Light drives the developmental progression of outer retinal function.

The complex nature of rod and cone photoreceptors and the light-evoked responsivity of bipolar cells in the mature rodent retina have been well characterized. However, little is known about the emergent light-evoked response properties of the mouse retina and the role light plays in shaping these emergent responses. We have previously demonstrated that the outer retina is responsive to green light as early as postnatal day 8 (P8). Here, we characterize the progression of both photoreceptors (rods and cones) and bipolar cell responses during development and into adulthood using ex vivo electroretinogram recordings. Our data show that the majority of photoreceptor response at P8 originates from cones and that these outputs drive second-order bipolar cell responses as early as P9. We find that the magnitude of the photoresponse increases concurrently with each passing day of postnatal development and that many functional properties of these responses, as well as the relative rod/cone contributions to the total light-evoked response, are age dependent. We compare these responses at eye opening and maturity to age-matched animals raised in darkness and found that the absence of light diminishes emergent and mature cone-to-bipolar cell signaling. Furthermore, we found cone-evoked responses to be significantly slower in dark-reared retinas. Together, this work characterizes the developmental photoresponsivity of the mouse retina while highlighting the importance of properly timed sensory input for the maturation of the first visual system synapse.

Ex vivo electroretinograms made easy: performing ERGs using 3D printed components.

KEY POINTS: Rod and cone photoreceptors convert light into electrochemical signals that are transferred to second order cells, initiating image-forming visual processing. Electroretinograms (ERGs) can detect the associated light-induced extracellular transretinal events, allowing for physiological assessment of cellular activity from morphologically intact retinas. We outline a method for economically configuring a traditional patch-clamp rig for performing high signal-to-noise ex vivo ERGs. We accomplish this by incorporating various 3D printed components and by modifying existing light pathways in a typical patch-clamp rig. This methodology provides an additional set of tools to labs interested in studying the physiological function of neuronal populations in isolated retinal tissue. ABSTRACT: Rod and cone photoreceptors of the retina are responsible for the initial stages in vision and convey sensory information regarding our visual world across a wide range of lighting conditions. These photoreceptors hyperpolarize in the presence of light and subsequently transmit signals to second-order bipolar and horizontal cells. The electrical components of these events are experimentally detectable, and in conjunction with pharmacological agents, can be further separated into their respective cellular contributions using electroretinograms (ERGs). Extracellular activity from populations of rods and cones generate the negative-going a-wave, while ON-bipolar cells generate positive-going b-waves. ERGs can be performed in vivo or alternatively using an ex vivo configuration, where retinas are isolated and transretinal photovoltages are recorded at high signal-to-noise ratios. However, most ERG set-ups require their own unique set of tools. We demonstrate how, at low cost, to reconfigure a typical patch-clamp rig for ERG recordings. The bulk of these modifications require implementation of various 3D printed components, which can alternatively aid in generating a stand-alone ERG set-up without a patch-rig. Further, we discuss how to configure an ERG system without a patch-clamp rig. Compared to in vivo ERGs, these are superior when measuring small responses, such as those that are cone-evoked or those from immature mouse retinae. This recording configuration provides high signal-to-noise detection of a-waves (300-600 µV) and b-waves (1-3 mV), and is ultimately capable of discerning small (1-2 µV) photovoltages from noise. These quick and economical modifications allow researchers to equip their technical arsenal with an interchangeable patch-clamp/ERG system.

The Development of Mid-Wavelength Photoresponsivity in the Mouse Retina.

PURPOSE: Photoreceptors in the mouse retina express much of the molecular machinery necessary for phototransduction and glutamatergic transmission prior to eye opening at postnatal day 13 (P13). Light responses have been observed collectively from rod and cone photoreceptors via electroretinogram recordings as early as P13 in mouse, and the responses are known to become more robust with maturation, reaching a mature state by P30. Photocurrents from single rod outer segments have been recorded at P12, but no earlier, and similar studies on cone photoreceptors have been done, but only in the adult mouse retina. In this study, we wanted to document the earliest time point in which outer retinal photoreceptors in the mouse retina begin to respond to mid-wavelength light. METHODS: Ex-vivo electroretinogram recordings were made from isolated mouse retinae at P7, P8, P9, P10, and P30 at seven different flash energies (561 nm). The a-wave was pharmacologically isolated and measured at each developmental time point across all flash energies. RESULTS: Outer-retinal photoreceptors generated a detectable response to mid-wavelength light as early as P8, but only at photopic flash energies. a-wave intensity response curves and kinetic response properties are similar to the mature retina as early as P10. CONCLUSION: These data represent the earliest recorded outer retinal light responses in the rodent. Photoreceptors are electrically functional and photoresponsive prior to eye opening, and much earlier than previously thought. Prior to eye opening, critical developmental processes occur that have been thought to be independent of outer retinal photic modulation. However, these data suggest light acting through outer-retinal photoreceptors has the potential to shape these critical developmental processes.