Age-Related RPE changes in Wildtype C57BL/6J Mice between 2 and 32 Months.

Purpose: This study provides a systematic evaluation of age-related changes in RPE cell structure and function using a morphometric approach. We aim to better capture nuanced predictive changes in cell heterogeneity that reflect loss of RPE integrity during normal aging. Using C57BL6/J mice ranging from P60-P730, we sought to evaluate how regional changes in RPE shape reflect incremental losses in RPE cell function with advancing age. We hypothesize that tracking global morphological changes in RPE is predictive of functional defects over time. Methods: We tested three groups of C57BL/6J mice (young: P60-180; Middle-aged: P365-729; aged: 730+) for function and structural defects using electroretinograms, immunofluorescence, and phagocytosis assays. Results: The largest changes in RPE morphology were evident between the young and aged groups, while the middle-aged group exhibited smaller but notable region-specific differences. We observed a 1.9-fold increase in cytoplasmic alpha-catenin expression specifically in the central-medial region of the eye between the young and aged group. There was an 8-fold increase in subretinal, IBA-1-positive immune cell recruitment and a significant decrease in visual function in aged mice compared to young mice. Functional defects in the RPE corroborated by changes in RPE phagocytotic capacity. Conclusions: The marked increase of cytoplasmic alpha-catenin expression and subretinal immune cell deposition, and decreased visual output coincide with regional changes in RPE cell morphometrics when stratified by age. These cumulative changes in the RPE morphology showed predictive regional patterns of stress associated with loss of RPE integrity.

Pentosan Polysulfate Sodium Causes Diminished Function and Subtle Morphological Changes in Retina and RPE of Mice.

Purpose: There are numerous reports of a distinctive maculopathy in adults exposed to pentosan polysulfate sodium (PPS), a drug prescribed to treat bladder discomfort associated with interstitial cystitis. We tested whether PPS treatment of mice injures RPE or retina to provide insight into the etiology of the human condition. Methods: Mice were fed PPS-supplemented chow over 14 months. RPE and retinal function was assessed by electroretinography (ERG) regularly. Following euthanasia, one eye was used for sagittal sectioning and histology, the contralateral for RPE flatmounting. ZO-1 positive RPE cell borders were imaged using confocal microscopy and cell morphology was analyzed using CellProfiler. Results: After 10 months of PPS treatment, we observed diminution of mean scotopic c-wave amplitudes. By 11 months, we additionally observed diminutions of mean scotopic a- and b-wave amplitudes. Analysis of flatmounts revealed altered RPE cell morphology and morphometrics in PPS-treated mice, including increased mean en face cell area and geometric eccentricity, decreased RPE cell solidity and extent, and cytosolic translocation of alpha-catenin, all markers of RPE cell stress. Sex and regional differences were seen in RPE flatmount measures. Shortened photoreceptor outer segments were also observed. Conclusions: PPS treatment reduced RPE and later retina function as measured by ERG, consistent with a primary RPE injury. Post-mortem analysis revealed extensive RPE pleomorphism and polymegathism and modest photoreceptor changes. We conclude that PPS treatment of mice causes slowly progressing RPE and photoreceptor damage and thus may provide a useful model for some retinal pathologies.

Changes in neuropeptide large dense core vesicle trafficking dynamics contribute to adaptive responses to a systemic homeostatic challenge.

Neuropeptides are packed into large dense core vesicles (LDCVs) that are transported from the soma out into their processes. Limited information exists regarding mechanisms regulating LDCV trafficking, particularly during challenges to bodily homeostasis. Addressing this gap, we used 2-photon imaging in an ex vivo preparation to study LDCVs trafficking dynamics in vasopressin (VP) neurons, which traffic and release neuropeptide from their dendrites and axons. We report a dynamic bidirectional trafficking of VP-LDCVs with important differences in speed and directionality between axons and dendrites. Acute, short-lasting stimuli known to alter VP firing activity and axonal/dendritic release caused modest changes in VP-LDCVs trafficking dynamics. Conversely, chronic/sustained systemic osmotic challenges upregulated VP-LDCVs trafficking dynamic, with a larger effect in dendrites. These results support differential regulation of dendritic and axonal LDCV trafficking, and that changes in trafficking dynamics constitute a novel mechanism by which peptidergic neurons can efficiently adapt to conditions of increased hormonal demand.

Protocols for measuring cold-evoked neural activity and cold tolerance in Drosophila larvae following fictive cold acclimation.

Here, we outline protocols to study cold acclimation in Drosophila from a neurobiological perspective, starting with fictive cold acclimation using a custom-built optogenetics-housing apparatus we call the OptoBox. We also provide detailed steps for single-unit electrophysiological recordings from larval cold nociceptors and a high-throughput cold-tolerance assay. These protocols expand the toolkit for the study of insect cold acclimation and nociception. For complete details on the use and execution of this protocol, please refer to Himmel et al. (2021).

