Mice with heterozygous alleles of the β2 subunit of the nicotinic acetylcholine receptor (β2+/−
) were kindly provided by Dr. Art Beaudet from Baylor College of Medicine (Xu et al., 1999
). These mice were bred after backcrossing onto a C57Bl/6 strain for many generations. We maintained colonies of heterozygous (β2+/−
) and homozygous (β2−/−
) mice from these breeding pairs. The genotypes were determined according to the published protocol (Xu et al., 1999
). Additional control experiments were performed on age-matched wild-type C57Bl/6 mice (Charles River Labs).
Physiological Preparation and Functional Imaging of Retinotopic Maps
All surgical procedures were approved by the University of California, San Francisco Committee on Animal Research. To image mouse cortical retinotopy, we followed the method developed by Kalatsky and Stryker (Kalatsky and Stryker, 2003
). Briefly, adult mice between 2 and 6 months of age were anesthetized with urethane (1.0 g/kg i.p.) supplemented by chlorprothixene (0.2 mg/mouse i.m.). Atropine (5 mg/kg) and dexamethasone (0.2 mg/mouse) were injected subcutaneously. A tracheotomy was performed, and a craniotomy was made over the visual area of the left hemisphere; the dura mater was left intact. For survival experiments to retrogradely label dLGN neurons, the animals were anesthetized using 1%–2% isoflurane in O2
, and no tracheotomy was performed. To image subcortical visual areas, the overlying cortex was aspirated, thereby exposing SC and dLGN.
Optical images of cortical intrinsic signal were obtained using a Dalsa 1M30 CCD camera (Dalsa, Waterloo, Canada) controlled by custom software. The surface vascular pattern or intrinsic signal images were visualized with illumination wavelengths set by a green (546 ± 10 nm) or red (610 ± 10 nm) interference filter, respectively. After acquisition of a surface image, the camera was focused 600 μm below the pial surface. An additional red filter was interposed between the brain and the CCD camera, and intrinsic signal images were acquired at the rate of 7.5 fps and were stored as 512 × 512 pixel images.
A high refresh rate monitor (Nokia Multigraph 445X, 1024 × 768 pixels, 120 Hz) was placed 25 cm away, to the right (contralateral to the hemisphere being imaged), with its left edge approximately aligned to the animal. Drifting thin bars (2° wide and full-screen length) were generated by a Matrox G450 board (Matrox Graphics, Inc., Quebec, Canada). The spatial frequency of the drifting bar was 1 cycle/80 degrees, and the temporal frequency was 1 cycle/ 8 s or 1 cycle/6 s. Animals were presented with thin bars drifting along the vertical or horizontal axes in order to stimulate the constant lines of elevation or azimuth, respectively. By extracting the optical signal at the stimulus frequency, we computed the response magnitudes and timing in reference to the stimulus cycle, which can then be converted to the location of the visual field. The absolute phase maps were then calculated by the method of “phase reversal” (Kalatsky and Stryker, 2003
Analysis of Retinotopic Maps
We used two measures to compare cortical maps. First, we determined the maximum magnitude of optical response (expressed as fractional change in reflectance × 104) from the absolute map. Second, to assess map quality, we selected the most responsive area within the primary visual cortex (V1) to compare the visual field positions of these pixels with those of their neighbors. Specifically, we used the elevation map to select the response area, because the azimuth maps of β2−/− mice are very weak. The 20,000 pixels (1.60 mm2 of cortical space) that had the greatest response magnitude in the elevation maps were selected. For each of these pixels, we calculated the difference between its position and the mean position of its surrounding 25 pixels. For maps of high quality, the position differences are quite small because of smooth progression. The standard deviation of the position difference was then used as an index of map quality. The quality of SC maps was similarly analyzed, using 5,000 pixels due to the smaller area of SC. Note that the compression of the SC map relative to the V1 map would cause any artifactual scatter that resulted from the measurement procedure to be greater in SC than in V1, ensuring that the greater measured scatter in β2−/− V1 is an underestimate.
Multiunit Recording of Cortical Neurons and Analysis of Receptive Fields
Guided by the optical maps, we recorded multiunit activity from V1 with 10 MΩ microelectrodes (Frederick Haer Company, Bowdoinham, ME). For each animal, three to six penetrations were made perpendicular to the pial surface across V1, and three to seven sites (>50 μm apart) were recorded in each penetration. The spikes were acquired using a System 3 workstation (Tucker Davis Technologies, FL) and analyzed using Matlab (The Mathworks, MA).
Single drifting bars 5° in width and 80° in length at the speed of 25°/sec were used to drive cortical cells. In each trial, the drifting bar was presented in four directions—rightward, leftward, upward, and downward—in a random sequence. Twenty to thirty of such trials were repeated to construct a peristimulus time histogram (PSTH) for each stimulus direction, using 5 ms bins. The PSTH was subsequently smoothed using a 10 ms window. The mean rate (Rb) and standard deviation (Stdb) of background firing activity were calculated from the period of PSTH when no stimulus was presented. A threshold was calculated as Rb + 3 * Stdb, and the bins in the PSTH above the threshold were used to calculate the mean timing weighted by their firing rate. Separately for each of the four directions of movement, the RF location was determined by converting the timing to position on the stimulus monitor, and the mean RF location of each penetration was calculated from all of the recording sites. The RF deviation of each recording site from the mean RF position was used to assay the precision of cortical RF organization.
