Restorative encoding memory integrative neural device: "REMIND".

Construction and application of a neural prosthesis device that enhances existing and replaces lost memory capacity in humans is the focus of research described here in rodents. A unique approach for the analysis and application of neural population firing has been developed to decipher the pattern in which information is successfully encoded by the hippocampus where mnemonic accuracy is critical. A nonlinear dynamic multi-input multi-output (MIMO) model is utilized to extract memory relevant firing patterns in CA3 and CA1 and to predict online what the consequences of the encoded firing patterns reflect for subsequent information retrieval for successful performance of delayed-nonmatch-to-sample (DNMS) memory task in rodents. The MIMO model has been tested successfully in a number of different contexts, each of which produced improved performance by a) utilizing online predicted codes to regulate task difficulty, b) employing electrical stimulation of CA1 output areas in the same pattern as successful cell firing, c) employing electrical stimulation to recover cell firing compromised by pharmacological agents and d) transferring and improving performance in naïve animals using the same stimulation patterns that are effective in fully trained animals. The results in rodents formed the basis for extension of the MIMO model to nonhuman primates in the same type of memory task that is now being tested in the last step prior to its application in humans.

Mitochondrial transport proteins of the brain.

In this study, cellular distribution and activity of glutamate and gamma-aminobutyric acid (GABA) transport as well as oxoglutarate transport across brain mitochondrial membranes were investigated. A goal was to establish cell-type-specific expression of key transporters and enzymes involved in neurotransmitter metabolism in order to estimate neurotransmitter and metabolite traffic between neurons and astrocytes. Two methods were used to isolate brain mitochondria. One method excludes synaptosomes and the organelles may therefore be enriched in astrocytic mitochondria. The other method isolates mitochondria derived from all regions of the brain. Immunological and enzymatic methods were used to measure enzymes and carriers in the different preparations, in addition to studying transport kinetics. Immunohistochemistry was also employed using brain slices to confirm cell type specificity of enzymes and carriers. The data suggest that the aspartate/glutamate carriers (AGC) are expressed predominantly in neurons, not astrocytes, and that one of two glutamate/hydroxyl carriers is expressed predominantly in astrocytes. The GABA carrier and the oxoglutarate carrier appear to be equally distributed in astrocytes and neurons. As expected, pyruvate carboxylase and branched-chain aminotransferase were predominantly astrocytic. Insofar as the aspartate/glutamate exchange carriers are required for the malate/aspartate shuttle and for reoxidation of cytosolic NADH, the data suggest a compartmentation of glucose metabolism in which astrocytes catalyze glycolytic conversion of glucose to lactate, whereas neurons are capable of oxidizing both lactate and glucose to CO(2) + H(2)O.

Regulation of microtubule-associated protein 1B (MAP1B) subunit composition.

The MAP1B and MAP1A genes each produce an mRNA that encodes a polyprotein. When cleaved, each polyprotein yields a single heavy chain and a single light chain, which become noncovalently associated. In previous work, it was found that the MAP1B light chains and heavy chains exist in a 2:1 ratio. Through use of quantitative immunoblot techniques, this finding was further examined in the developing rat brain. MAP1B heavy chain (HC) and light chain (LC1), as well as the light chain of MAP1A (LC2), were prepared in purified form for use as standards and/or immunogens for generation of antibodies for immunoblotting. Brain homogenates and microtubule-enriched fractions from developing rats were assayed for MAP1B HC and LC1 content. Results indicated that postnatal rat brain homogenates contain LC1 in a 6:1 to 8:1 molar ratio to the MAP1B HC. Purified microtubules also contain LC1 in excess of MAP1B HC, but at a ratio of 2:1. We propose that most of the excess LC1 in homogenates is either insoluble or not bound to microtubules. The findings raise the possibility of a function for the "excess" LC1 that does not require association with MAP1 HC and/or microtubules. Given a synthetic mechanism that produces MAP1B HC and LC1 in a 1:1 ratio at both transcription and translation steps, we propose that the "excess" LC1 in brain homogenates is a consequence of LC1 having a greater half-life than the MAP1B HC. Consistently with this hypothesis, a major pool of MAP1B HC is rapidly degraded after blocking protein synthesis with cycloheximide, whereas LC1 levels remain constant even after 24 hr of cycloheximide treatment.

