Microtubule reorganization is obligatory for growth cone turning

To examine the role of microtubules in growth cone turning, we have compared the microtubule organization in growth cones advancing on uniform laminin substrates with their organization in growth cones turning at a laminin–tenascin border. The majority (82%) of growth cones on laminin had a symmetrical microtubule organization, in which the microtubules entering the growth cone splay out toward the periphery of the growth cone. Growth cones at tenascin borders had symmetrically arranged microtubules in only 34% of cases, whereas in the majority of cases the microtubules were displaced toward one-half of the growth cone, presumably stabilizing in the direction of the turn along the tenascin border. These results suggest that reorganization of microtubules could underlie growth cone turning. Further evidence for the involvement of microtubule rearrangement in growth cone turning was provided by experiments in which growth cones approached tenascin borders in the presence of nanomolar concentrations of the microtubule stabilizing compound, Taxol. Taxol altered the organization of microtubules in growth cones growing on laminin by restricting their distribution to the proximal regions of the growth cone and increasing their bundling. Taxol did not stop growth cone advance on laminin. When growing in the presence of Taxol, growth cones at tenascin borders were not able to turn and grow along the laminin–tenascin border, and consequently stopped at the border. Growth cones were arrested at borders for as long as Taxol was present (up to 6 h) without showing any signs of drug toxicity. These effects of Taxol were reversible. Together, these results suggest that microtubule reorganization in growth cones is a necessary event in growth cone turning.

Circuitry for color coding in the primate retina.

Human color vision starts with the signals from three cone photoreceptor types, maximally sensitive to long (L-cone), middle (M-cone), and short (S-cone) wavelengths. Within the retina these signals combine in an antagonistic way to form red-green and blue-yellow spectral opponent pathways. In the classical model this antagonism is thought to arise from the convergence of cone type-specific excitatory and inhibitory inputs to retinal ganglion cells. The circuitry for spectral opponency is now being investigated using an in vitro preparation of the macaque monkey retina. Intracellular recording and staining has shown that blue-ON/yellow-OFF opponent responses arise from a distinctive bistratified ganglion cell type. Surprisingly, this cone opponency appears to arise by dual excitatory cone bipolar cell inputs: an ON bipolar cell that contacts only S-cones and an OFF bipolar cell that contacts L- and M-cones. Red-green spectral opponency has long been linked to the midget ganglion cells, but an underlying mechanism remains unclear. For example, receptive field mapping argues for segregation of L-and M-cone signals to the midget cell center and surround, but horizontal cell interneurons, believed to generate the inhibitory surround, lack opponency and cannot contribute selective L- or M-cone input to the midget cell surround. The solution to this color puzzle no doubt lies in the great diversity of cell types in the primate retina that still await discovery and analysis.

Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Effects of estrogen on growth plate senescence and epiphyseal fusion

Estrogen is critical for epiphyseal fusion in both young men and women. In this study, we explored the cellular mechanisms by which estrogen causes this phenomenon. Juvenile ovariectomized female rabbits received either 70 μg/kg estradiol cypionate or vehicle i.m. once a week. Growth plates from the proximal tibia, distal tibia, and distal femur were analyzed after 2, 4, 6, or 8 weeks of treatment. In vehicle-treated animals, there was a gradual senescent decline in tibial growth rate, rate of chondrocyte proliferation, growth plate height, number of proliferative chondrocytes, number of hypertrophic chondrocytes, size of terminal hypertrophic chondrocytes, and column density. Estrogen treatment accelerated the senescent decline in all of these parameters. In senescent growth plates, epiphyseal fusion was observed to be an abrupt event in which all remaining chondrocytes were rapidly replaced by bone elements. Fusion occurred when the rate of chondrocyte proliferation approached zero. Estrogen caused this proliferative exhaustion and fusion to occur earlier. Our data suggest that (i) epiphyseal fusion is triggered when the proliferative potential of growth plate chondrocytes is exhausted; and (ii) estrogen does not induce growth plate ossification directly; instead, estrogen accelerates the programmed senescence of the growth plate, thus causing earlier proliferative exhaustion and consequently earlier fusion.

A circadian clock regulates rod and cone input to fish retinal cone horizontal cells.

In the vertebrate retina, the light responses of post-receptor neurons depend on the ambient or background illumination. Using intracellular recording, we have found that a circadian clock regulates the light responses of dark-adapted fish cone horizontal cells. Goldfish were maintained on a 12-hr light/12-hr dark cycle. At different times of the day or night, retinas were superfused in darkness for 90 min ("prolonged darkness"), following which horizontal cells were impaled without the aid of any light flashes. In some of the experiments, fish were kept in constant darkness for 3-48 hr prior to surgery. After prolonged darkness during the night, but not during the day, the light responses of L-type cone horizontal cells resembled those of rod horizontal cells with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Following light sensitization during the night and day, the light responses of rod and cone horizontal cells were clearly different with respect to threshold, waveform, intensity-response functions, and spectral sensitivity. Under conditions of constant darkness for two full light/dark cycles, average responses of cone horizontal cells to a bright light stimulus during the subjective day were greater than during the subjective night. Prior reversal of the light/dark cycle reversed the 24-hr rhythm of cone horizontal cell responses to bright lights. In addition, following one full cycle of constant darkness, average cone horizontal cell spectral sensitivity during the subjective night closely matched that of rod horizontal cells, whereas average cone horizontal cell spectral sensitivity during the subjective day was similar to that of red (625 nm) cones. These results indicate that the effects of dark adaptation depend on the time of day and are regulated by a circadian clock so that cone input to cone horizontal cells predominates in the day and rod input predominates in the night.