Biomedical subjects
Publications and source records attributed to W A Harris.
Drosophila mutants transformed with a chimaeric gene that expresses the ocellar visual pigment in the major class of photoreceptor cells of the retina were used to investigate the properties of this minor pigment. The photoreceptor cells in which this opsin was misexpressed showed new spectral characteristics and physiology.
Xenopus embryos injected with tritiated thymidine throughout the stages of embryonic retinal neurogenesis showed that more than 95% of the embryonic retinal cells are born within a 25 hr period. While there are shallow central to peripheral, dorsal to ventral, and interlaminar gradients of neurogenesis in these eyes, throughout most of this 25 hr period, postmitotic cells are being added to all sectors and layers. Small clones of differentiated retinal neurons and glia derived from single neuroepithelial cells injected with HRP. These clones were elongated radially. They were also composed of many different combinations of cell types, suggesting a mechanism whereby determination is arbitrarily and independently assigned to postmitotic cells. Such a model, when tested statistically, fits our data very well. We present a scheme for cellular determination in the Xenopus retina in which a coherent group of clonally related cells stretch out radially as lamination begins. This brings different cells into different microenvironments. Local interactions in these microenvironments then lead the cells toward specific fates.
A glial cell line (XR1 cell line) derived from Xenopus retinal neuroepithelium was examined for neurite outgrowth promoting activity. A monolayer of the XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and freely elaborate neurites. The XR1 neurite outgrowth promoting activity is not secreted into the medium, but is laid down directly on the substrate where it remains active after lysing the cells by hypoosmotic shock. A polyclonal antiserum raised against membranes of the XR1 cells was effective in blocking neurite outgrowth on XR1 conditioned collagen. It is proposed that the neurite outgrowth promoting factors produced by the XR1 cells are associated with the extracellular matrix and possibly glial specific.
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Time-lapse video recordings were made of individual retinal ganglion cell fibres growing to and terminating in the optic tectum of Xenopus embryos. The fibres were stained by inserting a crystal of the lipophilic fluorescent dye, DiI, into the developing retina. Growth cones were observed in the optic tract and tectum using 20 ms flashes of light to induce fluorescence approximately once every minute. Fluorescent images were captured with a SIT camera, processed and saved on a time-lapse video recorder. The main conclusions from observing normal growing fibres are as follows. (1) Axons in the optic tract grow at a steady rate directly toward their targets without retracting or branching. (2) As axons approach the tectum they slow down and their growth cones become more complex. (3) Most terminal branches in the tectum are formed by back branching rather than by bifurcation of leading growth cones. In a second experiment, labelled growing axons were separated from their cell bodies by removing the retina. Such isolated axons continued to grow for up to 3 h in vivo and were capable of recognizing the tectum and arborizing there. This result shows that growth cones must contain the machinery needed to sense and respond to their specific pathways and targets.
Ocular dominance columns in the cat's visual cortex appear to develop out of an initially overlapping projection by a progressive segregation of the geniculocortical afferents serving the 2 eyes (reviewed in LeVay and Stryker, 1979). To determine whether electrical activity in the visual afferent pathway is involved in this normal, developmental rearrangement of synaptic connections, we blocked the discharge of retinal ganglion cells in both eyes by making repeated intravitreal injections of tetrodotoxin (TTX) during the period in which geniculocortical afferent segregation would normally be taking place. Control experiments for the side effects of the injection procedure, the systemic effects of TTX, and the effects of visual deprivation were carried out, and a series of normal animals of appropriate ages was also studied. We then examined the effects of retinal blockade and the various control procedures on the formation of ocular dominance columns using an anatomical assay, the autoradiographic labeling of geniculocortical afferent terminals in layer IV of the visual cortex by the transneuronal transport of tritiated proline injected into 1 eye, and a physiological assay, the ocular dominance of single cortical cells recorded extracellularly. After retinal TTX blockade, layer IV was labeled uniformly without periodic fluctuation in grain density, and nearly all cortical cells were driven well through both eyes. These assays thus indicated that retinal blockade completely blocked the formation of ocular dominance columns, unlike any of the control procedures, suggesting that the spontaneous maintained discharge of retinal ganglion cells may have an important role in the normal development of binocular connections in the visual cortex.
