The impact of intracerebral retinal transplants on types of behavior exhibited by host rats.

Retinae transplanted over the midbrain of newborn rats establish functional connections with host brain centers, which provide a substrate for several distinct visual functions. These responses provide insight into the relationship between anatomy and behavior under normal conditions and after brain injury, as well as into the strategies used by an animal to extract significant information from its visual environment.

Microchemical analysis of retina layers in pigmented and albino rats by Fourier transform infrared microspectroscopy.

Fourier transform infrared (FT-IR) microspectroscopy is a powerful technique that can be used to collect infrared spectra from microscopic regions of tissue sections. The infrared spectra are evaluated to chemically characterize the absorbing molecules. This technique can be applied to normal or diseased tissues. In the latter case, FT-IR microspectroscopy can reveal chemical changes that are associated with discrete regions of lesion sites, which can provide insights into the chemical mechanisms of disease processes. In the present study, FT-IR microspectroscopy was used to analyze sections of retina from normal (pigmented) and albino rats. The outer segments of retinas from pigmented animals were found to have unusually strong absorption values for C&z.dbnd6;C-H unsaturation and carbonyl functional groups. Docosahexaenoic acid (DHA), a major constituent of lipids in the outer segments, also had particularly high absorption values for these functional groups, which suggests that it is responsible for those enhanced absorption values. Absorbance values for the unsaturation and carbonyl functional groups were substantially reduced in the outer segments of retinas from albino animals. This finding, together with data from other studies on light-induced oxidative events in the retina, indicates a loss of DHA by a light-induced mechanism in albino animals. The outer nuclear layer had strong absorbance values for H-C-OH and P&z. dbnd6;O functional groups, which is likely due to the sugar phosphate backbone of DNA. The outer and inner plexiform layers were found to contain greater concentrations of CH(2) and C&z.dbnd6;O functional groups than the outer and inner nuclear layers, which is due to the high concentration of synaptic connections in the former layers. In summary, FT-IR microspectroscopy revealed a unique chemical profile in the outer segments compared to other retinal layers, and this profile was altered in albino animals.

Development of Light-activated Pupilloconstriction in Rats as Mediated by Normal and Transplanted Retinae.

The relationship between the development of the pupilloconstriction response to changes in light levels and retinal maturation was studied in normal rats and rats that had received intracranial retinal transplants at birth. A pupillary response to light was first observed between postnatal days 7 and 9 in normal rats, and was typically of small amplitude and sluggish. By the time the eyelids first open, 2 weeks after birth, the pupillary response had improved to near adult levels. The inception of the pupillary response correlates with the first appearance of conventional synaptic contacts in the inner and outer plexiform layers of the retina, while improved responses correlate with maturation of photoreceptor outer segments and formation of synaptic ribbons in the inner plexiform layer. When embryonic retinae were transplanted to intracranial locations in newborn hosts and the transplants later illuminated as the host matured, the onset of a pupillary response to transplant illumination was delayed in proportion to the developmental disparity between the transplant and the host. The pattern of anatomical development in transplanted retinae was also similar, but delayed in time, compared to normal retinae. This indicates that the limiting factors for expression of light-activated pupilloconstriction exist within the retina, rather than being intrinsic to the central nuclei or to the output pathway subserving the response.

Proximity as a factor in the innervation of host brain regions by retinal transplants.

Embryonic mouse retinae transplanted to a variety of locations within the rostral midbrain of neonatal rats exhibit selective innervation of host visual nuclei when studied at maturity. Some of these nuclei (superior colliculus, nucleus of the optic tract, dorsal terminal nucleus) usually receive extensive transplant projections, others are innervated partially (dorsal division of the lateral geniculate nucleus, olivary pretectal nucleus, medial terminal nucleus), while a few (ventral division of the lateral geniculate nucleus, suprachiasmatic nucleus, intergeniculate leaflet) are not innervated at all. The selectivity of this innervation is largely independent of the transplant's position within the rostral brainstem, while the density of innervation of individual nuclei depends in part upon the proximity of the transplant to the nucleus and upon whether the host retinal projection to that nucleus is present or absent. These findings provide a foundation for further studies of the behavioral capabilities of retinal transplants, for developmental studies of factors responsible for the establishment of normal neural projections, and for examination of the immunological consequences of transplantation.

The Internet and the nutritional scientist.

