Retinal ganglion cell anatomy and physiology after section of the optic nerve in mice overexpressing bcl-2.
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Animals↗
Biomedical subjects
E Strettoi
Publications and source records attributed to E Strettoi.
The number of unidentified amacrine cells in the mammalian retina.
The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit's retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.
Animals↗
Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2.
Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the photo-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we study in vivo the role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice. After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.
Animals↗
Long-term survival of retinal ganglion cells following optic nerve section in adult bcl-2 transgenic mice.
The bcl-2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl-2 in adult transgenic mice can protect the axotomized ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice approximately 65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, < 10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl-2 protein in central neurons is a very effective strategy to ensure long-term survival in axotomized cells.
Animals↗
The organization of the inner nuclear layer of the rabbit retina.
The initial goal of this study was to establish an accounting of the major classes of cells present in the inner nuclear layer (INL) of the rabbit's retina. Series of 80-100 radial sections 1 micron thick were cut from retinal blocks dissected at intervals along the vertical meridian. They were photographed at high magnification in the light microscope. By visualizing the initial segments of processes leaving the somata, we could identify each cell as a bipolar, amacrine, horizontal, or Müller cell. The identifications made by light microscopy were confirmed by electron microscopy of alternating ultrathin sections. On average, bipolar cells made up 41% of the total INL cells, amacrine cells 32%, horizontal cells 1.5%, and Müller cells 24%. These fractions varied relatively little across the retina or among different animals. We next immunolabeled the rod bipolar cells of whole-mounted retinas with antibodies against protein kinase C, using FITC as the visualizing agent. The same retinas were counterstained with a DNA-binding probe that fluoresces at longer wavelengths. Serial optical horizontal sections of the double-labeled wholemounts were made by confocal microscopy. On average, rod bipolars accounted for 10% of the total INL cells. By subtraction, the cone bipolars made up 31% of the total cells. We conclude that cone bipolars substantially outnumber rod bipolars, even in a retina in which rods outnumber cones by more than 20:1. Using the base of reference created here, a similar analysis can be carried out for other subclasses of retinal neuron. Because the analysis does not depend on absolute cell densities or corrections for shrinkage, data acquired by different histochemical techniques may be combined.
Animals↗
Cone bipolar cells as interneurons in the rod pathway of the rabbit retina.
In the mammalian retina, rod signals are transmitted by rod bipolars to the narrow-field, bistratified (AII) amacrine cell. This neuron, in turn, makes gap junctions with the axonal arborization of cone bipolar cells that reside in the vitreal half (sublamina b) of the inner plexiform layer (IPL). After examining rod bipolars and AII amacrines in the rabbit retina, we have now reconstructed from electron micrographs of continuous series of thin sections the synaptic connections of the axonal arborizations of cone bipolar cells that make the highest number of gap junctions with AII amacrines. These axonal arborizations were narrowly confined to stratum 4 (S4) of the IPL and made ribbon synapses to dyads of postsynaptic dendrites that belonged to either ganglion or amacrine cells. In the population of postsynaptic processes, 30% were ganglion cell dendrites. These dendrites were probably originating, at least in part, from on-center ganglion cells because their course was confined to sublamina b of the IPL. Of the remaining postsynaptic processes, 51.7% belonged to amacrine cells and 18.3% were not identified. Among the postsynaptic amacrine cell processes, 33.3% returned a reciprocal synapse onto the cone bipolar endings. These reciprocal synapses represented 21.3% of the total input onto the axonal arborizations, the remaining fraction (78.7%) arising from a heterogeneous population of amacrine dendrites that were purely presynaptic to the cone bipolars endings. Pre- and postsynaptic amacrines were part of several distinct microcircuits which suggest complex local processing of both rod and cone signals. Thus, the cone bipolars that make gap junctions with AII amacrines in sublamina b of the rabbit IPL exhibit a substantial output onto ganglion cells. This fact, in conjunction with our previous observations that in this sublamina ganglion cells receive negligible input from rod bipolars and AII amacrines, demonstrates that in the rabbit cone bipolars represent a necessary link in the pathway followed by rod signals to enter on-center ganglion cells. Thus, rod and cone signals ultimately share the same integrating mechanisms and converge onto the same set of ganglion cells.
Animals↗
The peripheral-type benzodiazepine receptor ligands [3H]Ro 5-4864 and [3H]PK 11195 bind to the retina of rabbit, but not of turtle.
The binding of [3H]flunitrazepam, [3H]Ro 5-4864, and [3H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [3H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [3H]Ro 5-4864 and [3H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (KD values of 24 +/- 2.3 and 2.2 +/- 0.8 nM, and Bmax values of 440 +/- 35 and 1,482 +/- 110 fmol/mg of protein, respectively). The binding was temperature- and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [3H]Ro 5-4864 and [3H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.
Animals↗
Synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the rabbit retina.
The synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the inner plexiform layer (IPL) of the rabbit retina were reconstructed from electron micrographs of continuous series of thin sections. The AII amacrine cell receives a large synaptic input from the axonal endings of rod bipolar cells in the most vitreal region of the IPL (sublamina b, S5) and a smaller input from axonal endings of cone bipolar cells in the scleral region of the IPL (sublamina a, S1-S2). Amacrine input, localized at multiple levels in the IPL, equals the total number of synapses received from bipolar cells. The axonal endings of cone bipolar cells represent the major target for the chemical output of the AII amacrine cell: these synapses are established by the lobular appendages in sublamina a (S1-S2). Ganglion cell dendrites represent only 4% of the output of the AII amacrine and most of them are also postsynaptic to the cone bipolars which receive AII input. The AII amacrine is not presynaptic to other amacrine cells. Finally, the AII amacrine makes gap junctions with the axonal arborizations of cone bipolars that stratify in sublamina b (S3-S4) as well as with other AII amacrine cells in S5. Therefore, in the rabbit retina 1) the rod pathway consists of five neurons arranged in series: rod-->rod bipolar-->AII amacrine-->cone bipolar-->ganglion cell; 2) it seems unlikely that a class of ganglion cells exists that is exclusively devoted to scotopic functions. In ventral, midperipheral retina, about nine rod bipolar cells converge onto a single AII amacrine, but one of them establishes a much higher proportion of synaptic contacts than the rest. Conversely, each rod bipolar cell diverges onto four AII amacrine cells, but one of them receives the largest fraction of synapses. Thus, within the pattern of convergence and divergence suggested by population studies, preferential synaptic pathways are established.
Animals↗
Synaptic connections of rod bipolar cells in the inner plexiform layer of the rabbit retina.
We have reconstructed from electron micrographs of a continuous series of thin sections the synaptic connections of the axonal arborizations of all the rod bipolar cells contained in a small region of the retina of the rabbit. We observed that all rod bipolars share the same pattern of connectivity and are probably functionally equivalent. As a rule, they do not contact ganglion cells. Their prevalent synaptic output is on narrow-field, bistratified, and indoleamine-accumulating amacrine cells. Their dominant inputs are the reciprocal synapses from the indoleamine-accumulating amacrines, but they also receive a sizable number of synaptic contacts from other, non-reciprocal, amacrine cells. The lateral spread of scotopic signals at the synapse between rod bipolars and narrow-field, bistratified amacrines is small. Finally, in the rabbit, as in the cat, a narrow-field, bistratified amacrine is inserted in series along the rod pathway.
Animals↗
Santiago Ramón y Cajal, the retina and the neuron theory.
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History, 19th Century↗
Subsurface cisternae in retinal double cones.
Double cones of tench and goldfish retina are characterized by extensive subsurface cisternae underlying the plasma membranes at the appositional area between the principal and accessory cone. Such a membrane system is absent in double cones of turtle and salamander retina. Measurements on both transverse and longitudinal sections gave a total appositional area of about 75 square microns, the subsurface cisterna in each element of the double cone being around 8-10% smaller due to multiple fenestrations at the level of the paraboloid. No gap junctions joining the inner segments of tench and goldfish double cones were detected, while gap junctions could be observed at the level of the ellipsoid and paraboloid of turtle double cones. The possible role of the subsurface cisternae in functional interactions between double cone elements is discussed.
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Intracellular recording from single and double cone cells isolated from the fish retina (Tinca tinca).
Intracellular recording were obtained from isolated single and double cone cells of the tench retina. Photoresponses show features characteristic of other species and behave linearly with very dim illumination. The cells spectral sensitivity matches their pigment absorption spectrum measured by previous authors. The principal and accessory members of double cones show a maximal sensitivity peak at about 644 and 547 nm, respectively. In addition, each of the two action spectra shows a secondary inflection at the peak wavelength of the adjacent member, suggesting functional coupling between the two members of double cones.
Animals↗
Appearance of cGMP-phosphodiesterase immunoreactivity parallels the morphological differentiation of photoreceptor outer segments in the rat retina.
We have investigated by immunofluorescence the appearance of immunoreactive guanosine 3'-5' cyclic monophosphate phosphodiesterase (cGMP-PDE) during the postnatal development of the retina of the pigmented rat. We show that a sudden increase in immunoreactivity takes place during postnatal day five (P5), when rod outer segments begin to form; immunoreactivity develops rapidly in the following days. Labeling is restricted to the developing photoreceptor outer segments, sparing other retinal cells, as confirmed by electron microscopy immunocytochemistry. In addition, cGMP-PDE immunoreactivity follows a center-to-periphery gradient paralleling photoreceptor differentiation. It appears that cGMP-PDE is expressed when the photoreceptor subcellular compartments are already formed, and represents a specific marker of late photoreceptor differentiation. The appearance of cGMP-PDE during development is temporally correlated with the appearance of other proteins of the phototransduction machinery.
3',5'-Cyclic-GMP Phosphodiesterases↗