Cone inputs to murine striate cortex
© Ekesten and Gouras; licensee BioMed Central Ltd. 2008
Received: 17 March 2008
Accepted: 14 November 2008
Published: 14 November 2008
We have recorded responses from single neurons in murine visual cortex to determine the effectiveness of the input from the two murine cone photoreceptor mechanisms and whether there is any unique selectivity for cone inputs at this higher region of the visual system that would support the possibility of colour vision in mice. Each eye was stimulated by diffuse light, either 370 (strong stimulus for the ultra-violet (UV) cone opsin) or 505 nm (exclusively stimulating the middle wavelength sensitive (M) cone opsin), obtained from light emitting diodes (LEDs) in the presence of a strong adapting light that suppressed the responses of rods.
Single cells responded to these diffuse stimuli in all areas of striate cortex. Two types of responsive cells were encountered. One type (135/323 – 42%) had little to no spontaneous activity and responded at either the on and/or the off phase of the light stimulus with a few impulses often of relatively large amplitude. A second type (166/323 – 51%) had spontaneous activity and responded tonically to light stimuli with impulses often of small amplitude. Most of the cells responded similarly to both spectral stimuli. A few (18/323 – 6%) responded strongly or exclusively to one or the other spectral stimulus and rarely in a spectrally opponent manner.
Most cells in murine striate cortex receive excitatory inputs from both UV- and M-cones. A small fraction shows either strong selectivity for one or the other cone mechanism and occasionally cone opponent responses. Cells that could underlie chromatic contrast detection are present but extremely rare in murine striate cortex.
The murine retina contains middle (M) and ultra-violet (UV) sensitive cone opsins, which could provide the mouse with a form of colour vision. These two cone opsins, however, are distributed in different regions of the retina; the M- and UV-cone opsins are more common in the superior and ventral retina, respectively, which complicates comparing their responses in the same areas of visual space, a logical requirement for colour vision. In addition, many murine cones seem to express both M- and UV-cone opsins [1, 2], which is also a handicap for colour vision. Nevertheless behavioural studies indicate that mice have colour vision  and anatomical evidence exists for UV cone specific bipolar cells , another requirement for colour vision. We have also found evidence that some retinal ganglion cells receive inputs exclusively from UV-cones  supporting the latter anatomical findings. Therefore the question of colour vision in the mouse deserves further study. To do this, we have recorded responses from single neurons in murine visual cortex to determine whether there is any unique selectivity for cone inputs at this higher region of the visual system that would support the possibility of colour vision in mice.
Physiological characteristics of cells in striate cortex
Cone selective cells
Cortical topography of cone inputs
We estimated the depth at which we encountered cells in each penetration by the reading of the micrometer drive. This is not an accurate method because it is often difficult to know exactly when the microelectrode has just entered the cortex and the angle of penetration is not always perpendicular to the cortical layers. Our overall estimate was that most of the cells encountered were in layers 2, 3 and 4.
Our results show that all areas of striate cortex have cells that respond to diffuse light stimulation. This presumably reflects reports that a high percentage of neurons in murine striate cortex do not show orientation selectivity [6–9]. These may be the cells that we studied, although it is possible that murine cortical cells could respond to full field stimuli and also show orientation selectivity to slits or bars. Whether the cell bodies of these cells were located in striate cortex is assumed and strengthened by occasional binocular responses. The main goal of our study was to determine if cells with exclusive selectivity for one cone mechanism exist in striate cortex and therefore this distinction as to whether a cell was an incoming geniculate axon or a cortical cell was not critical to this goal.
Our results reveal that most cells in murine striate cortex respond similarly to both wavelengths implying that they are receiving inputs from both UV- and M-cones. We assume that 505 nm light only affects M- and not UV-cone opsins, because the UV-cone opsin absorbs no light in this region of the spectrum . We also assume that 370 nm light affects these cells through UV-cones and not through the beta band of M-cone opsin. This assumption is strengthened by the fact that the sensitivity for 370 nm tended to be as great or greater than it was for 505 nm, which would not be the case if we were affecting these cells by the much less sensitive beta-band of M-cone opsin. A higher sensitivity to ultra-violet than to mid-spectral light agrees with recordings of the murine electroretinogram and retinal ganglion cell responses [3, 11]. The strong responsiveness to both wavelengths implies that most cells in murine striate cortex are using achromatic rather than colour contrast for vision. It also implies that the chromatic aberration inherent in ultra-violet versus mid-spectral images can be handled by the murine visual system. Chromatic aberration may be less of a problem due to greater depth of focus inherent in the small eye of the mouse and the structural properties of the lens [12, 13].
