Recordings were made from the visual cortex of 48 C57BL/J6 mice anesthetized by intra-peritoneal injections of 60 mg/kg pentobarbital sodium (Nembutal, Abbot Laboratories, North Chicago, IL) or a combination of 100 mg/kg ketamine (Ketaset, 100 mg/ml, Fort Dodge Animal Health, Fort Dodge, IO) and 10 mg/kg xylazine (AnaSed, 20 mg/ml, Lloyd Laboratories, Shenandoah, IO). The experiments were carried out following the guidelines of the ARVO guidelines for animal experimentation.
Recording and data acquisition system
The mouse's head was glued to a metal bar fixed to a stereotactic device. A 30-gage needle reference electrode was placed subcutaneously under the jaw; a similar ground electrode was placed on the back. The skin and bone overlying striate cortex was removed and the dura covered with hyaluronic acid (Healon, 10 mg/ml, AMO, Santa Ana, CA). The impulses of single neurons were detected by tungsten or 2 M KCl filled glass micropipette electrodes, the latter with resistances of 5–10 megohms. The tungsten microelectrode was introduced through the intact dura; glass micropipettes were introduced through a cut in the dura. The electrodes were slowly advanced through striate cortex by the micro-drive. Penetrations extended from the surface to a depth of about 1 mm as judged from the micrometer of an oil-coupled micro-drive. We usually made 4 or 5 penetrations in any one mouse monitoring responses with an oscilloscope, an audio amplifier and a digital data acquisition system. The responses were amplified and bandpass filtered (0.3 to 3 kHz) by a S-100 preamplifier (World Precision Instruments, Sarasota, FL) and stored on a Power Lab Chart system (AD Instruments, Colorado Springs, CO).
Both pupils were dilated using cyclopentolate eyedrops and the cornea was covered with hyaluronic acid (Healon, 10 mg/ml, AMO, Santa Ana, CA) to keep it moist. Either eye could be stimulated diffusely with either of two narrow band LEDs (370 nm, strong for UV cones and 505 nm, exclusively affecting M cones and rods). The energies of the stimuli produced by the two LEDs were measured with an IL-1700 radiometer (International Light Inc, Peabody, MA). The maximum energy of the 505 nm light emitting diode was 4.4 W/m2. The maximum energy produced by the 370 nm LED was 4.2 W/m2. These two LEDs appeared to produce relatively similar responses from most of the neurons encountered implying that there are similar numbers of these two cone types in murine retina as indicated by histological methods .
Eyes were light adapted with strong orange or white light to suppress rod activity. The contralateral eye was stimulated by either of these two wavelengths with light pulses of 100 to 500 milliseconds in duration, presented every 2.5 to 4 seconds. The ipsilateral eye could also be stimulated, but this was not routinely done.
201 penetrations were made into striate cortex of 48 mice following the maps of murine V1 [6, 20, 21]. In each penetration the impulse responses of a single neuron were compared to the two wavelengths. Sometimes there were multiple impulses from adjacent, responsive cells; in this case we concentrated on the unit of largest amplitude. We were able to monitor the waveform and amplitude of these impulses on an oscilloscope with a fast sweep speed in order to identify the particular unit being studied. We graded responses into five categories: cells excited exclusively by 505 nm; cells excited more strongly by 505 than 370 nm; cells affected equally; cells affected more by 370 than 505 nm and cells excited exclusively by 370 nm. We sometimes compared a cell's response to stimuli of reduced energies to determine the threshold of a cell's response.
We used a statistical software program (JMP 7.0, SAS Institute Inc., Cary, NC) to combine our estimates of UV-versus M-cone strength influencing each cell and the location of the cell in the striate cortex to produce a colour coded contour plot showing the topography of the cone inputs.