Microtubule-associated protein 1 subunit expression in primary cultures of rat brain.

Microtubule-associated protein 1A (MAP1A) and MAP1B are developmentally regulated proteins linked to axon formation. They each consist of a unique heavy chain and three common light chains. We used immunofluorescence microscopy to qualitatively assess the variability in MAP1 subunit expression between individual cells. The ratio of light chain 1 to MAP1 heavy chain varies greatly between cells with some cells expressing MAP1A heavy chain in the apparent absence of light chain 1. The results imply the existence of MAP1 molecules that differ in light chain composition. Transfection experiments indicate that the light chains differ in microtubule binding activity and subcellular targeting activity. This further suggests that the regulation of MAP1 light chain content can control MAP1 function.

Wound healing following anterior keratectomy and lamellar keratoplasty in the pig.

PURPOSE: To examine corneal wound healing in an animal model of two types of mechanical lamellar keratectomy. METHODS: One eye from each of 28 pigs was studied. Using a motorized keratome, corneas were subjected to an anterior lamellar keratectomy with removal of anterior stroma and epithelium, or to automated lamellar keratoplasty (ALK) with reapposition of a corneal flap. The exposed stromal surfaces were labeled intraoperatively with a fluorescent dye (DTAF) to assess deposition of stromal components during subsequent wound healing. Examination before surgery and enucleation included measurement of corneal curvature and intraocular pressure, and assessment of corneal haze. Eyes were prepared for histological examination, fluorescence microscopy, and for fibronectin immunohistochemistry. RESULTS: Both keratectomy procedures produced flattening of corneas by up to 3.80 diopters, 28 days after surgery. Corneal haze was more pronounced in eyes from which epithelium was removed (anterior lamellar keratectomy group). The increased haze in this group was associated histologically with appearance of many reactive keratocytes and inflammatory cells, deposition of new stromal material, and more widespread appearance of fibronectin immunoreactivity. In the lamellar keratoplasty group, only the edges of the corneal wound showed significant reactivity, and included keratocyte activation and epithelial ingrowth. CONCLUSIONS: The pig provides a useful model for studies of refractive surgical techniques using procedures and instruments designed for use in humans. Mechanized keratectomy procedures that minimize disruption of the epithelium and Bowman's layer produce a less reactive corneal wound than procedures in which an expanse of epithelium and anterior stroma are removed.

Corneal wound-associated glycoconjugates analyzed by lectin histochemistry.

PURPOSE: To examine changes in corneal glycoconjugates during wound healing in a pig model of refractive surgery. METHODS: Pig corneas were wounded using a mechanized microkeratome. Lamellar keratectomy, without replacement of a corneal cap, or automated lamellar keratoplasty, with replacement of a corneal cap, was performed. Corneas were prepared for microscopy 1, 7, or 28 days after surgery. Cryosections of corneas were probed with lectins recognizing a variety of carbohydrate moieties. Glycoconjugates were characterized by glycosidase digestion and chemical extraction. RESULTS: Of the lectins used, only that from Dolichus biflorus (DBA), specific for terminal N-acetylgalactosamine, exhibited a dramatic change in staining pattern after wounding. In unwounded corneas, DBA labeled the superficial epithelium, Bowman's layer, and occasional keratocytes. After wounding, DBA intensely labeled exposed stromal surfaces and keratocytes near wound sites. RESULTS of glycosidase treatments indicated that the DBA-binding material contains alpha-linked N-acetylgalactosamine. After extraction with lipid solvents, stromal DBA labeling was unchanged, while that of keratocytes was greatly diminished. CONCLUSIONS: Corneal wounding results in the accumulation of specific glycoconjugates at surfaces and in keratocytes. After keratectomy, the exposed stromal surface is infiltrated with ocular surface components, including DBA-binding glycoproteins. The presence of these components may influence the course of wound healing. In addition, changes in keratocyte glycoconjugates may accompany their mobilization to the wound-repair phenotype.

Heat shock protein 70 in the retina of Xenopus laevis, in vivo and in vitro: effect of metabolic stress.