That the effectiveness of these two wavelengths was relatively similar throughout striate cortex was surprising in comparison to the representation of these two cone mechanisms across the retina . We expected that the caudal part of striate cortex, reflecting the inferior retinal projection, would be more obviously dominated by UV-cone inputs and the rostral part more representative of M-cones. This difference was not apparent, although there was a tendency for UV-cone strength to be greater caudally. Perhaps this lack of representation of cone mechanisms according to their retinal representation reflects co-expression of M-cone opsin in many UV-cones. Another possibility could be that our full field stimulus drives a cell system, which has a large projection area in striate cortex and tends to receive an input from both cone mechanisms. This more global system may inhibit a more local system that reflects the retinal anatomy more closely.
There were only a few cells that responded strongly or only to one of these two wavelengths and very few that responded in an opponent fashion. It would be valuable to have more information about the receptive field organization of these rare cone opponent cells, although this is somewhat difficult to do with ultra-violet stimuli that the investigators cannot see. The rarity of such cone selective cells could reflect the fact that chromatic contrast has an inherently lower spatial resolution than achromatic contrast and therefore may involve fewer cortical cells for the processing of colour vision. This feature appeared in studies of monkey striate cortex where most cortical cells showed no evidence of cone opponency, while many cells in the lateral geniculate nucleus did. Colour opponent cells also exist in cat retina and lateral geniculate nucleus[15, 16], but they have never been detected in cat striate cortex, an area extensively studied. Again, the ratio of chromatic to achromatic selective cells seems to diminish in striate cortex. An important aspect of chromatic processing in monkeys is the existence of small, local areas called "blobs" , where cells with cone opponent behaviour are concentrated and therefore processing the same area of visual space. In murine cortex, most of the cells selective for UV stimuli were located at intermediate and caudal-lateral areas while cells selective for M-opsin stimuli were commonly located rostrally, which seems inappropriate for colour vision where comparisons of cone strengths must be determined within the same areas of visual space. It is interesting, however, that the three cone opponent cells we encountered were found in different areas, perhaps also reflecting small, local sites of chromatic processing.
We found two different types of neurons responding to our stimuli. One had little to no spontaneous activity and responded briefly and at high frequency to both wavelengths of stimulation and tended to have relatively large impulse amplitudes. The other class of neurons had more spontaneous activity, responded in a more prolonged way to our stimuli and usually had impulses of relatively small amplitude. Both of these cell types were recorded next to each other in most penetrations implying that they are similarly distributed. We found a similar dichotomy among retinal ganglion cells , but such a distinction has not been reported by other investigators of murine retinal ganglion cells . Recordings from the murine lateral geniculate nucleus indicate the presence of burst-tonic responses from many cells, but there was no mention of a class of phasic cells . There is some evidence of two classes of cells without orientation selectivity in murine striate cortex, one with conventional center/surround receptive fields and a second class with large fields best excited by fast moving stimuli [8, 9]. More research will be needed to determine if two different cell systems target murine striate cortex.
There were two varieties of cell responses. One type of cell, phasic cells, had little to no spontaneous activity and responded with a few impulses of large amplitude and high frequency either at the on and/or the off phase of the stimulus (42%). Another type of cell, which was more frequent (51%) had more spontaneous activity, impulses of smaller amplitude and tended to respond tonically.
Most cells in murine striate cortex receive excitatory inputs from both UV- and M-cones. A small fraction shows either strong selectivity for one or the other cone mechanism and occasionally cone opponent responses. The latter supports the hypothesis that mice may have some form of colour vision.
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.
This research was supported in part by Formas Grant 221-2006-2192 and Research to Prevent Blindness Inc.
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