Subcellular topography of visually driven dendritic activity in the vertebrate visual system.

Preparation Albino Xenopus tadpoles (stage 46–48) ( Nieuwkoop and Faber, 1967 ) were anaesthetized (0.02%, MS222) and mounted dorsal side up in a Sylgard dish containing external solution (in mM: 115 NaCl, 2 KCl, 2.5 CaCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, 0.01 glycine [pH 7.3], osmolality 260 mosmo/kg). d-tubocurarine (0.1 mM) or α-bungarotoxin (2 µg/ml) was added to prevent occasional twitching of muscle fibers. The optic tectum was cut along the dorsal midline, and one tectal lobe was dissected out to obtain access to the periventricular aspect of the remaining lobe for electroporation and patch-clamp recordings ( Zhang et al., 1998 ). In some cases, a glass micropipette was micropositioned rostrally in the tectal lobe to minimize vertical drift of the preparation during recordings. All experiments were approved by Harvard University’s Standing Committee on the Use of Animals in Research and Training. Single-Cell Dye Fills Individual tectal neurons were filled with Oregon-Green-Bapta-1 (OGB-1, Invitrogen, USA) by using the single-cell electroporation technique ( Haas et al., 2001 ; Nevian and Helmchen, 2007 ; Kitamura et al., 2008 ). Glass micropipettes (open tip diameter ca 1–2 µm) were filled with an internal solution containing (in mM): 110 K-gluconate, 10 KCl, 5 NaCl, 1.5 MgCl 2 , 20 HEPES, 2 Mg-ATP, 0.3 Na-GTP, and OGB-1 (2–4 mM) ([pH 7.3], osmolality 255 mosmo/kg). Under infrared-visual control (see below), the pipette was brought into contact with a tectal soma. A 200 Hz train of voltage pulses (amplitude 2–10 V, pulse width of 4 ms) was applied for 250 ms to electroporate the somatic membrane. Alternatively, brief whole-cell patch-clamp recordings were used to fill individual neurons with OGB-1 and Alexa 594 (Invitrogen, USA). Patch pipettes were filled with an internal solution as described above, but with different dye concentrations (OGB-1: 0.625 mM; Alexa 594: 1.25 mM), and were used to dialyze the indicators into the tectal cell. The pipette was retracted ~30 s after rupturing the tight-sealed membrane patch, and an outside-out membrane patch was formed, suggesting that the somatic membrane resealed as well. With both techniques, dye was allowed to diffuse throughout the dendritic tree for ~1 hr before dendritic imaging was started. In one set of experiments, NMDAR channels were blocked by applying APV (0.1–0.2 mM, Tocris), either in the bath or locally through a micropipette positioned in the tectal neuropil ( Figure S4 ). In a different set of experiments after electroporation with OGB-1, somatic loose-patch recordings were performed to record visually driven action potential firing of electroporated cells, which was used to calibrate ΔF/F signals in the proximal dendritic tree to the number of action potentials per burst ( Figure 5 ). After recording, the membrane patch was ruptured and the neuron was filled with Alexa 594 from the recording pipette to verify that the dendritic Ca 2+ recording and the somatic spike recording were from the same neuron.

Whole-Cell Patch-Clamp Recordings

Recordings were performed by using micropipettes pulled from borosilicate glass capillaries (Kimax) with an open tip resistance of 6–10 MΩ and an Axo-patch 200B amplifier (Molecular Devices). Pipettes were filled with the same internal solution as described above, but with reduced dye concentrations (OGB-1: 0.1–0.2 mM; Alexa 594: 0.2–0.4 mM). When measuring dendritic Ca 2+ signals in response to somatic current injection ( Figure 5 ), d-APV (0.1–0.2 mM), CNQX (0.05 mM), SR95531 (0.01 mM, all from Tocris), and strychnine (0.06 mM, Sigma) were added to the external solution to block glutamatergic, GABAergic, and glycinergic transmission, respectively. The cell was held in current clamp at −60 to −70 mV, and action potential trains were evoked by repetitive current injection into the soma (4 ms duration, 50 Hz). Functional Two-Photon Ca 2+ Imaging In Vivo A custom-built multiphoton confocal microscope was used to record dendritic Ca 2+ signals. A Ti:Sapphire laser (MaiTai, Newport Corp) tuned to 940–950 nm and focused through a water-immersion objective (20×, NA 0.95) (Olympus, Japan) was used to image the morphology and record dendritic Ca 2+ concentration changes in tectal neurons ( Denk et al., 1990 ; Euler et al., 2002 ; Zelles et al., 2006 ). The detection pathway consisted of a dichroic mirror (690 dcxxr; Chroma) and a bandpass (e700sp-2p; Chroma) to separate fluorescence emission from the infrared excitation. Fluorescence emission was further split into two channels by a dichroic beam splitter (585 dcxr; Chroma) and a green (HQ522/40 m; Chroma) and a red (628/40; Semrock, USA) bandpass, respectively, and recorded by two photomultiplier tubes (R3896, Hamamatsu, Japan). Furthermore, infrared laser light was recorded with a fast photodiode (DET100A, Thorlabs, USA) in trans -illumination mode, which can be used to generate an infrared contrast image of the tectum (e.g., Figures 6A and 7A ) to distinguish between the periventricular cell body layer and the neuropil. Similarly, positioning of micropipettes during electroporation and patch-clamp recordings of tectal cells were aided by simultaneously recording (resolution 256 × 256, 2.5 Hz) the infrared contrast image and the negative stain image, which is transiently created by a fluorescent indicator ejected into the extracellular space while the micropipette approaches the cell body ( Figures 1C and 1D ). During experiments, the three-dimensional dendritic structure of one or a few dye-filled neurons was first visualized in image stacks at a resolution of 512 × 512 pixels. Fast area scans (typically 64 × 64 pixels at 14 Hz frame rate) or line scans (256 pixels, at 864 Hz) were subsequently performed at selected dendritic regions in the primary dendritic branch and at more distal dendritic elements in the tectal neuropil during visual stimulation.

