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We have used recent measurements of mammalian cone light responses and voltage-gated currents to calculate cone ATP utilization and compare it to that of rods. The largest expenditure of ATP results from ion transport, particularly from removal of Na⁺ entering outer segment light-dependent channels and inner segment hyperpolarization-activated cyclic nucleotide-gated channels, and from ATP-dependent pumping of Ca2+ entering voltage-gated channels at the synaptic terminal. Single cones expend nearly twice as much energy as single rods in darkness, largely because they make more synapses with second-order retinal cells and thus must extrude more Ca2+. In daylight, cone ATP utilization per cell remains high because cones never remain saturated and must continue to export Na⁺ and synaptic Ca2+ even in bright illumination. In mouse and human retina, rods greatly outnumber cones and consume more energy overall even in background light. In primates, however, the high density of cones in the fovea produces a pronounced peak of ATP utilization, which becomes particularly prominent in daylight and may make this part of the retina especially sensitive to changes in energy availability.

The light absorbing chromophore in opsin visual pigments is the protonated Schiff base of 11- cis- retinaldehyde (11cRAL). Absorption of a photon isomerizes 11cRAL to all- trans- retinaldehyde (atRAL), briefly activating the pigment before it dissociates. Light sensitivity is restored when apo-opsin combines with another 11cRAL to form a new visual pigment. Conversion of atRAL to 11cRAL is carried out by enzyme pathways in neighboring cells. Here we show that blue (450-nm) light converts atRAL specifically to 11cRAL through a retinyl-phospholipid intermediate in photoreceptor membranes. The quantum efficiency of this photoconversion is similar to rhodopsin. Photoreceptor membranes synthesize 11cRAL chromophore faster under blue light than in darkness. Live mice regenerate rhodopsin more rapidly in blue light. Finally, whole retinas and isolated cone cells show increased photosensitivity following exposure to blue light. These results indicate that light contributes to visual-pigment renewal in mammalian rods and cones through a non-enzymatic process involving retinyl-phospholipids. It is currently thought that visual pigments in vertebrate photoreceptors are regenerated exclusively through enzymatic cycles. Here the authors show that mammalian photoreceptors also regenerate opsin pigments in light through photoisomerization of N -ret-PE ( N -retinylidene-phosphatidylethanolamine.

Computation in neural circuits relies on the judicious use of nonlinear circuit components. In many cases, multiple nonlinear components work collectively to control circuit outputs. Separating the contributions of these different components is difficult, and this limits our understanding of the mechanistic basis of many important computations. Here, we introduce a tool that permits the design of light stimuli that predictably alter rod and cone phototransduction currents – including stimuli that compensate for nonlinear properties such as light adaptation. This tool, based on well-established models for the rod and cone phototransduction cascade, permits the separation of nonlinearities in phototransduction from those in downstream circuits. This will allow, for example, direct tests of how adaptation in rod and cone phototransduction affects downstream visual signals and perception.

The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo.

The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that [alpha]7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the [alpha]7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured [alpha]7.sup.+/+ and [alpha]7.sup.lacZ/lacZ postnatal cerebral cortical neurons with [alpha]7.sup.+/+ or [alpha]7.sup.lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of [alpha]7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many [alpha]7.sup.+/+ OECs migrated through laminin-coated transwells compared to [alpha]7.sup.+/+ OECs on poly-L-lysine (PLL). This is in contrast to [alpha]7.sup.lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express [alpha]7 integrin, and that laminin and its [alpha]7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo.

Computation in neural circuits relies on the judicious use of nonlinear circuit components. In many cases, multiple nonlinear components work collectively to control circuit outputs. Separating the contributions of these different components is difficult, and this limits our understanding of the mechanistic basis of many important computations. Here, we introduce a tool that permits the design of light stimuli that predictably alter rod and cone phototransduction currents - including stimuli that compensate for nonlinear properties such as light adaptation. This tool, based on well-established models for the rod and cone phototransduction cascade, permits the separation of nonlinearities in phototransduction from those in downstream circuits. This will allow, for example, direct tests of how adaptation in rod and cone phototransduction affects downstream visual signals and perception.

Computation in neural circuits relies on the judicious use of nonlinear circuit components. In many cases, multiple nonlinear components work collectively to control circuit outputs. Separating the contributions of these different components is difficult, and this limits our understanding of the mechanistic basis of many important computations. Here, we introduce a tool that permits the design of light stimuli that predictably alter rod and cone phototransduction currents – including stimuli that compensate for nonlinear properties such as light adaptation. This tool, based on well-established models for the rod and cone phototransduction cascade, permits the separation of nonlinearities in phototransduction from those in downstream circuits. This will allow, for example, direct tests of how adaptation in rod and cone phototransduction affects downstream visual signals and perception.

Genetic engineering in mammals is furthest developed in Mus musculus and has facilitated great strides towards understanding the molecular and cellular mechanisms underlying human biology and disease. Despite the advantages afforded through genetic manipulations, studies involving retinal photoreceptors have been largely constrained to rods due to the technical challenges of isolating cones. This dissertation describes the methodology I have developed to reliably identify unlabeled mouse cone somata, and using whole-cell patch clamp, record their conductance and voltage changes in response to diverse stimuli. I made highly resolved measurements of cone dark current, membrane capacitance, and resting membrane potentials. Photoresponses were recorded with brief and steady-light stimulation protocols, and I characterized parameters describing response sensitivity and kinetics. Wild-type cones showed evidence of lateral electrical coupling to rod photoreceptors (i.e. the rod secondary pathway). The loss of calcium- sensitive proteins affected waveform of the responses to brief and steady-light stimulation. Notably, when compared to controls, the loss of guanylyl cyclase accelerating proteins caused cones to be more sensitive to a given light stimulus and to reopen fewer light-sensitive channels. The ability to manipulate the cone membrane potential enabled the biophysical characterization of multiple inner segment ion conductances. Synaptic calcium and the hyperpolarization-activated rectifying conductances were isolated with pharmacology. By combining voltage stimulation with light flashes, I also studied the reversal potential of the light response for the first time in any mammalian species. The inner segment calcium- and calcium-activated anion conductances were significantly large in the mouse cones and had to be blocked in order to isolate the light-sensitive conductance. Unlike rods, cones must remain active in brighter ambient light. In addition to calcium-dependent adaptation that adjusts the phototransduction machinery, the cone must replenish large fractions of photopigment. We identified a light-driven, non- enzymatic pathway in which all-trans-retinal does not even leave the photoreceptor. We show that when bound to a retinyl phosopholipid-complex in the membrane and exposed to blue light, all-trans-retinal can be preferential photoconverted back to 11-cis- retinal. All the mechanisms employed to keep cones functional and out of saturation are for naught if well defined synaptic connections are not made properly. We show that cones lacking a synaptic adaptor protein LRIT1 responded to light with normal characteristics. Cone bipolar cells, however, responded to light flashes with altered sensitivities. Thus, visual perception relies not only on high-fidelity encoding of light stimuli, but also on precise circuitry and signal transmission.