Latent TGF-ß binding protein-2 is essential for the development of ciliary zonule microfibrils.

Latent TGF-ß-binding protein-2 (LTBP-2) is an extracellular matrix protein associated with microfibrils. Homozygous mutations in LTBP2 have been found in humans with genetic eye diseases such as congenital glaucoma and microspherophakia, indicating a critical role of the protein in eye development, although the function of LTBP-2 in vivo has not been well understood. In this study, we explore the in vivo function of LTBP-2 by generating Ltbp2(-/-) mice. Ltbp2(-/-) mice survived to adulthood but developed lens luxation caused by compromised ciliary zonule formation without a typical phenotype related to glaucoma, suggesting that LTBP-2 deficiency primarily causes lens dislocation but not glaucoma. The suppression of LTBP2 expression in cultured human ciliary epithelial cells by siRNA disrupted the formation of the microfibril meshwork by the cells. Supplementation of recombinant LTBP-2 in culture medium not only rescued the microfibril meshwork formation in LTBP2-suppressed ciliary epithelial cells but also restored unfragmented and bundled ciliary zonules in Ltbp2(-/-) mouse eyes under organ culture. Although several reported human mutant LTBP-2 proteins retain normal domain structure and keep the fibrillin-1-binding site intact, none of these mutant proteins were secreted from their producing cells, suggesting secretion arrest occurred to the LTBP-2 mutants owing to conformational alteration. The findings of this study suggest that LTBP-2 is an essential component for the formation of microfibril bundles in ciliary zonules.

Layer specific regulation of critical period timing and maturation of mouse visual cortex by endocannabinoids.

Plasticity during the critical period is important for the functional maturation of cortical neurons. While characteristics of plasticity are diverse among cortical layers, it is unknown whether critical period timing is controlled by a common or unique molecular mechanism among them. We here clarified layer-specific regulation of the critical period timing of ocular dominance plasticity in the primary visual cortex. Mice lacking the endocannabinoid synthesis enzyme diacylglycerol lipase-α exhibited precocious critical period timing, earlier maturation of inhibitory synaptic function in layers 2/3 and 4, and impaired development of the binocular matching of orientation selectivity exclusively in layer 2/3. Activation of cannabinoid receptor restored ocular dominance plasticity at the normal critical period in layer 2/3. Suppression of GABAA receptor rescued precocious ocular dominance plasticity in layer 4. Therefore, endocannabinoids regulate critical period timing and maturation of visual function partly through the development of inhibitory synaptic functions in a layer-dependent manner.

Difference in binocularity and ocular dominance plasticity between GABAergic and excitatory cortical neurons.

Neuronal circuits in the cerebral cortex consist mainly of glutamatergic/excitatory and GABAergic/inhibitory neurons. In the visual cortex, the binocular responsiveness of neurons is modified by monocular visual deprivation during the critical period of postnatal development. Although GABAergic neurons are considered to play a key role in the expression of the critical period, it is not known whether their binocular responsiveness and ocular dominance plasticity are different from those of excitatory neurons. Recently, the end of the critical period was found to be not strict so that cortical neurons in the adult still have some ocular dominance plasticity. It is not known, however, which type of neurons or both maintain such plasticity in adulthood. To address these issues, we applied in vivo two-photon functional Ca(2+) imaging to transgenic mice whose GABAergic neurons express a yellow fluorescent protein called Venus. We found that GABAergic neurons are more binocular than excitatory neurons in the normal visual cortex, and both types of neurons show the same degree of modifiability to monocular visual deprivation during the critical period, but the modifiability of GABAergic neurons is stronger than that of excitatory neurons after the end of the critical period.

Dark Rearing Promotes the Recovery of Visual Cortical Responses but Not the Morphology of Geniculocortical Axons in Amblyopic Cat.

Monocular deprivation (MD) of vision during early postnatal life induces amblyopia, and most neurons in the primary visual cortex lose their responses to the closed eye. Anatomically, the somata of neurons in the closed-eye recipient layer of the lateral geniculate nucleus (LGN) shrink and their axons projecting to the visual cortex retract. Although it has been difficult to restore visual acuity after maturation, recent studies in rodents and cats showed that a period of exposure to complete darkness could promote recovery from amblyopia induced by prior MD. However, in cats, which have an organization of central visual pathways similar to humans, the effect of dark rearing only improves monocular vision and does not restore binocular depth perception. To determine whether dark rearing can completely restore the visual pathway, we examined its effect on the three major concomitants of MD in individual visual neurons, eye preference of visual cortical neurons and soma size and axon morphology of LGN neurons. Dark rearing improved the recovery of visual cortical responses to the closed eye compared with the recovery under binocular conditions. However, geniculocortical axons serving the closed eye remained retracted after dark rearing, whereas reopening the closed eye restored the soma size of LGN neurons. These results indicate that dark rearing incompletely restores the visual pathway, and thus exerts a limited restorative effect on visual function.

