The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

No abstract

ABSTRACTlonotropic receptors for y-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAc responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated rpl and rp2 after their previously identifled human homologues. When coexpressed in Xenopus oocytes, rpl/rp2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by rp2 subunits since homomeric rpl receptors were sensitive to PTX; rp2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in rp2 in place of a threonine in rpl) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.

Whole cell voltage-clamp recordings were performed to investigate voltage-activated Ca(2+) currents in acutely isolated retinal bipolar cells of rats. Two groups of morphologically different bipolar cells were observed. Bipolar cells of the first group, which represent the majority of isolated bipolar cells, were immunoreactive to protein kinase C (PKC) and, therefore likely to be rod bipolar cells. Bipolar cells of the second group, which represent only a small population of isolated bipolar cells, did not show PKC immunoreactivity and were likely to be cone bipolar cells. The validity of morphological identification of bipolar cells was further confirmed by the presence of GABA(C) responses in these cells. Bipolar cells of both groups displayed low-voltage-activated (LVA) Ca(2+) currents with similar voltage dependence of activation and steady-state inactivation. However, the activation, inactivation, and deactivation kinetics of the LVA Ca(2+) currents between rod and cone bipolar cells differed. Particularly, the LVA Ca(2+) currents of rod bipolar cells displayed both transient and sustained components. In contrast, the LVA Ca(2+) currents of cone bipolar cells were mainly transient. In addition, the LVA Ca(2+) channels of rod bipolar cells were more permeable to Ba(2+) than to Ca(2+), whereas those of cone bipolar cells were equally or less permeable to Ba(2+) than to Ca(2+). The LVA Ca(2+) currents of both rod and cone bipolar cells were antagonized by high concentrations of nimodipine with IC(50) of 17 and 23 microM, respectively, but largely resistant to Cd(2+) and Ni(2+). Bipolar cells of both groups also displayed high-voltage-activated (HVA) Ca(2+) currents. The HVA Ca(2+) currents were, at least in part, to be L-type that were potentiated by BayK-8644 (1 microM) and largely antagonized by low concentrations of nimodipine (5 microM). The L-type Ca(2+) channels were almost exclusively located at the axon terminals of rod bipolar cells but expressed at least in the cell soma of cone bipolar cells. Results of this study indicate that rod and cone bipolar cells of the mammalian retina differentially express at least two types of LVA Ca(2+) channels. Rod and cone bipolar cells also show different spatial distribution of L-type Ca(2+) channels.