The Internet provides many opportunities to learn, to educate, and to communicate new ideas. This article introduces concepts and terms that will facilitate the use of electronic information media by nutritional scientists. A vast array of sites on the Internet are relevant to the nutritional scientist, including those developed by government, industry, and educational sources, professional societies, and individuals. Using the wide variety of electronic sources that make up the Internet in an efficient and effective manner is an important skill not only for locating specific information, but also for keeping abreast of novel developments and new concepts as they are introduced and discussed. Uncritical acceptance of information appearing in the electronic media, however, is problematic; electronic publishing may occur without the rigorous peer-review process common for publishing in scientific journals. Those intending to publish material electronically must accept responsibility for the veracity of the information, realizing that anyone, from the lay consumer to the professional, may have access to that information. The Internet and its electronic relatives (eg, the World Wide Web and newsgroups) can become invaluable tools for nutritional scientists, extending beyond traditional sources of information (eg, the library) to support research and educational efforts, but use of this new technology must be tempered with knowledge of its limitations as well as potentials.

Anatomical and functional consequences of induced rejection of intracranial retinal transplants.

Retinae from embryonic rats transplanted over the midbrain of newborn host rats establish connections with visual centres of the host brain, which mediate a pupilloconstrictor response in the host eye when the transplant is stimulated by light. The changes in the size of the host pupil can be measured accurately with a pupillometry system. We have taken advantage of the additional observation that while grafts between rat strains, as between Long Evans and Sprague-Dawley strains, may survive indefinitely, they can be induced to reject by skin grafting from the strain providing the donor retinal tissue. Combining pupillometry with skin grafting provides a useful way of examining correlated anatomical and behavioural changes associated with graft rejection from its earliest stage to the point of overt destruction. Even within three days of skin grafting, the amplitude and speed of constriction as well as the response latency all showed significant enhancement from normal, and this was sustained for a further week or more. Response deterioration followed during the second week post-skin grafting, but the exact timing varied considerably among animals. Anatomical observations of the process of retinal rejection showed the first invasion of lymphocytes to occur between days 5 and 7 and total degeneration of the retinal transplant and its projections to occur by two to three weeks post-skin grafting. The lymphocytic infiltration was preceded by upregulation of microglia, which expressed both class I and II major histocompatibility antigens and by activation of astrocytes identified by their expression of glial fibrillary acidic protein. Within the target region of retinal transplant axons, major histocompatibility antigen expression and astrocytic responses preceded degeneration of transplant derived axons (demonstrated by the Fink-Heimer stain) and there was no evidence for any lymphocytic lymphocytic infiltration during transplant rejection. These observations show that the earliest stages of microglial activation are accompanied by an enhancement of response parameters, but that the functional failure finally occurs only at an advanced stage of graft destruction. The absence of lymphocytic infiltration into areas receiving terminals from axons of transplant origin, even though these contain significant numbers of reactive microglia, suggests that the terminal axonal processes are not a primary target for the immune response.

The pupillary light response: functional and anatomical interaction among inputs to the pretectum from transplanted retinae and host eyes.

Pupilloconstriction to light can be mediated in rats through direct illumination of retinae previously transplanted to intracranial locations. Transplant-driven and normal pupillary light responses are stable under optimal testing conditions, and parameters describing the response can be quantified precisely. The present study demonstrates the interaction between transplant-driven and normal pupillary response patterns. When stimuli are presented concurrently to a transplanted retina and to the remaining eye in host rats, a greater degree of pupilloconstriction occurs than when either the transplanted or the host eye is illuminated independently. This suggests that transplant and host retinal inputs act in concert to determine pupil diameter. The second portion of this study investigates the pattern of retinal input to the pretectum to determine if a structural basis for such functional interactions may exist. Crossed and uncrossed retinal projections to the olivary pretectal nucleus occupy non-overlapping regions of this bilaterally represented nucleus in normal rats, with a greater number of optic axons directed to the contralateral olivary pretectal nucleus. Retinae transplanted to the midbrain of neonatal rats, from whom the contralateral eye had been removed, also project to the olivary pretectal nucleus at maturity. By contrast with the normal pattern of segregated retinal inputs, however, the terminal fields of transplant axons were found to overlap extensively with the retinal projection from the remaining host eye. In addition, the relative proportion of transplant axons directed to the ipsilateral and contralateral olivary pretectal nucleus varied among animals. The lack of spatial segregation between inputs from transplant and host sources and the relative proportion of ipsilateral and contralateral transplant axons together may represent a structural basis for the observed functional interactoin of these inputs to the neural circuit subserving pupilloconstriction to light. These features may also relate to the marked improvements in transplant-mediated responses that frequently occur when optic input from the remaining host eye is eliminated. The results presented here, together with our previous transplant studies, show that this preparation can be used to provide insight into more general questions as to the dynamic interactions that occur between converging sensory inputs in the generation of integrated output responses.

The pupillary light response: assessment of function mediated by intracranial retinal transplants.

