Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina.

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

Gating of Ca2+-activated K+ channels controls fast inhibitory synaptic transmission at auditory outer hair cells.

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.

R- and B-cadherin expression defines subpopulations of glial cells involved in axonal guidance in the optic nerve head of the chicken.

Glial cells play a crucial role in the organization and function of the nervous system. Cell-cell adhesion receptors of the cadherin family have been shown to participate in distinct morphogenetic processes throughout the development of the CNS, but little is known about glial expression of cadherins. Applying immunofluorescence and confocal laser scanning microscopy, we investigated R- and B-cadherin expression in relation to the glial cell differentiation in the optic nerve head and pecten oculi of developing chicken. Throughout embryonic development, R- and B-cadherin were expressed in distinct cell populations, which differentiated into distinct subtypes of glial cells. R-cadherin was located in the glia limitans perivascularis et superficialis of the optic nerve and in cells bordering the optic nerve head, where it comes in contact with the retina. B-cadherin was located in the glia limitans perivascularis et superficialis of the pecten oculi and in a subset of cells at the retinal border. R-cadherin-expressing cells differentiated unequivocally into a glial fibrillary acidic protein (GFAP)-positive but glutamine synthetase (GS)-negative phenotype, whereas B-cadherin-expressing glia developed into a GS-positive but GFAP-negative phenotype. In addition, the B-cadherin-positive population developed into a highly pigmented cell type, which was consistently associated with pecten-type capillaries. By contrast, the R-cadherin-positive glia remained unpigmented and surrounded normal brain-phenotype capillaries. These data suggest that glial cells, like neurons, may use the expression of different cadherins to segregate and differentiate into distinct subtypes, which goes hand in hand with their involvement in special functions and morphogenetic processes. To address this issue, we selectively lysed both glial subtypes in developing embryos by microinjection of R- and B-cadherin antibodies with complement. First evidence is presented for R-cadherin-positive glial cells as crucial to the organization of the optic nerve and axonal guidance at its lateral margin. B-cadherin-positive cells are involved in the axonal guidance at the pecteneal margin, avoiding the ingrowth of axons into the pecten.

The peripapillary glia of the optic nerve head in the chicken retina.

The eye of reptiles and birds is characterized by an avascular retina and a vascular convolute called conus papillaris in reptiles and pecten oculi in birds which arises from the papilla nervi optici (PNO) or optic nerve head into the vitreous. At least in birds, this central part of the retina is the site of a heterogeneous population of glial cells. Müller cells reside in the retina, astrocytes in the optic nerve, and pecteneal glial cells in the pecten. The latter are developmentally related to the pigment epithelial cells. In addition to these established types of cells, there is a population of glial cells lining the base of the pecten oculi. In the present study, we investigated both the morphology and the development of these glial cells of the PNO in a series of chicken embryos. These cells were called peripapillary glial cells. They were characterized by their morphology and by their spatiotemporal expression of antigens typical of glial cells (intermediate filaments and glutamine synthetase). They reside at the border between the retina and the optic nerve and at the innermost border of the ventricular cleft representing transitional forms among Müller cells, astrocytes, and pigment epithelial cells. The developmental data suggest a migration of the perikarya of the peripapillary glia in vitread direction, which may coincide with that of the pecteneal glia. Whereas the pecteneal glial cells differentiate morphologically from E16 on, the peripapillary glia retain characteristics of radial glia by spanning the distance from the vitreous to the ventricular cleft. Blood vessels only occurred in the optic nerve head and the pecten oculi. No capillaries were found in the retinal tissue, beyond the peripapillary glia, leading us to suggest that these cells may play a role in demarcating the outer limit of vascularization. The functional properties of these cells are unknown but were discussed to include prevention of vessel growth into the avascular retina and/or axonal guidance during development.

Differentiation of a unique macroglial cell type in the pecten oculi of the chicken.

Glial cells in the CNS of vertebrates serve specialized functions in close interaction with surrounding neurons and blood vessels. In the avian eye, the neural tissue (retina) and the supporting vascular structure (pecten oculi) are spatially separated and comprise distinct glial cell types, i.e., the Müller glia and the pecteneal glia, respectively. In the present study we combined morphological and immunocytochemical investigations on the differentiation of the pecteneal glia in comparison to the retinal Müller glia, the retinal pigment epithelium, and the astrocytic cells of the optic nerve head in order to elucidate the nature, origin, and function of the pecteneal glia. Conventional transmission electron microscopy and freeze-fracture imaging revealed striking similarities between the pecteneal glia and retinal pigment epithelial cells at the transition zone to the optic nerve head. Immunofluorescence investigation identified specific labeling for vimentin and glutamine synthetase (GS) but not for glial fibrillary acidic protein (GFAP) in the mature pecteneal glia. Immunogold labeling confirmed the cellular specificity. GS labeling was weak during embryonic development but increasingly strong after hatching. Surprisingly, the intraneuroectodermal endothelial cells were highly immunopositive for GS throughout embryonic development and lost GS expression after hatching. GS expression in the pecteneal glia may participate in pH-regulation of the avian eye. Endothelial GS expression in the developing CNS may detoxify detrimental ammonium concentrations resulting from egg yolk degradation.