Identification of a neural basis for cold acclimation in Drosophila larvae.

Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. Herein, we describe cold-nociceptive behavior among 11 drosophilid species; we find that the predominant cold-evoked larval response is a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. We therefore tested the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation. We found that Drosophila melanogaster Class III nociceptors are sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.

Age-Related Retinal Changes in Wild-Type C57BL/6J Mice Between 2 and 32 Months.

Purpose: The purpose of this study was to extend our understanding of how aging affects normal retina function and morphology in wild-type C57BL/6J mice, by analyzing electrophysiological recordings and in vivo and post mortem anatomy. Methods: Electroretinograms (ERGs), spectral domain optical coherence tomography (SD-OCT), and confocal scanning laser ophthalmoscope (cSLO) in vivo images were obtained from mice between the ages of 2 and 32 months in four groups: group 1 (<0.5 years), group 2 (1.0-1.5 years), group 3 (1.5-2.0 years), and group 4 (>2.0 years). Afterward, mouse bodies and eyes were weighed. Eyes were stained with hematoxylin and eosin (H&E) and cell nuclei were quantified. Results: With aging, mice showed a significant reduction in both a- and b-wave ERG amplitudes in scotopic and photopic conditions. Additionally, total retina and outer nuclear layer (ONL) thickness, as measured by SD-OCT images, were significantly reduced in older groups. The cSLO images showed an increase in auto-fluorescence at the photoreceptor-RPE interface as age increases. H&E cell nuclei quantification showed significant reduction in the ONL in older ages, but no differences in the inner nuclear layer (INL) or ganglion cell layer (GCL). Conclusions: By using multiple age groups and extending the upper age limit of our animals to approximately 2.65 years (P970), we found that natural aging causes negative effects on retinal function and morphology in a gradual, rather than abrupt, process. Future studies should investigate the exact mechanisms that contribute to these gradual declines in order to discover pathways that could potentially serve as therapeutic targets.

Morphometric Analysis of Retinal Pigment Epithelial Cells From C57BL/6J Mice During Aging.

Purpose: To quantitatively evaluate the changes in orientation and morphometric features of mouse retinal pigment epithelial (RPE) cells in different regions of the eye during aging. Methods: We segmented individual RPE cells from whole RPE flatmount images of C57BL/6J mice (postnatal days 30 to 720) using a machine-learning method and evaluated changes in morphometric features, including our newly developed metric combining alignment and shape of RPE cells during aging. Results: Mainly, the anterior part of the RPE sheet grows during aging, while the posterior part remains constant. Changes in size and shape of the peripheral RPE cells are prominent with aging as cells become larger, elongated, and concave. Conversely, the central RPE cells maintain relatively constant size and numbers with aging. Cell count in the central area and the overall cell count (approximately 50,000) were relatively constant over different age groups. RPE cells also present a specific orientation concordance that matches the shape of the specific region of the eyeball. Those cells near the optic disc or equator have a circumferential orientation to cover the round shape of the eyeball, whereas those cells in the periphery have a radial orientation and corresponding radial elongation, the extent of which increases with aging and matches with axial elongation of the eyeball. Conclusions: These results suggest that the fluid RPE morphology reflects various growth rates of underlying eyeball, and RPE cells could be classified into four regional classes (near the optic disc, central, equatorial, and peripheral) according to their morphometric features.

Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes.

OBJECTIVE: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. METHODS: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. RESULTS: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. CONCLUSIONS: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Comparison of histologic findings in age-related macular degeneration with RPE flatmount images.

Purpose: To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD. Methods: Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images. Results: Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0­51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60­93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (<63 µm), medium (63.0­124 µm), and large ( ≥ 125 µm) drusen. The RPE was arranged in rosette-like structures overlying small drusen, attenuated overlying medium-sized drusen, and consisted of large multinucleated cells overlying large drusen. The RPE in the area of geographic atrophy was attenuated and depigmented. Conclusions: Confocal images of flatmounts from eyes with AMD showed RPE patterns overlying various types of drusen and geographic atrophy that correlated with histologic characteristics. We propose RPE repair mechanisms that may result in the patterns that we observed.

Differential Exon Expression in a Large Family of Retinal Genes Is Regulated by a Single Trans Locus.

Transcription and RNA processing can generate many variant mRNAs (isoforms) from a given genomic locus. The more we learn about RNA processing the more we realize how complex it can be. Examining the expression profiles of individual exons, we observed that specific exons were differentially expressed across a large number of genes in mice. We found that each isoform or exon is independently expressed compared to other exons from the same gene and regulated separately in trans. Each trans locus was identified by mapping using linkage analysis in a large mouse recombinant inbred strain set. We present evidence for a limited number of these master regulatory loci in the retina. One major locus controls about half the expression of the individual exons and resides on Chromosome 4, between 133 and 136 Mb.