The outgrowth of axons from the early retina in vivo is compared with that from retinal explants in two types of culture systems. The normal time course of axonal growth along the primordial optic pathway to the optic tectum is characterized, using tritiated proline and horseradish peroxidase (HRP) as anterograde tracers. The rate of axonal elongation in vivo is estimated to be about 32 micron/hr at 22 degrees C. The HRP technique allows visualization of retinal growth cones in vivo. Observations can thus be made on their microanatomy and on the environment through which they navigate. The growth cones of retinal ganglion cells in the embryo have lamellipodia and fairly short filopodia (approximately 10 micron) which are directed forward. The growth cones are found near the pial surface of the brain but do not seem to maintain contact with it. Two culture systems were developed to investigate axonal pathways in vitro. In the first, different substrates and culture media were explored. Results indicate that growth cones prefer a polyornithine substrate over a collagen one. The media that promotes the best neurite outgrowth consists of L15 (60%), fetal calf serum (10%) and Xenopus embryo extract (1 mg/ml). Time-lapse video monitoring of substrate cultures reveals an average rate of outgrowth of about 18 micron/hr with great variability. The growth cones in these cultures are large, flattened, and complex compared to those in vivo, and their filopodia extend in many different directions. The second culture system is a collagen gel infiltrated with growth medium. In these conditions neurite outgrowth more closely mimics that in vivo. The rate is faster than on substrates, and the growth cones appear morphologically similar to those in the embryo. Preliminary experiments using the gel culture system to test for chemotaxis of retinal axons toward their targets failed to demonstrate such an effect.
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This study describes a classification of overall retinopathy severity based on the presence and severity of lesions graded in the Airlie House classification of diabetic retinopathy, and demonstrates its use in assessing progression of retinopathy over a 6-year period in a group of insulin-taking patients. One hundred and ninety-one insulin-taking patients with diabetes of at least 5 years' duration were identified from their doctors' records in 1970-1971. Patients were seen again in 1972-1973 and 1976-1977. Stereoscopic color fundus photographs of 7 fields in each eye were taken at each visit. All photos were graded using a modification of the Airlie House classification, which specified 6 levels of retinopathy for a given eye; when both eyes are considered an 11-step grading scheme results. Of all patients seen at the 2-year visit whose retinopathy was at risk of progressing, 41.2% showed progression of one level or more on the scale, 19.2% progression of two or more levels. At the 6-year examination comparable rates were 75.0% and 58.4%. Of patients whose retinopathy was level 4 in each eye (nonproliferative retinopathy of "moderate" severity) at the baseline visit, 13/21 (72%) had progressed to proliferative retinopathy in at least one eye at the 6-year examination. The classification scheme proposed appears useful for characterizing overall retinopathy severity of patients on the basis of gradings of fundus photographs. The data presented may be of help in planning trials of treatment aimed at slowing the development or progression of retinopathy.
Embryonic eye primordia were transplanted from the anuran, Xenopus laevis, to the urodele, Ambystoma mexicanum, in order to assess whether retinal axons of one species could grow to appropriate central targets in a distantly related species. To trace the early retinal projections, Xenopus primordia were removed, incubated for 20 min in a solution containing [3H]proline, washed, and then transplanted to host axolotl embryos. Genetically eyeless hosts were used in some of the experiments so that the Xenopus optic fibers would not be guided to their targets by the host's own optic nerve projections. Xenopus eyes were usually able to differentiate into small eyes in the axolotl host. Shortly after primary differentiation, the development of these eyes seemed to arrest. Autoradiography on paraffin sections of the central nervous system of the host revealed that in some cases Xenopus retinal ganglion cells were indeed able to send axons to the tectum of the axolotl host. This result suggests that the mechanisms of axonal navigation for this particular central projection are evolutionarily conserved.