Distribution of mitochondria within Müller cells--II. Post-natal development of the rabbit retinal periphery in vivo and in vitro: dependence on oxygen supply.

The occurrence and localization of mitochondria within glial (Müller) cells and neurons of the peripheral (avascular) rabbit retina was studied electron microscopically and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). Post-natal development in vivo was compared with development of organ cultures from neonatal rabbit retinae, grown over 2 weeks in vitro. The adult pattern of mitochondrial localization (restriction to the sclerad end of the cells) was observed from the beginning of enzyme expression at early post-natal stages. However, when neonatal retinal pieces were grown in vitro with their vitread surface exposed to the air, their Müller cells contained mitochondria along most of their length. When functionally developed retinae from postnatal day 14 were explanted in vitro, they retained their sclerad mitochondrial distribution for almost 24 h but thereafter the inner portions of their cytoplasm became occupied by mitochondria within a few hours. This was achieved mainly by mitochondrial migration rather than by formation of new mitochondria because it was not prevented by cycloheximide-induced inhibition of protein synthesis. These data support the following hypotheses: (1) the mitochondrial distribution in Müller cells is determined by the local cytoplasmic O2 pressure (pO2), (2) existing mitochondria move towards cytoplasmic regions of sufficient pO2 by rather rapid migration and (3) the start of this migration is delayed by almost 24 h due to the action of as yet unknown control mechanisms. In contrast, the mitochondrial content of retinal ganglion and amacrine cells in the vitread retinal layers was virtually independent of the source and level of oxygen supply.

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species.

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.

Endocrine and hemodynamic effects of stress versus systemic CRF in alcoholics during early and medium term abstinence.

In alcoholics, disturbances of the autonomic nervous system as well as of the hypothalamic-pituitary-adrenal axis (HPA) are known. However, these two systems have never been analyzed, under stimulated conditions, in parallel in the same patients. Moreover, studies using intravenous (i.v.) corticotropin releasing factor (CRF) to assess neuroendocrine function bypass the hypothalamic component of the HPA axis. Therefore, i.v. human (h) CRF (pituitary stimulation/exogenous CRF) and a multifaceted stress test (hypothalamic activation/endogenous CRF) were compared with respect to their effects on hemodynamics as well as plasma norepinephrine (NE), epinephrine (E), ACTH, and cortisol in abstinent alcoholics (n = 11) versus healthy men (n = 10). Each stimulus was tested twice, 12 weeks apart, in two separate experimental blocks (I and II). Alcoholics entered block 18 days after the last ethanol ingestion and were controlled for abstinence up to block II. hCRF caused a fall in mean arterial pressure (MAP), most pronounced in alcoholics, particularly in block II. In contrast, stress testing raised MAP in both groups and blocks. A sustained increase in ACTH, cortisol, and NE occurred after hCRF, although the ACTH response in alcoholics was blunted in both blocks. Stress testing elevated NE in both groups and blocks, while raising plasma ACTH and cortisol during block I only in controls. However, unlike the persistently blunted ACTH response to i.v. CRF, a normalization of the stress-induced ACTH output occurred in alcoholics after 12 weeks of abstinence. During block I, basal E levels were elevated in alcoholics whereas NE levels tended to be lower than in controls, resulting in a significantly decreased NE/E ratio that returned to near control values in block II. Neither CRF nor stress had any effect on circulating E in either group or block. To conclude: (1) Normalization of the ACTH response to stress, but not to i.v. CRF, after 12 weeks of abstinence, suggests that other ACTH secretagogues may be compensating for CRF dysfunction in alcoholics. (2) Despite the dramatically lowered plasma NE/E ratio in alcoholics, the NE response to stimuli was unaffected. (3) The exaggerated hypotensive reaction and blunted ACTH response to i.v. CRF may reveal a long-term dissociative dysregulation of CRF actions in alcoholics.

Quantitative methods in research on complementary and alternative medicine. A methodological manifesto. NIH Office of Alternative Medicine.

OBJECTIVES: This article summarizes the deliberations of the Quantitative Methods Working Group convened by the National Institutes of Health (NIH) in support of the NIH Office of Alternative Medicine. METHODS: The working group was charged with identifying methods of study design and data analysis that can be applied to empirical research on complementary and alternative medicine. This charge was broad and inclusive and addressed the evaluation of alternative therapies, the investigation of the basic science of complementary medical systems, studies of health promotion and disease prevention, and health services research. RESULTS: The working group produced a "methodological manifesto," a summary list of seven recommended methodological guidelines for research on alternative medicine. These recommendations emphasize the robustness of existing research methods and analytic procedures despite the substantive unconventionality of alternative medicine. CONCLUSIONS: Contrary to the assertions of many researchers and alternative practitioners, established methodologies (eg, experimental trials, observational epidemiology, social survey research) and data-analytic procedures (eg, analysis of variance, logistic regression, multivariate modeling techniques) are quite satisfactory for addressing the majority of study questions related to alternative medicine, from clinical research on therapeutic efficacy to basic science research on mechanisms of pathogenesis and recovery.

