RNAscope

Pleasant touch provides emotional and psychological support that helps mitigate social isolation and stress. However, the underlying mechanisms remain poorly understood. Using a pleasant touch-conditioned place preference (PT-CPP) test, we show that genetic ablation of spinal excitatory interneurons expressing prokineticin receptor 2 (PROKR2), or its ligand PROK2 in sensory neurons, abolishes PT-CPP without impairing pain and itch behaviors in mice. Mutant mice display profound impairments in stress response and prosocial behaviors.

RESULTS : In rodent eye (both rat and mouse), CFH mRNA is strongly expressed in the retinal pigment epithelium with some expression also found in inner nuclear (INL) and retinal ganglion cell (RGC) layers of the retina. C3 mRNA is expressed mainly in RGC, INL of retina, ciliary body, corneal epithelium with some expression is also found in rodent retinal pigment epithelium layer. However, in human eye, CFH and C3 mRNA are strongly expressed in the choroid. Some expression is also found in RGC, INL layer of retina, ONH, sclera, cornea endothelial and stroma; and ciliary body.

METHODS : To visualize the BRB _in vivo, _we utilized the transgenic _Tg(l-fabp:DBP-EGFP) _zebrafish model that expresses vitamin D binding protein (a member of the albumin gene family) tagged to GFP. This model displays the integrity of the BRB with GFP-tagged protein localized within the retinal vasculature by 3 days post-fertilization. Breakdown of the BRB is visualized as “leaking” of GFP outside the vasculature.

RESULTS : Cell clustering revealed that Myrf deficiency altered cell type distributions with reductions in RPE cells at all timepoints. Cell cycle dynamics were stable, consistent with increased cell death in mutants. There was also a compensatory increase in retinal progenitor (RPC) population at P0, without alteration in overall cell cycle dynamics. Differential gene expression analysis and PANTHER gene ontology-term analysis revealed down regulation of key pathways in mutant RPE cells, including melanosome biogenesis, cytoskeleton, and extracellular matrix.

METHODS : 8-week wild-type mice were used to determine gene (_Htr1b_) expression. RNAscope _in situ_ hybridization (ISH) was performed on retinal cryosections and imaged using confocal microscopy. Whole field flash electroretinograms (ERGs) were used to record scotopic and photopic amplitudes in 22 mice (8 _Htr1b_-/-; 8 _Htr1b_+/-; 6 WT). Positive scotopic threshold response (pSTR), b-wave, and a-wave amplitudes were recorded. Visual behavior was evaluated in _Htr1b_-/- mice and controls by assessing the scotopic and photopic optokinetic response.

RESULTS : PR-specific expression of _PRLΔE1_ was observed in the following canine models of progressive inherited retinal degeneration (IRD): _RPGR_-XLPRA1 and _NPHP5_-LCA. In _RPGR_-XLPRA2 carrier retinas that undergo random X-inactivation, patches of_ PRLΔE1 _expression correlated with patches of PR degeneration. However, we did not observe expression of _PRLΔE1_ 24 hrs and 2 wks after light exposure that triggers acute rod loss in the canine RHO-T4R model of adRP. No _PRLΔE1 _expression was seen either in the _CNGB3_-ACHM3 retina that undergoes extremely slow cone degeneration.

PURPOSE : Methylenetetrahydrofolate reductase (_MTHFR_) is a critical enzyme in the folate/methionine/homocysteine pathway. Variants in _MTHFR, _notably _677C>T,_ have_ _been associated with glaucoma as well as Alzheimer’s disease and vascular dementia, suggesting an overlapping mechanism in brain and eye. However, mechanisms driving increased risk are not known, hindering the development of new treatments. Approximately 30% of individuals carry at least one copy of _MTHFR677C>T_, causing a 50% decrease in MTHFR enzyme efficiency.

RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells.

METHODS : Retinal, optic nerve head (ONH) and distal optic nerve (ON) tissues from 8 juvenile 10-12 week-old cats (4 males and 4 females) with feline congenital glaucoma (FCG) and 5 age-matched normal control cats (3 males and 2 females) were used. Data for weekly intraocular pressure (IOP) and optic nerve axon counts were available for all subjects. Protein and gene expression in tissue cryosections were examined by immunofluorescence labeling (IF) and RNAscope in situ hybridization (ISH), respectively. Retinal tissue was IF labeled for myeloid cell marker, IBA-1 and flat-mounted.

RESULTS : After quality control and data integration, 17,401 nuclei were isolated from 26,471 original droplets, derived from macular samples of 4 patients without retinal disease and 3 patients with POAG. The proportion of retinal ganglion cells in glaucomatous retina was significantly lower than that in healthy retina (p=0.024). An activated subpopulation of Müller glia was identified in both healthy and glaucomatous retina by cell clustering. Cross-species analysis comparing zebrafish and humans identified YAP1 activation as a differentiator between zebrafish and human glial activation.