We utilized the frog eyecup as an in vitro model to compare heat-shock protein 70 (hsp70) synthesis in untreated retinas and in hyperthermia-, arsenite-, or glutamate-treated retinas. Hsp70-like immunoreactivity in vivo was concentrated in the photoreceptors in a pattern that was basically unchanged throughout the light/dark cycle. Retinas from eyecups in culture displayed the same immunoreactivity pattern as those in vivo except for a rapid, transient increase in immunoreactivity surrounding the photoreceptor nuclei. The immunoreactivity pattern in heat-treated retinas was similar to that of controls, but overall intensity was greatest in the outer plexiform layer. Arsenite-treated retinas displayed hsp70-like immunoreactivity in a pattern that was also like that of control retinas. Glutamate exposure resulted in increased hsp70-like immunoreactivity not only in the inner segments and outer plexiform layer, but also in photoreceptor nuclei. Gel fluorography of 35S-methionine-labeled proteins from heat- and arsenite-stressed retinas demonstrated increased synthesis of one or two proteins of approximately 70 kD and one protein of approximately 90 kD. Exposure of eyecups to glutamate did not result in detectable changes in protein synthesis. Following exposure to heat or glutamate, the retinas displayed swelling of the inner plexiform layer (IPL) as well as pyknotic nuclei in the inner nuclear layer. Exposure of eyecups to arsenite caused clumping of the melanin granules of the retinal pigmented epithelium (RPE) but not IPL swelling or pyknotic nuclei. We have shown that the stress response can be manipulated successfully in the in vitro Xenopus retina and that the pattern of the response depends on the nature of the stressor.

The human cornea has a high incidence of acquired chromosome abnormalities.

Structurally and functionally, the human cornea is a highly specialized tissue. The corneal stromal collagen matrix is uniquely transparent and yet maintains a mechanically tough and chemically impermeable barrier between the eye and environment. We report for the first time that stromal keratocytes of the human cornea show cytogenetic abnormalities with a frequency that is unprecedented among normal tissues. The abnormalities are acquired, clonal and nonclonal, primarily aneuploid in nature, and present in normal as well as diseased corneas.

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PURPOSE: To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. METHODS: Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. RESULTS: ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. CONCLUSIONS: The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.

Cellular origins of tenascin in the developing nervous system.

We have used in situ hybridization and reverse transcriptase polymerase chain reaction (PCR) to study the origins of the extracellular matrix glycoprotein tenascin during the development of the central and peripheral nervous systems. Previous studies have shown that neural crest cells migrate along pathways that are lined with tenascin. In situ hybridization, PCR, and western blotting reveal that these cells themselves are a major source of tenascin both in vitro and in the embryo. Thus, tenascin is probably not acting as a guidance molecule but is more likely to be promoting neural crest cell motility in a more general way. Similarly, subpopulations of proliferating and migrating glia make tenascin in the developing central nervous system, as do the radial glia that are used as a substratum for migrating neuronal cell bodies. In the adult, tenascin continues to be expressed in the cerebellum by Golgi epithelial cells. This expression, as well as the expression of tenascin in connective tissue, indicates that this molecule may also be playing a role in regulating differentiation. Finally, the distribution of tenascin transcripts in the developing brain and spinal cord is similar to the distribution of mRNAs encoding receptors for platelet-derived growth factor-AA and basic fibroblast growth factor. In vitro studies indicate that both of these factors are potential regulators of tenascin expression.

Immunohistochemical evidence for transient expression of fibronectin in the developing dorsal lateral geniculate nucleus of the ferret.

In recent years, the important role of the extracellular matrix in neural development has been increasingly recognized. In order to begin to examine what role might be played by the extracellular matrix in the developing dorsal lateral geniculate nucleus (dLGN), the present study used immunocytochemistry to assess the distribution of a recognized extracellular matrix molecule, fibronectin (FN), during postnatal development of the ferret dLGN. Prior to the segregation of cell layers, no clear pattern of FN distribution can be distinguished within the dLGN. By P16, when layers A and A1 are separated by an interlaminar space, FN is localized in that space. By P24, FN is present not only between layers A and A1, but also within layers A and A1 as bands parallel to the laminar borders. These bands appear to correspond spatially and temporally to the development of sublaminar boundaries in the ferret (Hahm and Sur, Neurosci Abstr 14:460, 1988). By the end of the first postnatal month, immunoreactivity is diminished but still present. FN is no longer present at P44 or in the adult. Adjacent sections were incubated with antiserum to glial fibrillary acid protein (GFAP). When a laminar pattern of GFAP can be distinguished, it coincides with the interlaminar distribution of FN. The findings described here are consistent with a role for FN in the development of a laminar organization in the dLGN.

