . (b) The head is in its frontal view and rotated around its vertical axis at 15° and 30° in (c, d) , respectively. Scale bar: 500 µm.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining

Journal: Scientific Reports

Article Title: A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The induced fluorescent pseudopupil is a good tool for estimating the sampling resolution of a compound eye. (a) Projections of the interommatidial angle, Δ φ , (average of horizontal and vertical components) and (b) facet diameter (values in µm) of a single forager honeybee, Apis mellifera . The highest resolution is found in the frontal part of the eye where there is an averaged Δ φ of 1.3°. Note that the location of this minimum value does not coincide with the location in the eye having the largest facet diameter. The boundary of the visual field ( dashed lines ) is reconstructed from . Dotted lines show 0° azimuth. F frontal, D dorsal, L lateral.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Sampling

Journal: The Journal of Neuroscience

Article Title: Synaptic Currents Generating the Inhibitory Surround of Ganglion Cells in the Mammalian Retina

doi: 10.1523/JNEUROSCI.21-13-04852.2001

Figure Lengend Snippet: Drawings of four ganglion cells from whole-mounted rabbit retinas, viewed from the ganglion cell side. The cells were filled with Neurobiotin during patch-clamp recordings. The axons are indicated by the arrows, and the horizontal axis is parallel to the visual streak. A, α ganglion cell from the center of the streak. Recordings from this cell are shown in Figure​Figure6.6. B, δ-Like ganglion cell from an eccentricity (distance from the center of the streak) of 800 μm. Recordings from this cell are shown in Figure ​Figure8.8. C, Bistratified ON–OFF direction-selective ganglion cell. The solid dendrites stratify in the inner IPL, and the dotted dendrites branch 9 μm farther, toward the outer IPL. The cell was from an eccentricity of 200 μm. D, Sluggish concentric ganglion cell from an eccentricity of 1.2 mm. Recordings from this cell are shown in Figures ​Figures22A and 5. Scale bar, 100 μm.

Article Snippet: Neurobiotin (Linaris GmbH, Bettingen, Germany) was also added to the internal solution at final concentrations ranging from 0.1 to 0.5%.

Techniques: Patch Clamp