PMC

Rod bipolar cell—dye fills. (A) A single rod bipolar cell filled with Neurobiotin (green), focus on the dendrites in the OPL. The cell body is blurred because it is out of focus. This is a perfect fill which provides complete details of the dendritic tree. The major dendritic branches terminate at approximately 100 rod spherules. (B) Enlarged view of square in (A) to show two cone pedicles labeled for GluR5 (blue). The left cone pedicle, circled, receives a branch from the dye-filled rod bipolar cell. (C) Enlarged view of square in (A) to show the rod bipolar cell contact with a cone terminates at a fine mGluR6 (red) puncta within the cone pedicle, circled. On the right side, the rod bipolar cell dendrites appear to approach another cone pedicle but terminate at two close-by rod spherules. This can be seen because the rod bipolar dendrites terminate at one half of an mGluR6 doublet, arrow. This pattern is seen throughout the dendritic tree because normally rod spherules receive input from two different rod bipolar cells. (D–F) An example from another Neurobiotin filled rod bipolar cell where a rod bipolar dendrite reaches to the center of a cone pedicle, circled. Again, except for the cone contact, the rod bipolar dendrites terminate at half an mGluR6 doublet marking the site of rod spherules.

Localization of rod bipolar cell postsynaptic proteins and demonstration of rod divergence. (a) Deconvolved confocal image of P82 rod bipolar cell expressing tdTomato and stained for mGluR6, TRPM1, and Ribeye. (b) The same rod bipolar cell without synaptic protein staining and with the soma cutoff to highlight the dendritic tips. (c) Magnification of a single dendritic process (white dashed box in a and b) showing the tdTomato expression (top left), tdTomato and mGluR6 (top right), tdTomato and TRPM1 (bottom left), and tdTomato, mGluR6, TRPM1, and Ribeye (bottom right). Note that the rod bipolar cell process coincides with one punctum of the mGluR6/ TPRM1 ‘doublet’. (d) Confocal image of a tdTomato-expressing P82 rod bipolar cell. (e) PKCα staining showing all rod bipolar cells. (f) Overlay of the tdTomato and PKCα. (g) Cell from D at the level of dendritic tips. (h) Magnification of one dendritic tip (white dashed box in g) showing the tdTomato expression (top left); PKCα (top right); tdTomato, PKCα, and Ribeye (bottom left); and tdTomato, PKCα, Ribeye, and mGluR6 (bottom right). Example of divergence of the rod (Ribeye) to two different rod bipolar cell processes (white dashed outlines 1 and 2). Only one tdTomato rod bipolar cell process coincides with one of the PKCα-positive dendrites and one of the mGluR6 ‘doublet’ puncta

Multiple rod pathways in the mammalian retina. A, In the primary rod pathway, the rod-mediated signals are transmitted to rod bipolar cells and subsequently to AII amacrine cells. AII amacrine cells then make sign-conserving electrical synapses with ON cone bipolar cell axon terminals as well as sign-inverting chemical synapses with OFF cone bipolar cell axon terminals in the inner plexiform layer. In turn, the ON and OFF cone bipolar cells transmit the rod signals to ON and OFF ganglion cells, respectively. In all panels, the asterisks indicate electrical synapses, arrowheads indicate chemical synapses, and shaded areas mark the elements of the particular rod pathway. CB, Cone bipolar cell; RB, rod bipolar cell; AII, AII amacrine cell; GC, ganglion cell. B, In the secondary rod pathway, the rod-mediated signals move directly from rod to cone photoreceptors via interconnecting gap junctions. Then the rod signals are relayed to ON and OFF cone bipolar cells, which carry the signals to ganglion cells in the inner retina. C, A tertiary rod pathway provides an additional route for rod-mediated signals to reach OFF ganglion cells. In this pathway, the rod photoreceptors make direct, sign-conserving chemical synapses with a type of OFF cone bipolar cell, which, in turn, transmits rod signals to OFF ganglion cells. The large arrow indicates the direct chemical contact between rods and OFF bipolar cells.

Characterization of rod cell death. (A, B, D) One-μm optical sections of isolated rod cells fluorescently labeled with AO and EtdBr obtained by confocal microscopy. (C) Nomarski optics image of an isolated rod cell treated with high K⁺ Ringer's solution demonstrating rod cell morphology after loss of membrane integrity. (A) Live rod cells had green (AO), homogeneously stained chromatin. (B) Early apoptotic rod cells had green (AO), condensed chromatin. (C) Rod cells that had lost membrane integrity could be identified by their rectangular, elongated ellipsoid. *, ellipsoid; N, nucleus. (D) Late apoptotic cells with permeant membranes had red EtdBr, condensed chromatin. (Bar = 10 μm.) (E) Percent change in the number of apoptotic rod cells after treatment with light and 10 μM 9-cis-retinal. Photoactivation of reconstituted isorhodopsin increased apoptotic rod cell death by 49%. In n = 5 experiments from two animals, a total of 83 apoptotic rod cells was counted; *, P < 0.01, means ± SEM. (F) Percent change in the number of apoptotic rod cells after treatment with 2.5 μM β-ionone. β-ionone treatment increased apoptotic rod cell death by 46%. In n = 9 experiments from three animals, a total of 120 apoptotic rod cells was counted; *, P < 0.01, means ± SEM. (G) The effect of β-ionone on rod cell death was blocked by preincubation and cotreatment with 1 μM DEVD-fmk. In n = 11 experiments from four animals, a total of 4,417 rod cells was counted; *, P < 0.01 compared with control; **, P < 0.01 compared with β-ionone treated; unpaired t test, means ± SEM.