Phospholipase C b 4 is involved in modulating the visual response in mice ( animal behavior y vision y transgenic mice y retinal function y visual response )

Expression of G protein-regulated phospholipase C (PLC)b4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC b4 may play a role in the mammalian visual process. A mouse line that lacks PLC b4 was generated and the physiological significance of PLC b4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC b4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC b4-null mice showed 4-fold reduction in the maximal amplitude of the rod aand b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC b4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC b4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC b4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell–cell interactions between rods and other retinal cells. Many hormones, neurotransmitters, and other biologically active molecules transduce signals by activating phospholipase C (PLC) through heterotrimeric GTP-binding proteins (G protein) (1, 2). PLC hydrolyzes phosphatidylinositol 4,5bisphosphate to generate two important second messengers: inositol triphosphate and diacylglycerol (3). The Gq class of G proteins, including Gaq, Ga11, Ga14, Ga15, and Ga16, activate the b class of PLC enzymes (4–12). Molecular cloning has revealed four isoforms, PLC b1-4. The b4 isoform is found in the retina (13, 14) and in the brain (13, 15). PLC b4 has been localized to the photoreceptors, bipolar cells, horizontal cells, and ganglion cells of the bovine retina by immunohistochemical staining (14). Intriguingly, PLC b4 shares closer homology than PLC b1-3 with the NorpA protein, which mediates the phototransduction cascade in Drosophila photoreceptors (16). However, the role of PLC b4 in mammalian visual processes is not yet clear; hydrolysis of cGMP by G protein-regulated phosphodiesterase is established as the signal transduction pathway that mediates visual signal processing in photoreceptors (17). We have examined the effects of disrupting the PLC b4 gene on vision; changes in electroretinograms (ERGs) and in behavior suggest that PLC b4 plays a role in a step in visual processing that occurs after the initial photocascade in the rod outer segment and might feedback to effect the retinal response in situ. MATERIALS AND METHODS Shuttle Box Test. The shuttle box test was performed basically as described (18). The mice were placed in a shuttle box, which is divided into two compartments. An initial conditioned stimulus (CS, sound or light flash) was applied followed after 10 sec by an unconditioned stimulus (US, electric foot-shock, 0.5 mA). One hundred trials were performed each day. The mice with auditory and visual abilities can learn to associate sound and light, respectively, with the unconditional stimulus, and cross into the other compartment within 10 sec of conditional stimulus onset without receiving a foot-shock. ERG Recording. For recording methods and intensity calibrations see Lyubarsky and Pugh (19). The mice were anesthetized and their pupils were dilated. Light flashes were calculated to produce, from dimmest to most intense, the numbers of photoisomerizations as shown in the figures. Average responses of wild-type and PLC b4-null rods to 10 msec flashes of increasing strength. Stimulus timing is shown by flashmonitor below records. Responses were normalized by the saturating response amplitudes, rmax (Table 1). Flash strengths were: wild-type, 8.34, 53.7, 99.4, 361, and 1210 photonsymm2; PLC b4-null, 13.3, 44.2, 98.1, 161, 297, and 3640 photonsymm2 at 500 nm. Retinal Morphology.Wild-type and PLC b4-null mice used for ERG recordings were deeply anesthetized, and their eyes were enucleated. The eye was incised at the equator, and fixed in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffer. A central square (about 1.4 3 1.4 mm2 around the optic disk) was cut out, dehydrated, embedded in Epon, and sectioned radially. Sections (0.5–0.7 mm) were collected every 100 mm at the periphery and every 25 mm near the center; they were stained with toluidine blue, and retinal thickness at different locations was measured.