Differential Scaling of Synaptic Molecules within Functional Zones of an Excitatory Synapse during Homeostatic Plasticity.

Homeostatic scaling is a form of synaptic plasticity where individual synapses scale their strengths to compensate for global suppression or elevation of neuronal activity. This process can be studied by measuring miniature EPSP (mEPSP) amplitudes and frequencies following the regulation of activity in neuronal cultures. Here, we demonstrate a quantitative approach to characterize multiplicative synaptic scaling using immunolabelling of hippocampal neuronal cultures treated with tetrodotoxin (TTX) or bicuculline to extract scaling factors for various synaptic proteins. This approach allowed us to directly examine the scaling of presynaptic and postsynaptic scaffolding molecules along with neurotransmitter receptors in primary cultures from mouse and rat hippocampal neurons. We show robust multiplicative scaling of synaptic scaffolding molecules namely, Shank2, PSD95, Bassoon, and AMPA receptor subunits and quantify their scaling factors. We use super-resolution microscopy to calculate scaling factors of surface expressed GluA2 within functional zones of the synapse and show that there is differential and correlated scaling of GluA2 levels within the spine, the postsynaptic density (PSD), and the perisynaptic regions. Our method opens a novel paradigm to quantify relative molecular changes of synaptic proteins within distinct subsynaptic compartments from a large number of synapses in response to alteration of neuronal activity, providing anatomic insights into the intricacies of variability in strength of individual synapses.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

UNLABELLED: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable. SIGNIFICANCE: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: long-term survival analysis.

BACKGROUND: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. MATERIALS AND METHODS: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. RESULTS: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan-Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1-87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. CONCLUSIONS: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.

PURPOSE: Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. METHODS: This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10-14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patient's affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. RESULTS: A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3 ± 1.6 (range: 1-7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. CONCLUSIONS: Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

Binocular interaction in post-LASIK subjects with unsatisfactory outcome.

PURPOSE: A shift from binocular summation to binocular inhibition has been observed when there is a significant decrease in contrast sensitivity or reduced retinal illuminance in one eye compared to the other as in cases of unilateral cataract and amblyopia. This study aims to determine how binocular function in post-LASIK subjects with unsatisfactory outcomes is influenced by differences between the two eyes in visual acuity and contrast sensitivity. MATERIALS AND METHODS: A retrospective analysis of monocular and binocular visual acuity (VA) and contrast sensitivity (CS) results was performed for 23 LASIK subjects who previously underwent a Functional Vision Test battery at Vision Sciences Research Corp. (San Ramon, CA). Photopic VA was measured with ETDRS chart and sine-wave grating CS was measured with FACT chart. RESULTS: The difference between binocular and better monocular VA and CS was calculated for each subject and referred to as binocular difference. The interocular difference was calculated as better eye-worse eye for VA and CS. Significant negative correlations (P < 0.01) were observed between binocular difference and interocular difference for VA (r = -0.84) and CS at each spatial frequency of 1.5 (r = -0.90), 3 (r = -0.85), 6 (r = -0.79), 12 (r = -0.68), and 18c/d (r = -0.72). The greater the difference between the eyes, the lesser the binocular summation. Linear regression predicted a 1-line decrease in binocular summation for a 4-line increase in interocular acuity difference and a 35% decrease in binocular summation for a 50% increase in interocular difference of contrast sensitivity at 3c/d (ANOVA, P < 0.01). CONCLUSIONS: Binocular inhibition in post-LASIK subjects increases as the visual sensitivity difference between the two eyes increases. Since the peak of human contrast sensitivity function is at the middle spatial frequencies, the decrease in binocular CS at 3c/d due to reduced CS in one eye is functionally significant.

Closing gaps in the human genome with fosmid resources generated from multiple individuals.

The human genome sequence has been finished to very high standards; however, more than 340 gaps remained when the finished genome was published by the International Human Genome Sequencing Consortium in 2004. Using fosmid resources generated from multiple individuals, we targeted gaps in the euchromatic part of the human genome. Here we report 2,488,842 bp of previously unknown euchromatic sequence, 363,114 bp of which close 26 of 250 euchromatic gaps, or 10%, including two remaining euchromatic gaps on chromosome 19. Eight (30.7%) of the closed gaps were found to be polymorphic. These sequences allow complete annotation of several human genes as well as the assignment of mRNAs. The gap sequences are 2.3-fold enriched in segmentally duplicated sequences compared to the whole genome. Our analysis confirms that not all gaps within 'finished' genomes are recalcitrant to subcloning and suggests that the paired-end-sequenced fosmid libraries could prove to be a rich resource for completion of the human euchromatic genome.

Gas permeable (GP) versus non-GP lens wearers: accuracy of orthokeratology in myopia reduction.

PURPOSE: The primary objective of this study is to determine whether there are significant differences in visual and refractive outcomes between gas permeable (GP) and non-GP wearers following a 1-month period of overnight orthokeratology (OK). METHODS: The study included 14 subjects between the ages of 18 and 42 years. Group 1 consisted of six subjects wearing GP lenses for the correction of myopia for, at minimum, 1 year. Group 2 consisted of eight subjects wearing soft contact lenses or spectacles for the correction of myopia. All subjects were fit into the BE design in Boston XO material and lenses were worn for a period of 1 month. Unaided visual acuity using high (90%) and low (10%) contrast log MAR Bailey-Lovie vision charts, subjective refraction, corneal topography, and slit lamp evaluation were performed. Subjects were evaluated at day 1, 7, 14, 21, and 30. RESULTS: One eye of each subject was considered for analysis; the eye with the better response was chosen based on post-OK measures. The mean post-OK spherical equivalent was 0.29 +/- 0.55 D in the GP group and 0.37 +/- 0.46 D in the non-GP group; the difference was statistically significant (p = 0.03). Baseline astigmatism decreased in the non-GP group after OK while there was no significant change in the GP group. The mean high contrast acuities were 0.06 +/- 0.12 in the GP group and 0.17 +/- 0.07 in the non-GP group (p = 0.05), whereas the low contrast acuities were 0.18 +/- 0.17 in the GP and 0.02 +/- 0.09 in the non-GP group (p = 0.01). CONCLUSIONS: Although the non-GP group has higher post-OK visual acuity and spherical equivalent statistically, the GP group has attained an average unaided acuity of >20/20 and residual myopia <0.5 D. Clinically, this shows that OK can be a promising technique in GP wearers.