Causal modeling and alternative medicine.

Many problems of health promotion or prevention call for an understanding of relations among variables embedded in complex causal webs that may include psychosocial, cultural, or environmental factors as well as biological dysfunction. Experimental investigation of these kinds of research problems is frequently impossible or not feasible. Causal modeling, particularly latent variable structural modeling, can provide a useful alternative to manipulative experimentation when one is trying to build and test explanatory models in a rigorous and systematic fashion. A hypothetical model of fibromyalgia is presented to illustrate how latent variable models can prove useful when the malady under investigation is of relatively complex multifactorial origin. Topics discussed include the fundamental notion underlying causal models, how such models solve problems due to measurement error, and why both cross-sectional and longitudinal models deserve consideration. A number of applications in medical research are described.

Backward masking, information processing, and schizophrenia.

Many researchers have used backward masking to examine information-processing speed in schizophrenic subjects. The validity of this approach rests upon two main assumptions. One is that the mask effectively limits the time a previously presented stimulus is available for processing. The other is that the components of the masking mechanism in schizophrenic subjects are comparable to those in control groups. It is argued that the masking procedures used in these studies fail to meet either assumption. Alternative interpretations of these investigations, however, suggest important hypotheses for further research in our quest to understand information-processing deficits associated with schizophrenia.

Construct validity of various memory testing procedures.

The construct validity of Wechsler Memory Scale Logical Memory, Paired Associate Learning and Visual Reproduction subtests was evaluated, as well as the validity of the Benton Visual Retention Test, and the two memory factor scales of the Luria-Nebraska Neuropsychological Battery (LNNB). The results of a series of factor analyses based on test performances of 102 subjects indicated that delayed reproduction measures of visual memory were more valid than the traditional immediate reproduction administrations, which were more closely associated with visual-perceptual-motor abilities. Construct validity was also demonstrated for Logical Memory and Paired Associate Learning. Although both LNNB measures loaded on a memory factor, item heterogeneity and brief sampling of items raised serious questions about the clinical utility of these scales.

Gender differences in schizophrenia.

Lewine (Psychol. Bull., 90: 432-444, 1981) has proposed sex differences, specific to schizophrenics, in age at first psychiatric hospitalization, age at first reported symptoms, and premorbid social competence. To evaluate Lewine's hypothesis we collected data on 64 schizophrenic and 30 nonschizophrenic psychiatric outpatients. As no interaction between sex and diagnostic groups was found, our data failed to demonstrate sex differences specific and unique to schizophrenia. It is possible that Lewine's evidence is due to a cultural artifact.

Factor analysis of the WAIS and Wechsler Memory Scale: an analysis of the construct validity of the Wechsler Memory Scale.

The WAIS and Wechsler Memory Scale subtest scores of 256 neurologic and nonneurologic subjects were factor analyzed. The results supported the construct validity of the Wechsler Memory Scale as a measure of verbal learning and memory, attention and concentration, and orientation. Construct validity was not demonstrated for the Visual Reproduction subtest as a measure of visual memory. Suggestions are offered for future development and research on measures of visual memory.

Proprioception in schizophrenia.

B. Ritzler (J. Abnorm. Psychol., 86: 501-504, 1977) failed to replicate previous weight-lifting studies showing a proprioceptive deficit in schizophrenics. However, Ritzler did not use the same standard weights that the previous studies employed. Could this difference in procedure have caused his failure to replicate? Two experiments were completed to provide additional information about this question and related issues. In Experiment I, chronic hospitalized schizophrenics and normals were tested with light and heavy weights. Rosenbaum's original modified method of limits and the method of constant stimuli were utilized. Both methods provided comparable results, which, like Ritzler's data, failed to show a differential impairment in the discrimination of the light weights for paranoid schizophrenics. In Experiment II, outpatient paranoid schizophrenics, outpatient nonparanoid schizophrenics, outpatient psychiatric patients, and aged normals were evaluated utilizing the same psychophysical procedure and standard weights employed in the original studies. No differential impairment was found for the light weights for any group. These results provide strong support for Ritzler's position that a proprioceptive deficit is not unique to schizophrenia.

Growth of anchorage dependent mammalian cells on glycine-derivatized polystyrene in suspension culture.

Glycine-derivatized polystyrene beads were prepared and used as microcarriers to grow normal cells of human embryonic kidney, rhesus monkey kidney, an human foreskin fibroblasts in suspension cultures. Growth of the cells on polystyrene beads derivatized with other amino acids, peptides, and carboxylic acids also was investigated.

Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells.

Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.