The Influence of Illumination Color on the Subjective Visual Recognition of Biological Specimens.

BACKGROUND: Visual examination by the naked eye is integral to medical diagnosis and surgery. The illumination in conditioned color is widely used for visual inspection in the industry but has not been introduced to the biomedical context. The color that can enhance the visual recognition of individual tissues is still unknown. Therefore, we carried out a visual recognition experiment on biological specimens to determine the subjective preference for illumination color based on questionnaires. METHODS: Twenty healthy subjects were asked to compare the visual recognizability of several rat tissues between the illuminations in test colors and white. The rats were anesthetized, and the femoral vein and abdominal cavity were exposed. Seven tissues were selected for a visual recognition test. Illumination was generated using a multi-color LED light. The subjects observed the tissues under the illuminations of white and one of the test colors alternately and reported which illumination is suitable for visual recognition using a questionnaire. RESULTS: The analysis of the questionnaires showed that the blue test color was more effective than white illumination in the visual recognition of fine structures such as the branching of blood vessels and nerves, and red illumination disturbed the visual recognizability of the same tissues. On the other hand, the red but not the blue illumination improved the visual recognizability of the vein beneath the intact skin. As to the recognition of individual tissues in the abdominal cavity, the white illumination gave a better visual recognizability compared to every other test color. CONCLUSION: This study shows that the illumination color influences the visual recognition of biological specimens and the adequate color for the visual recognition of specific tissue parts is distinct among biological specimens. Using the lighting system to make fine adjustments to the illumination color may be useful in medical diagnosis and surgery.

GABAergic neurons are less selective to stimulus orientation than excitatory neurons in layer II/III of visual cortex, as revealed by in vivo functional Ca2+ imaging in transgenic mice.

Most neurons in the visual cortex are selectively responsive to visual stimulation of a narrow range of orientations, and GABAergic neurons are considered to play a role in the formation of such orientation selectivity. This suggests that response properties of GABAergic neurons may be different from those of excitatory neurons. This view remains unproved, however. To address this issue, we applied in vivo two-photon functional Ca2+ imaging to transgenic mice, in which GABAergic neurons express enhanced green fluorescent protein. Astroglia were stained by an astrocyte-specific dye. The three types of cells, GABAergic neurons, excitatory neurons, and astrocytes, in layer II/III of the visual cortex were differentially identified by using different wavelengths of excitation light and a dichroic mirror for emitted fluorescence, and their responses to moving visual stimuli at different orientations were measured with changes in the intensity of fluorescence of a Ca2+-sensitive dye. We found that almost all GABAergic neurons have orientation-insensitive responses, whereas most of excitatory neurons have orientation-selective responses.

Recovery of binocular responses after brief monocular deprivation in kittens.

Monocular deprivation induces a rapid ocular dominance change in the developing visual cortex. The early phase of the change is supposed to be labile and stabilized later by consolidation processes. To test the stability of early ocular dominance change, we examined whether binocular responses of cortical neurons can recover after a brief monocular deprivation in anesthetized and paralyzed kittens in which ocular dominance plasticity does not operate. After the 24-h monocular deprivation, most cortical neurons lost their responses to the deprived eye. The deprived eye responses, however, recovered following 2-3 days interval under anesthetized and paralyzed conditions. Visual stimulation did not facilitate the recovery. These results suggest that the early phase of ocular dominance plasticity is labile and declines passively.

Developmental and visual input-dependent regulation of the CB1 cannabinoid receptor in the mouse visual cortex.

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.

Chronic electrical stimulation of afferents from one eye changes ocular dominance of visual cortical neurons in kittens.

Binocular visual responsiveness of neurons in visual cortex of the cat can be changed by monocular visual deprivation in the critical period of postnatal development. It is hypothesized that afferents from each eye compete with one another for synaptic connections with cortical neurons so that less active afferents from the deprived eye fail to maintain the connections. This hypothesis predicts that an increase in inputs from one eye instead of decrease due to deprivation should also change binocular responsiveness of cortical neurons. However, the hypothesis has not successfully been tested with experimental activation of afferents from one eye. In the present study, we activated one of the optic nerves by chronic electrical stimulation of theta-burst type in behaving kittens for 2 days. After such a monocular activation, visual cortical neurons showed a significant ocular dominance shift in favor of the electrically activated eye, although neurons in the activated and nonactivated layers of the dorsal lateral geniculate nucleus had no biased visual responses. Also, we found no detectable difference between activated and nonactivated eye responses of cortical neurons in other response properties such as orientation selectivity. These results support the hypothesis that the balance between activities of both afferents is critical for formation or consolidation of each eye-specific pathway.