Visual Stimulation

Two-dimensional visual stimulus patterns were displayed on the exit face of a rigid image conduit (3 mm diameter, Edmund Optics), micropositioned to a fixed distance (1.5 mm) in front of the eye. Orientation of the eye and alignment with the visual stimulator were carefully adjusted under visual control through the eye piece of the microscope. Stimulus patterns were generated in LabView (National Instruments, USA), and projected with a DLP projector (Optoma, Taiwan) and an air objective lens (10×, NA 0.3) onto a flexible plastic fiberoptic bundle (Nanoptics, USA), which was connected to the image conduit. Stimulus light was in the yellow range, filtered with a bandpass (575/10 nm; Omega Optical, USA). Dimming squares (subtending ~10° visual angle) or bars (10° × 70°) were flashed on a bright background. Topographic bias in the dorsoventral axis of the visual field was tested by using five horizontal bars in different vertical positions (−30°, −15°, 0°, 15°, 30°, Figures 2 – 4 ). Dimming bars were shown in a pseudorandomized order, with a dimming bar presented for 0.5 s every 5 s. Contrast was set in the range between 30% and 90%, typically 88% for dimming bars. The angular distribution of light intensity emitted from the image conduit was measured and used to implement a radially symmetric increase in background light and stimulus intensities toward the periphery of the image conduit in order to keep the effective stimulus contrast ratio and background illumination constant across the visual field.

Data

Acquisition and Analysis Two-photon Ca 2+ imaging data and electrophysiological recordings were acquired and analyzed offline by using custom-written software in LabView (National Instruments, USA), Igor Pro (Wavemetrics, USA), and Matlab (Math-works, USA). Functionally imaged neurons used to analyze dendritic topography ( Figures 2 – 4 ) exhibited a robust Ca 2+ signal in the primary dendrite and most parts of the distal dendritic tree in response to visual subfield stimulation. Visually evoked ΔF/F transients were measured as peak amplitudes averaged in a 150 ms window surrounding the maximum fluorescence value after the stimulus and subtracted with the baseline value averaged in a 1 s interval preceding the stimulus. Subcellular dendritic topography was determined by comparing the mean-subtracted center of mass of tuning curves (ΔR) with the relative position of the dendritic region (Δu′ in Figure 2 ; Δu in Figures 3 and 4 ) within the dendritic tree of a neuron. The center of mass of a tuning curve was calculated by taking the sum of vertical stimulus positions weighted by the ΔF/F amplitude measured in response to that stimulus. This was done either from the ΔF/F responses averaged across multiple trials in the same dendritic region ( Figure 2D ) or from the individual ΔF/F responses measured within a line scan ( Figures 3 and 4 ). Specifically in frame scan mode, where dendritic regions in different z-planes were sequentially scanned, Δu′ was determined by projecting the ROI coordinates of scanned regions against the u′ axis (blue arrow in Figure 2A ). The u′ coordinate of the midpoint between the maximum and minimum u′ coordinate of all scanned dendritic regions within a neuron was taken as the origin. Specifically, in line scan mode ( Figures 3 and 4 ), where different dendritic regions in the same z-plane were scanned simultaneously, the coordinate of each dendritic region was projected against the medial-to-lateral tectal axis (u; arrow in Figure 3B ) and measured relative to the midpoint between the most medial and lateral dendritic region in each scan (”0” in Figure 3B ). Three-dimensional image stacks were filtered with an anisotropic diffusion filter ( Broser et al., 2004 ) for display purposes. For reconstruction and measurement of dendritic length, the software tool ”Neuromantic” by D. Myatt, University of Reading was used.

Retinal Dye Injections

RGC axonal arbors were labeled by injection of Alexa Fluor 488-Dextran (10,000 MW) and Alexa Fluor 594-Dextran (10,000 MW) into the ventral and dorsal retina of stage-46/47 tadpoles. The tip of a glass micropipette was broken to yield a 10–20 µm tip opening, and the pipette was filled with a 50% (w/v) solution of the indicator dissolved in Xenopus external solution. A total of 18–24 hr after dye injection, the contralateral tectal lobe was screened for labeled axons in a fluorescence dissecting microscope. Tadpoles with labeled retinotectal projections were dissected as in the physiological Ca 2+ imaging experiments such that the location and orientation of the tectal neuropil could be compared between functional and anatomical imaging data. To quantify differences in the tectal location of dorsally and ventrally derived RGC axons, the extent of the tectal neuropil was outlined and divided into a medial and a lateral half ( Figure 6A ), and the fractional intensity of the green and red channel was measured for the two hemifields.

Supplementary Material supp