We have adapted a pupillometry measurement system to test the functional efficacy of retinae previously transplanted over the midbrain of neonatal rats in mediating a pupillary light reflex in the host eye. This has permitted us to examine factors influencing various parameters of the response, and to study transplant-mediated responses in comparison with responses mediated by way of the normal consensual pathway. Despite the unusual location of these transplanted retinae and the absence of supportive tissues normally associated with retinae in situ, it is clear that pupilloconstriction in the host eye can be elicited by transplant illumination. Under the optimal conditions here defined, response parameters for individual animals were stable with repeated testing over extended periods. When considered as individual cases, response amplitude, constriction rate and response latency were intensity dependent, although responses elicited by transplant illumination were less sensitive than normal, typically by 2-3 log units. Large-amplitude transplant-mediated pupillary responses could, however, be elicited repeatedly throughout long trains of stimuli, unlike normal responses, which rapidly failed to recover to baseline under similar test conditions. Finally, even though some cellular elements of the visual cycle are absent in transplanted retinae, pupilloconstriction in the host eye could be elicited repeatedly by transplant illumination as long as two years after transplantation took place. These observations indicate the applicability of this preparation as an assay for the effects of experimental manipulations on information processing and response plasticity in the visual system, and as a tool for examining, in general, the necessary conditions for optimal function of grafts that work by synthesizing and relaying neural signals.

Axon-target interactions maintain synaptic gene expression in retinae transplanted to intracranial regions of the rat.

In the present study we examined the effects of optic axon-CNS target interactions on gene expression in the rat retina. These studies took advantage of a transplantation paradigm that allowed us to assay gene expression in retinae transplanted to different intracranial locations in the neonatal rat that either promoted (dorsal midbrain) or precluded (cerebral cortex) the formation of retino-collicular connections. Using in situ hybridization experiments, we observed that transplantation to the dorsal midbrain resulted in a relatively normal pattern of nicotinic acetylcholine receptor (nAChR) beta-3 subunit and glutamate receptor 3 (GluR3) gene expression. In contrast, retinae transplanted to the cerebral cortex (which did not result in normal retino-collicular interactions) showed a dramatic reduction in nAChR beta-3 subunit and GluR3 gene expression. These results agree with those obtained in the adult goldfish retina, where it has been demonstrated that an optic nerve-optic tectum interaction is responsible for the re-induction nAChR and NMDA receptor gene expression during optic nerve regeneration. Taken together, these results support the hypothesis that proper axon-target interactions are required for maintenance of nAChR and glutamate receptor gene expression in the mature vertebrate retina.

Time-course of ultrastructural changes in regenerated optic fiber terminals of goldfish.

The ultrastructure of regenerated optic fiber terminals differs from normal terminals during the first 12 months following optic nerve crush. The area of the regenerated terminals occupied by axoplasm initially increases (1 month postcrush, mpc), then declines to a below normal level (8-12 mpc) and eventually returns to the normal level (16 mpc). The density of vesicles within the regenerated terminals remains initially the same (1 mpc), then increases (4-12 mpc) and finally returns to normal values by 16 mpc. The multiplicity of reestablished retino-tectal synapses gradually increased from an initially lower value at 1 mpc to the normal value by 4 mpc whereas the length of their synaptic contacts decreased from an initial elongation (1 mpc) to the normal length (4 mpc).

Opsin gene expression and regulation in retinal transplants.

In the present study we investigated the expression and regulation of the opsin gene in retinal transplants. Embryonic retinae were transplanted to intracranial locations in neonatal rodents in which they either reliably projected to the superior colliculus, or in locations (such as the cerebral cortex) in which they did not project to subcortical visual nuclei. Our results show that, regardless of the graft location, the developmental schedule of opsin gene expression in the outer nuclear layer was similar to normal, and that it was maintained in transplants for at least 6 months. To test if ambient light affected opsin gene expression, we dark-reared rats containing a retinal transplant for up to 26 days before assaying for opsin transcripts. In situ hybridization experiments showed that opsin gene expression in the transplants of these dark-reared recipients was not different either from transplants in animals reared in cyclic light conditions, or from the retina in situ. These observations support the hypothesis that the opsin gene is activated and maintained by molecular mechanisms intrinsic to the photoreceptor.

Rapid enhancement of transplant-mediated pupilloconstriction after elimination of competing host optic input.

Retinae transplanted over the brainstem, when directly illuminated, cause pupilloconstriction in the host eye. This occurs even when the host eye is maintained in darkness. If the host optic nerve is then severed, the transplant-mediated response is substantially augmented within 1-2 days. It is suggested that this is due to enhancement of the signal-to-noise ratio resulting from elimination of dark discharge in the host optic nerve.