Retinal axons were challenged to grow to their targets both along abnormal pathways and in the absence of impulse activity. Eye primordia were first transplanted from normal to ectopic sites in axolotl embryos. Most of the hosts were genetically eyeless, others were enucleated normal embryos. These axolotl embryos were then parabiotically joined to California newt embryos. Both operations were completed by stage 28, which is before axons have left the eye. The result of the parabiosis was a paralysis of the "eyeless" axolotl twin due to the newt's tetrodotoxin (TTX), while the newt twin remained normally active. When the axolotl twin reached early larval stage, about 1 week later, the projection from the silent transplanted retina was assessed using horseradish peroxidase (HRP) injections into the retina, after which the animals were killed and prepared histologically to reveal the presence of HRP in neuronal processes. The results from 17 such cases show normal topographic retinotectal projections: the dorsal retina projecting to the ventrolateral tectum, and the ventral retina projecting to the dorsomedial tectum. Unusual pathways were often taken to achieve these destinations. Control animals, both normal axolotl larvae developing alone and normal axolotl larvae parabiosed to newts, also showed the normal retinotectal projection patterns. These results indicate that the retinal projections in the experimental group were basically normal. Thus, fibers need neither impulse activity nor a particular pathway to navigate to their correct targets during development. Both factors can be eliminated simultaneously, yet retinal axons still find their way to the tectum and make an ordered map. This indicates that other factors, such as the chemoaffinity mechanisms proposed by Sperry (Sperry, R.W. (1963) Proc. Natl. Acad. Sci. U.S.A. 50: 703-710), may play a more major role in axonal pathfinding in this system.
Retinal nerve fibres form an orderly map of visual space in several centres in the vertebrate brain. Such topographic maps are a common feature of central nervous system organization, yet the way in which they develop is poorly understood. Early nerve projections in the fetal and neonatal mammalian brain have been found in several cases to be less restricted than those in the adult, suggesting that nerve fibres may initially form a diffuse set of connections in their target structure from which the adult map is sculpted by the elimination of terminals. Indeed, previous electrophysiological data indicate that the retinotectal map in Xenopus laevis might be initially disorganized. We report here, however, that the retinotectal projection is ordered from the beginning of tectal innervation (stage 39/40). We demonstrate this first autoradiographically by tracing groups of growing ganglion cell axons which we labelled by incubating sectors of eye rudiments, before axonal outgrowth, in 3H-proline and replacing them orthotopically. Separate labelling of dorsal and ventral parts of the initial projection showed that retinal fibres are organized topographically, as in the adult, in the tectal rudiment and throughout much of the pathway. Second, we show that visual responses are ordered in the tectum from the first stage that they can be mapped (stage 40). We conclude that the topographic ordering of retinotectal connections develops as a result of directed axonal outgrowth.
The plasticity of somatosensory afferents in the adult axolotl tectum was studied in two ways. First, normal adult axolotls were monocularly enucleated, and second, adult animals which had been monocular since embryogenesis had the tectum contralateral to the remaining eye excised. After a survival time of about one year the brains of these animals were studied electrophysiologically and histochemically. In the enucleated adults, the deprived tectum lacked visual activity, and the acetylcholinesterase staining, dense in the superficial neuropil of the normally innervated tectum, was much reduced. Somatosensory units remained deep in the tectal neuropil, as in normally innervated tecta. Serotonergic terminals associated with somatosensory responses were correspondingly largely restricted to the deeper tectal neuropil in these animals. This result is in contrast to that of embryonic enucleation where somatosensory activity and serotonergic terminals are found in the superficial tectal neuropil of the adult. Autoradiographic results from the tectal excision experiments showed that retinal axons in adults regrew to innervate the ipsilateral tectum when deprived of their normal targets. Associated with the ipsilateral innervation both visual units and acetylcholinesterase staining were found in the superficial tectum. In spite of this change in the visual innervation of the tectum, there was little difference in the distribution of somatosensory responses or serotonergic terminals; both remained relatively superficial and appeared to be unaffected by the late ingrowing retinal axons. This result is in contrast to the findings from embryonic transplants to genetically eyeless embryos which, as adults, show somatosensory responses and serotonergic endings restricted to the deeper tectal neuropil.
