9 research outputs found
Development of On and Off retinal pathways and retinogeniculate projections
A fundamental functional feature of the visual system, one recognized in the very first electrophysiological retinal recordings ever made, is that some cells respond to light increments (On cells) while others are activated by light decrements (Off cells). The circuitry underlying On and Off responses in the mature retina have been well-established. In particular, it is known that the dendrites of On- and Off-center retinal ganglion cells (RGCs) stratify in different sublamina of the inner plexiform layer (IPL), where they are innervated by spatially segregated On- and Off-cone bipolar cell inputs. Also, segregated into On and Off sublaminae of the IPL are the processes of starburst amacrine cells. In some species (notably ferret and mink) the retinogeniculate projections are also segregated into sublayers of the dorsal lateral geniculate nucleus (dlgn). The mature organizational features summarized above arise gradually during the course of normal development. Thus, the dendrites of immature RGCs initially ramify throughout the IPL before becoming stratified into On or Off sublamina. This developmental event is regulated by the release of glutamate by developing bipolar cells. Treating the developing retina with the glutamate analog 2-amino-4-phosphonobutyric acid (APB) has been found to prevent the stratification of RGC dendrites. In the mature retina APB binds with mGluR6 receptors expressed by On cone and rod bipolar cells which hyperpolarizes these retinal interneurons and blocks their release of glutamate. The effects of short-term APB treatment are reversible by subsequent normal visual experience, while those of long-term treatment appear to be permanent. At the time that developing RGCs are multistratified they respond to both light onset as well as light offset, suggesting that these neurons are initially functionally innervated by On as well as Off-cone bipolar cells. In the dark-adapted state, On-Off responses of immature multistratified RGCs are completely blocked by APB, while at maturity only On responses are APB-sensitive. This suggests that an APB-resistant Off pathway (possibly from rods to Off-cone bipolar cells) is formed relatively late in development, after RGCs attain their stratified state. In contrast to the activity-regulated refinement of stratified On and Off RGCs, the segregated ingrowth of On- and Off-cone bipolar cells occurs in a highly specific manner, and is not dependent on the presence of either RGCs or cholinergic amacrine cells. It is suggested that the directed ingrowth of bipolar cell axons is guided by molecular cues expressed in the extracellular matrix whose identity is yet to be established. There is also evidence that the later formation of segregated On and Off retinogeniculate projections in the ferret is regulated by an activity-dependent Hebbian type mechanism. Blockade of RGC discharges as well as NMDA receptors in the dlgn perturbs the formation of such segregated inputs. Moreover, On and Off RGCs show distinct correlated firing patterns during the developmental period when the intermingled projections of these neurons are being sorted into sign specific sublayers. Collectively, the available evidence indicates that different developmental mechanisms operate on the different components of retinal and retinogeniculate On and Off pathways to attain the segregated state characteristic of the mature visual system. © 2003 Elsevier Ltd. All rights reserved
Segregation of On and Off bipolar cell axonal arbors in the absence of retinal ganglion cells
Retinal cells that respond selectively to light onset or offset are segregated into On and Off pathways. Here, we describe the development of cone bipolar cells whose axonal arbors at maturity synapse onto ganglion cell dendrites confined to On and Off strata of the inner plexiform layer (IPL). In particular, we sought to determine whether the formation of this segregated pattern is dependent on the presence of ganglion cells. Developing bipolar cells were visualized using an antibody against recoverin, the calcium binding protein that labels On and Off cone bipolar cells in the adult rat retina. Recoverin-positive cells were apparent in the ventricular zone on the day of birth [postnatal day 0 (P0)], before bipolar cells begin to migrate to the inner nuclear layer. Two distinct strata were first apparent in the IPL at P8, with the Off pathway maturing earlier than the On pathway. There was no indication of exuberant bipolar cell projections. Throughout development, there were also a small number of recoverin-positive cells of unknown origin in the ganglion cell layer. To assess whether the formation of On and Off cone bipolar cell projections is dependent on the presence of ganglion cells, these target neurons were eliminated by unilateral section of the optic nerve. This was done on the day of birth, resulting in a total loss of ganglion cells 5-6 d before bipolar cell axons innervate the IPL. In retinas with optic nerve sections, On and Off cone bipolar cells were present, albeit at a lower than normal density, and the axonal arbors of these interneurons were organized into two distinct strata. This indicates that ganglion cells are not essential for the formation of segregated On and Off bipolar cell inputs. These results lend support to the hypothesis that specific ingrowth patterns of bipolar cell terminal arbors could regulate the formation of stratified retinal ganglion cell dendrites
Ectopic photoreceptors and cone bipolar cells in the developing and mature retina
An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and -50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer
Depletion of cholinergic amacrine cells by a novel immunotoxin does not perturb the formation of segregated On and Off cone bipolar cell projections
Cone bipolar cells are the first retinal neurons that respond in a differential manner to light onset and offset. In the mature retina, the terminal arbors of On and Off cone bipolar cells terminate in different sublaminas of the inner plexiform layer (IPL) where they form synapses with the dendrites of On and Off retinal ganglion cells and with the stratified processes of cholinergic amacrine cells. Here we first show that cholinergic processes within the On and Off sublaminas of the IPL are present early in development, being evident in the rat on the day of birth, ∼10 d before the formation of segregated cone bipolar cell axons. This temporal sequence, as well as our previous finding that the segregation of On and Off cone bipolar cell inputs occurs in the absence of retinal ganglion cells, suggested that cholinergic amacrine cells could provide a scaffold for the subsequent in-growth of bipolar cell axons. To test this hypothesis directly, a new cholinergic cell immunotoxin was constructed by conjugating saporin, the ribosome-inactivating protein toxin, to an antibody against the vesicular acetylcholine transporter. A single intraocular injection of the immunotoxin caused a rapid, complete, and selective loss of cholinergic amacrine cells from the developing rat retina. On and Off cone bipolar cells were visualized using an antibody against recoverin, the calcium-binding protein that labels the soma and processes of these interneurons. After complete depletion of cholinergic amacrine cells, cone bipolar cell axon terminals still formed their two characteristic strata within the IPL. These findings demonstrate that the presence of cholinergic amacrine cells is not required for the segregation of recoverin-positive On and Off cone bipolar cell projections
Developmental Changes in the Electrophysiological Properties of Brain Stem Trigeminal Neurons During Pattern (Barrelette) Formation
Expression and purification of cysteine introduced recombinant saporin
Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity. © 2007 Elsevier Inc. All rights reserved