M. Carter Cornwall, Ph.D.
Professor of Physiology & Biophysics
- Title Professor of Physiology & Biophysics
- Office W402A
- Email firstname.lastname@example.org
- Phone (617) 358-8466
- Education B.S. University of Utah
Ph.D. University of Utah
Dark Adaptation in Vertebrate Photoreceptors
The work in my laboratory is focused on understanding the response of the vertebrate eye to and recovery from the effects of bright light. Our principal approach is to make electrophysiological measurements of rod and cone photoreceptors of cold-blooded vertebrate animals, and to correlate these physiological responses to microspectrophotomeric measurements of the visual pigments as well as microfluorometric measurements of the concentration of vitamin A and Ca2+ contained within the cells. The cartoon in figure 1 illustrates the principal reactions that take place as the chromophore, 11-cis retinal in the visual pigment is photoisomerized by light to its all trans form light, how retinal is subsequently processed to recycle chromophore in the retinal pigment epithelium, and how it returns to the outers segments of rods to condense with opsin to newly form visual pigment.
Figure 2 illustrates two of the techniques which I use in my laboratory. Shown in the center is a schematic diagram of a microspectrophotometer which is used to measure the absorbance spectra of isolated rod and cone photoreceptors that have been dissociated from vertebrate retina. The inset at the right shows spectral absorbance data taken from a field of mouse rod photoreceptor outer segments. Rod outer segments contain the visual pigment, rhodopsin, The absorbance spectrum of mouse rhodopsin is shown by the blue trace. After exposure to bright green light, the spectrum in red is changed to show that most of this pigment has been photo-activated (bleached). The normal dark adapted spectrum can be restored following treatment of these cells with 11-cis retinal (vitamin A aldehyde). These data illustrate how we can manipulate the visual cycle (see above) under in vitro conditions. The data inset to the left of this figure illustrates an example of visual responses of these same photoreceptors to short flashes of green light. Here, a series of flashes of increasing intensity elicit responses that first are very small but then increase in amplitude and then saturate at high levels of intensity. Data illustrated in the left and right panels of the figure show how we can make measurements of the visual pigment in photoreceptors and at the same time make measurements from single cells of their sensitivity under different conditions of illumination.
Examples of visual pigment spectra taken from a variety of different mammalian photoreceptors is shown in figure 3. These data illustrate different spectra from both rod and cone photoreceptors can be obtained from the outer segments of such cells. Details of these spectral recordings are shown in the figure legend.
Other work in our laboratory has focused on the time course of changes in retinol fluorescence intensity following a large bleach of the visual pigment. As illustrated in Fig 1, the reduction of all trans retinal to all trans retinol is the first step in the visual cycle. Representative data to investigate this reaction are shown in the figure below. The bright field image at top left shows cellular fragments as well as one intact cone (lower left) and one intact red rod (upper right). The cells were suspended in a bath that had been mounted on the stage of the fluorescence microscope. The second field on the top row shows a fluorescence image of the cells obtained following exposure to UV fluorescence excitation light light before visual pigment was photoactivated (bleached) (T = 0.00 min). It is apparent that both intact cells exhibit a large amount of fluorescence in their ellipsoid regions. This fluorescence is consistent with the large concentration of mitochondria located there, and likely results from the high concentration of NADH in mitochondria.
The cell was then exposed to sufficient bright green (500 nm) light to bleach in excess of 99% of the visual pigment contained in the outer segments of both cells. The top right panel (T = 0.52 min.) shows a large uniform increase in fluorescence in the region of the cone outer segment, and a small amount of fluorescence beginning to appear in the most proximal part of the outer segment of the rod. The bottom panel on the left (T = 13.09 min.) shows that by this time, much of the outer segment fluorescence in the cone had dissipated; however, the fluorescence in the rod outer segment continued to increase, and appeared to uniformly fill the outer segment of the rod. The image in the second panel from the left, bottom row (T = 37.80 min.) shows that the fluorescence in the cone outer segment by this time was very low, but that in the outer segment of the rod had achieved a maximum level. At this time 2 :m bovine IRBP was added to the bath. Thereafter, the fluorescence in the rod outer segment was observed to decline. The last measurement of fluorescence was made 87.82 min following the initial pigment bleach.
A complete list of publications from the laboratory can be obtained at PubMed.
Department of Physiology & Biophysics
Boston University School of Medicine
700 Albany Street, W402A
Boston MA 02118-2526
Phone: (617) 358-8466