Cellular retinoic acid-binding protein in rat lacrimal gland.

We employed a monoclonal antibody to cellular retinoic acid-binding protein (CRABP) to assess the presence and localization of this retinoid-binding protein in the lacrimal gland of the rat. Immunoblots of extracts of rat lacrimal gland showed specific immunostaining of lacrimal CRABP in the region 14-16 kDa. Sections of rat lacrimal glands that were stained with anti-CRABP antibodies showed reaction product in the cytoplasm of the acinar cells. Retinoic acid may play a role in maintaining the proper function of lacrimal gland cells.

Immunolocalization of N-acetylgalactosaminylphosphotransferase in the adult retina and subretinal space.

The cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase) modulates N-cadherin-mediated adhesion among embryonic chick retinal cells (Balsamo et al., 1990). We are investigating the potential role of this transferase in modulating adhesive interactions in the adult retina. Using a previously characterized monoclonal anti-GalNAcPTase, we have used immunohistochemical and immunoblot techniques to localize and characterize the transferase in the retinas of the post metamorphic frog (Xenopus laevis), adult cow, and adult cynomolgus macaque. On frozen sections, anti-GalNAcPTase specifically labels the outer segments of photoreceptors in all species. Immunolabel appears at the surface of, or between rod outer segments in all species. The nerve fiber layer also shows high immunoreactivity in all species. Monkey cone outer segments are also highly immunoreactive. Photoreceptor inner segments are clearly immunoreactive in the cow retina. Immunoblots of purified cow rod outer segments show immunolabeled bands near 220 kDa, which is the molecular mass of the GalNAcPTase used as immunogen. Purified Xenopus rod outer segments are not immunoreactive on blots, while soluble interphotoreceptor matrix (IPM) shows immunoreactive bands principally at 113-130, and 166 kDa. Cow soluble IPM shows immunoreactivity at 180 kDa. Based on these findings, we propose that the GalNAcPTase, or a fragment thereof, is a component of the IPM, and perhaps of the photoreceptor outer segment as well.

Effects of acute and chronic stress on the neural retina of young, mid-age, and aged Fischer-344 rats.

Male Fischer-344 rats at 5 (young), 11 (mid-age) and 18 (aged) months of age were exposed either to a single, 1-h period of acute stress, or to daily 4-h periods (chronic) of escapable footshock stress for 6 months, and subsequently allowed a one month interval without stress. The influence of age and exposure to stress on the neural retina was examined by histopathologic and morphometric methods. Age changes in the retina of unstressed control animals included reduction in the thickness of the outer nuclear layer (ONL; photoreceptor nuclei) and of the retina, especially in the peripheral areas. The superior hemisphere was more severely affected than the inferior retina. Exposure to acute stress did not influence retinal histopathology. However, in mid-age and aged rats exposed to chronic stress, the ONL and retinal thicknesses were reduced significantly. Our results indicate for the first time that exposure of rats to chronic stress produces changes in retinal morphology that are associated commonly with aging, such as extensive loss of peripheral photoreceptor cells. In addition, the results show that the effects of chronic stress exposure are greatest in aged rats. The effect of light exposure on the aging retina was not investigated since all rats were exposed to the same total photoperiod.

Gender and chronic stress effects on the neural retina of young and mid-aged Fischer-344 rats.

Young (5 months) and mid-aged (11 months) male and female Fischer rats were exposed to daily (5 days/week) chronic escapable foot-shock stress for 6 months. Following a subsequent 1-month rest period, by which time the animals were 12 and 18 months old, neural retinas were evaluated histopathologically and morphometrically. A significant reduction in the thickness of the retina occurred in the mid-aged, as compared to the young animals. A severe age-related loss in photoreceptor cells, particularly in the peripheral zones of the retina, occurred in a pattern resembling that described for aging humans. The effect of stress was to increase photoreceptor loss in a pattern resembling that of age-related cell loss. Stress-associated photoreceptor cell death was observed in males and females of both ages, but was more pronounced and statistically significant for mid-aged males (a five-fold increase in cell loss over females). The results demonstrated that exposure of rats to chronic escapable foot-shock stress exacerbates retinal changes commonly associated with aging and that the deleterious effects of chronic stress exposure were greater in the older age, male group.