Orbitofrontal Cortex Neurons Respond to Sound and Activate Primary Auditory Cortex Neurons.

Sensory environments change over a wide dynamic range and sensory processing can change rapidly to facilitate stable perception. While rapid changes may occur throughout the sensory processing pathway, cortical changes are believed to profoundly influence perception. Prior stimulation studies showed that orbitofrontal cortex (OFC) can modify receptive fields and sensory coding in A1, but the engagement of OFC during listening and the pathways mediating OFC influences on A1 are unknown. We show in mice that OFC neurons respond to sounds consistent with a role of OFC in audition. We then show in vitro that OFC axons are present in A1 and excite pyramidal and GABAergic cells in all layers of A1 via glutamatergic synapses. Optogenetic stimulation of OFC terminals in A1 in vivo evokes short-latency neural activity in A1 and pairing activation of OFC projections in A1 with sounds alters sound-evoked A1 responses. Together, our results identify a direct connection from OFC to A1 that can excite A1 neurons at the earliest stage of cortical processing, and thereby sculpt A1 receptive fields. These results are consistent with a role for OFC in adjusting to changing behavioral relevance of sensory inputs and modulating A1 receptive fields to enhance sound processing.

IRBP deficiency permits precocious ocular development and myopia.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is abundant in the subretinal space and binds retinoids and lipophilic molecules. The expression of IRBP begins precociously early in mouse eye development. IRBP-deficient (KO) mice show less cell death in the inner retinal layers of the retina before eyelid opening compared to wild-type C57BL/6J (WT) controls and eventually develop profound myopia. Thus, IRBP may play a role in eye development before visually-driven phenomena. We report comparative observations during the course of the natural development of eyes in WT and congenic IRBP KO mice that suggest IRBP is necessary at the early stages of mouse eye development for correct function and development to exist in later stages. METHODS: We observed the natural development of congenic WT and IRBP KO mice, monitoring several markers of eye size and development, including haze and clarity of optical components in the eye, eye size, axial length, immunohistological markers of differentiation and eye development, visually guided behavior, and levels of a putative eye growth stop signal, dopamine. We conducted these measurements at several ages. Slit-lamp examinations were conducted at post-natal day (P)21. Fundus and spectral domain optical coherence tomography (SD-OCT) images were compared at P15, P30, P45, and P80. Enucleated eyes from P5 to P10 were measured for weight, and ocular dimensions were measured with a noncontact light-emitting diode (LED) micrometer. We counted the cells that expressed tyrosine hydroxylase (TH-positive cells) at P23-P36 using immunohistochemistry on retinal flatmounts. High-performance liquid chromatography (HPLC) was used to analyze the amounts of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) at P7-P60. Monocular form deprivation in the right eye was induced using head-mounted goggles from P28 to P56. RESULTS: Eye elongation and eye size in the IRBP KO mice began to increase at P7 compared to the WT mice. This difference increased until P12, and the difference was maintained thereafter. SD-OCT images in live mice confirmed previously reported retinal thinning of the outer nuclear layer in the IRBP KO mice compared to the WT mice from P15 to P80. Slit-lamp and fundoscopy examination outcomes did not differ between the WT and KO mice. SD-OCT measurements of the optical axis components showed that the only factor contributing to excess optical axis length was the depth of the vitreous body. No other component of optical axis length (including corneal thickness, anterior chamber depth, and lens thickness) was different from that of the WT mouse. The refractive power of the IRBP KO mice did not change in response to form deprivation. The number of retinal TH-positive cells was 28% greater in the IRBP KO retinas compared to the WT mice at P30. No significant differences were observed in the steady-state retinal DA or DOPAC levels or in the DOPAC/DA ratios between the WT and IRBP KO mice. CONCLUSIONS: The IRBP KO mouse eye underwent precocious development and rapid eye size growth temporally about a day sooner than the WT mouse eye. Eye size began to differ between the WT and KO mice before eyelid opening, indicating no requirement for focus-dependent vision, and suggesting a developmental abnormality in the IRBP KO mouse eye that precedes form vision-dependent emmetropization. Additionally, the profoundly myopic KO eye did not respond to form deprivation compared to the non-deprived contralateral eye. Too much growth occurred in some parts of the eye, possibly upsetting a balance among size, differentiation, and focus-dependent growth suppression. Thus, the loss of IRBP may simply cause growth that is too rapid, possibly due to a lack of sequestration or buffering of morphogens that normally would bind to IRBP but are unbound in the IRBP KO eye. Despite the development of profound myopia, the DA levels in the IRBP KO mice were not statistically different from those in the WT mice, even with the excess of TH-positive cells in the IRBP KO mice compared to the WT mice. Overall, these data suggest that abnormal eye elongation in the IRBP KO mouse is independent of, precedes, and is epistatic to the process(es) of visually-driven refractive development.