Plasticity in innervation of the rat superior colliculus by transplanted retinae as a result of eye removal at maturity.

When the superior colliculus of a rat is innervated by inputs from both eyes as well as a retina transplanted intracranially over one tectum at birth, the tectal projection from the transplant is confined mainly to the superficial surface of the superior colliculus. The transplant-derived fibers possess a simple morphology, lacking terminal arborizations. If the contralateral eye is removed one month after transplantation, these fibers can be induced to arborize into the denervated portion of the superior colliculus over the next month. This demonstration of sprouting in a mature sensory relay system raises the possibility that an enhancement of behavioral responses mediated by transplanted retina might also occur. In turn, this may provide an ideal system to study the correlation between anatomical changes in transplant axons and changes in behaviors mediated by transplant activity.

Light-evoked cortical activity produced by illumination of intracranial retinal transplants: experimental studies in rats.

We have investigated whether information relayed through intracranial retinal transplants can elicit responses in the visual cortex of host rats. Embryonic retinae were transplanted over the midbrain of neonatal rats. Four to eight weeks later, the transplants were exposed and stimulated with light flashes. This photic stimulation elicited both evoked responses and multi-unit activity in area 18a of the visual cortex. Pathway tracing studies using horseradish peroxidase showed that these responses are transmitted to the cortex via the superior colliculus, the lateral division of the lateral posterior nucleus of the thalamus, and possibly the medial portion of the dorsal lateral geniculate nucleus. It is suggested that this pathway may be involved in complex transplant-mediated visual behaviors.

Chemical changes in the photoreceptor outer segments due to iron induced oxidative stress: analysis by Fourier transform infrared (FT-IR) microspectroscopy.

Oxidative stress is thought to be an important pathogenic mechanism in many diseases of the retina. The purpose of this study was to investigate the chemical changes that are present in the photoreceptor outer segments of the retina following exposure to oxidative stress. Fourier transform infrared (FT-IR) microspectroscopy enables the characterization and semi-quantitation of chemical functional groups in microscopic regions of tissue sections. This technique was used to evaluate the chemical changes in the outer segments following exposure to ferrous sulfate, which promotes oxidative tissue damage. A reduction of C=C-H and C=O functional groups was observed in the outer segments of iron-injected eyes compared to vehicle-injected eyes at 3 days following injection, which is prior to major histological changes that occur by 7 days. These functional groups are found in docosahexaenoic acid (DHA), which is present at a high concentration in the outer segments. DHA contains a series of six cis-conjugated double bonds, which are vulnerable to free radical attack, and the reduction of these unsaturation group absorptions suggests that DHA was degraded and/or removed from the outer segments. An unexpected finding was that several other chemical functional groups increased in concentration over time in the outer segments of vehicle-injected eyes compared to non-injected eyes. These increases generally did not include C=C-H or C=O, which suggests that either DHA was being degraded while other organic molecules were being concentrated, or that production of DHA failed to be upregulated in vehicle-injected eyes. In summary, there was a loss of both C=C-H and C=O functional group concentrations in the outer segments of iron-injected eyes, and there was an increased concentration of several other chemical functional groups following trauma induced by vehicle injection.

Partial rescue of the ocular retardation phenotype by genetic modifiers.

The or(J) allele of the murine ocular retardation mutation is caused by a premature stop codon in the homeodomain of the Chx10 gene. When expressed on an inbred 129/Sv strain, the or(J) phenotype is characterized by microphthalmia and a thin, poorly differentiated retina in which the peripheral portion is affected to a greater extent than the central portion. Such mutant retinae lack differentiated bipolar cells and the optic nerve typically fails to form, leading to blindness. Here, we show that progeny from an outcrossed backcross between 129/Sv-or(J) /or(J) and Mus musculus castaneus produce animals that are homozygous for the or(J) mutation and exhibit a much ameliorated eye phenotype. Although not of normal size, such modified or(J) eyes are significantly larger than those in 129/Sv-or(J) /or(J) mice, and contain a better organized retina which includes bipolar cells. Furthermore, optic nerves are frequently present, and the eyes show a degree of function as reflected by electroretinogram and pupillary response. As in 129/Sv-or(J) /or(J) mice, however, modified or(J) eyes show incomplete growth and a lack of cell differentiation in the periphery of the retina. The selective, and apparently nonmodifiable, effect of the ocular retardation phenotype on the periphery of the retina indicates that Chx10 plays an important role in the central-to-peripheral gradient of retinal development. These findings demonstrate that the ocular retardation phenotype can be greatly modified by the genetic background, and help to define a role for Chx10 in ocular development.