Systemic circulatory responses to atracurium (0.2 and 0.4 mg/kg) were studied in 15 healthy (ASA I or II) adult patients during enflurane (1.0 to 1.25% inspired) and nitrous oxide (70% inspired) anesthesia. All patients were premedicated with intramuscular morphine (10-15 mg) and glycopyrrolate (0.2 mg). Compared with control measurements during enflurane-nitrous oxide anesthesia, heart rate, cardiac and stroke index, central venous pressure, and systemic mean arterial pressure remained unchanged at 2, 5, and 10 min after administration of both doses of atracurium. Systemic vascular resistance was minimally decreased (7% compared to control) (0.01 less than P less than 0.05) at 10 min following both doses of atracurium. No patient demonstrated a decrease in systemic mean arterial pressure greater than 6 mmHg. The authors conclude that atracurium in doses which produce adequate skeletal muscle relaxation during steady-state enflurane anesthesia produces no clinically significant alteration in hemodynamic variables.
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Myxamoebae of Dictyostelium discoideum from exponentially growing cultures showed altered ornithine decarboxylase activity upon external osmotic perturbation. On transfer to hypotonic NaCl solutions (20 mosmol/kg), cells showed high enzyme activity which was relatively independent of the concentration of the coenzyme pyridoxal phosphate (assay concentrations, 5 and 200 microM). In hypertonic solution (400 mosmol/kg) cells had a reduced level of ornithine decarboxylase activity which was dependent on the coenzyme concentration. The changes in activity were freely reversible in further external osmotic manipulation. The response to osmotic change occurred rapidly, within a few minutes. The changes still occurred at 7 degrees C but 2 mM sodium azide prevented the formation of the high activity form, although this effect was reversed when azide was removed. Cycloheximide had no effect on the osmotically induced changes. Addition of putrescine caused ornithine decarboxylase eventually to the converted to the low-activity form regardless of the osmolality of the solution. The characteristic cofactor concentration dependence of the high- and low-activity form were retained on storage of the cell extracts. No evidence was found for diffusible effectors which stabilized one or the other form of the activity. The enzymes responsible for the two forms were of the same molecular size as judged by gel filtration, and the activities had similar thermostabilities. The results are interpreted in terms of an osmotically induced interconversion of two forms of a single ornithine decarboxylase.
Eyes were transplanted from normal axolotls to eyeless mutants, and several anatomical and physiological observations were made on the central visual centers in these animals. Some central projections were bilateral to the optic centers of the thalamus and midbrain, some traveled ipsilaterally to the same centers, and the rest grew down the spinal cord. This is similar to what has been found in eyes transplanted to normal hosts. The type of projection made in eyeless hosts correlated with the site of nerve entry into the CNS as in control hosts. Thus, the transplanted projection did not appear to be influenced by the host's optic nerves and tracts or lack of them. In spite of the transplanted optic fibers' taking abnormal paths, they made normally organized topographic maps on the host tecta. The visual and somatosensory topographic projections to the tectum were found to be in near perfect register normally, but in eyeless mutants to which rotated eyes had been transplanted, they were not. Acetylcholinesterase activity, found in the primary optic neuropil in normal animals, was greatly diminished in eyeless mutants, yet normal mutants with grafted eyes. Finally, transplantation of an eye to an eyeless mutant corrected the abnormally dark pigmentation caused by eyelessness but only in those cases of bilateral central innervation.