Retrograde Labeling of Thalamocortical Projections and Image Analysis
Cholera toxin subunit B (CTB) conjugated to Alexa Fluor (Molecular Probes, OR), CTB-488 (green), and CTB-594 (red) were injected into the cortex, guided by the retinotopic maps or stereotaxic coordinates to retrogradely label dLGN neurons. A small amount of 2 mg/ml solution of each CTB in PBS was injected by Nanoject (Drummond Scientific Company, PA), using a glass pipette with a 20–30 μm tip opening. The Nanoject was set to inject 32.2 nl of the dye (18.4 nl for P8 mice), but small variations in injection volume were unavoidable. Mice were sacrificed and intercardially perfused with 4% paraformaldehyde in PBS 48 hr later. The brains were fixed overnight before sectioning coronally at 100 μm using a vibratome (Lancer, MO). Images of the dLGN and injection sites in the cortex were captured using a confocal microscope (Biorad 1024, CA).
To analyze the patterns of retrogradely labeled cells in the dLGN, we first calculated the background signal as the mean signal of an area within the dLGN where no labeled cells were seen. The image was then thresholded separately for each color at the level of 1.5 times the background. (A wide range of threshold from 1.2 to 1.8× was tested and similar results were obtained.) We then calculated the position of the center of mass for all of the labeled pixels within the dLGN. The percentage of labeled pixels within the dLGN as a function of the distance from the center was then computed. The above procedure was repeated for all sections of the dLGN (six to seven 100 μm sections per animal). The sections at the rostral and caudal ends were excluded due to the small number of labeled pixels. Finally, we calculated the mean of the percentage of labeled pixels in all sections of each animal. To quantify the differences between the control and β2−/− mice, we used the radius of the circle within which 80% of the labeled pixels were found to represent the degree of the dispersion.
To calculate the actual size of injection, we reconstructed the injection sites from the 100 μm coronal sections. From each section, we measured the width of the dye at the level of layer IV, i.e., 400 μm from the pial surface. The area of injection in this section is then the product of the width of the dye and the section thickness (100 μm). The total area was then calculated as the sum of all sections.
Multielectrode Extracellular Recording from Retina
P4 WT mice were sacrificed by decapitation. Retinal dissection was performed in a bicarbonate-based extracellular saline under normal room illumination. The saline consisted of: 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 22.2 mM glucose; the pH was maintained at 7.3–7.4 by bubbling with a mixture of 95%O2/5% CO2. The retina was placed in a multielectrode chamber (MEA-60 system; MultiChannel Systems, Germany) ganglion cell-side down. Retinas were perfused for 30 min at room temperature and then for 30 min at 34°C before recordings were started, and the temperature was maintained at 34°C throughout the recordings.
Multielectrode data were analyzed using OfflineSorter software (v.1.3; Plexon Inc., TX) as described previously (Tian and Copenhagen, 2003
). Correlation indices of these units, as described in Wong et al. (1993)
, were calculated with IGOR Pro (v.4.0; WaveMetrics, OR) using custom macros. The correlation index for a given pair of neurons indicates the fold increase of the firing rate of one neuron within a given time window of a spike from the other neuron. It was calculated using the number of spikes from one neuron that were detected within the time window (100 ms) of a spike from the other neuron. This value was scaled to the mean firing rate of the first neuron and the firing rate of the other neuron during the time window. The correlation index increases with the likelihood of the simultaneous firing of the two neurons (Demas et al., 2003
In Vivo Intravitreous Application of Epibatidine
Every 48 hr from P1 to P7, mice were anesthetized, and 0.5–1 μl of 1 mM epibatidine (Sigma, MO) in sterile saline, or sterile saline alone, was injected intravitreously into both eyes. We confirmed that the effects of epibatidine injections were limited to the eye and did not reach the CNS via the circulation, by systemic administration of the same dose in other animals, which produced CNS signs within 3 min. Eye injections at P1 and P3 were performed through the intact eyelids, and at P5 and P7 the eyelids were cut open. A 33G needle attached to a Hamilton (Reno, NV) microsyringe was used to inject the solution at the rate of about 1 μl/min into the vitreous humor at the ora serrata. The needle is withdrawn after holding it in place for 30 s to 1 min. The animals were allowed to survive to P40–P50 before the dLGN neurons were retrogradely labeled with CTBs. Functional imaging was not performed in these animals, and the CTBs were injected at the same stereotaxic coordinates as in the mice defined by imaging. The retinal morphology was examined in both epibatidine- and saline-treated mice to confirm that their retinas were not damaged by the intravitreous injections.
The statistical test used was Student’s t test and the results were expressed as the mean ± standard error, unless otherwise indicated.