The Contribution of TRPA1 to Corneal Thermosensitivity and Blink Regulation in Young and Aged Mice.

The role of TRPA1 in the thermosensitivity of the corneal cold thermoreceptor nerve endings was studied in young and aged mice. The contribution of the TRPA1-dependent activity to basal tearing and thermally-evoked blink was also explored. The corneal cold thermoreceptors' activity was recorded extracellularly in young (5-month-old) and aged (18-month-old) C57BL/6WT (WT) and TRPA1-/- knockout (TRPA1-KO) mice at basal temperature (34 °C) and during cooling (15 °C) and heating (45 °C) ramps. The blink response to cold and heat stimulation of the ocular surface and the basal tearing rate were also measured in young animals using orbicularis oculi muscle electromyography (OOemg) and phenol red threads, respectively. The background activity at 34 °C and the cooling- and heating-evoked responses of the cold thermoreceptors were similar in WT and TRPA1-KO animals, no matter the age. Similar to the aged WT mice, in the young and aged TRPA1-KO mice, most of the cold thermoreceptors presented low frequency background activity, a low cooling threshold, and a sluggish response to heating. The amplitude and duration of the OOemg signals correlated with the magnitude of the induced thermal change in the WT but not in the TRPA1-KO mice. The basal tearing was similar in the TRPA1-KO and WT mice. The electrophysiological data suggest that the TRPA1-dependent nerve activity, which declines with age, contributes to detecting the warming of the ocular surface and also to integrating the thermally-evoked reflex blink.

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder.

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for sustaining life would be possible without the ability to transition from one position to another. Unfortunately, motor skills can be compromised in a wide array of diseases. Therefore, investigating the mechanisms of motor functions at the cellular, molecular, and circuit levels, as well as understanding the symptoms, causes, and progression of motor disorders, is crucial for developing effective treatments. Mouse models are frequently employed for this purpose. This article describes a protocol that allows the monitoring of various aspects of motor performance and learning in mice using an automated tool called the Erasmus Ladder. The assay involves two phases: an initial phase where mice are trained to navigate a horizontal ladder built of irregular rungs ("fine motor learning"), and a second phase where an obstacle is presented in the path of the moving animal. The perturbation can be unexpected ("challenged motor learning") or preceded by an auditory tone ("associative motor learning"). The task is easy to conduct and is fully supported by automated software. This report shows how different readouts from the test, when analyzed with sensitive statistical methods, allow fine monitoring of mouse motor skills using a small cohort of mice. We propose that the method will be highly sensitive to evaluate motor adaptations driven by environmental modifications as well as early-stage subtle motor deficits in mutant mice with compromised motor functions.

Deciphering the Action of Perfluorohexyloctane Eye Drops to Reduce Ocular Discomfort and Pain.

Perfluorohexyloctane (F6H8) eyedrops have been recently introduced in Europe as a product to treat dry eye disease, based on its ability to reduce tear film instability in Meibomian gland dysfunction and evaporative dry eye disease, although its mechanism of action is still unknown. In the present pilot study, we evaluated the effects of the ocular instillation of a single drop of commercial F6H8 eyedrops in 20 healthy humans (9 women/11 men), measuring: (a) Corneal surface temperature (CST) from infrared video images; (b) tear volume using phenol red threads; (c) blinking frequency; and (d) ocular surface sensations (cold, dryness, pricking, foreign body, burning, itching, gritty, eye fatigue, watering eyes, and light-evoked discomfort sensations; scored using 10 cm Visual Analog Scales), before and 5-60 min after F6H8 or saline treatment. CST decreased and tearing and blinking frequency increased significantly after F6H8 but not after saline solution. When applied unilaterally, CST decreased only in the F6H8-treated eye. No sensations were evoked after F6H8 or saline. The corneal surface temperature reduction produced by topical F6H8 does not evoke conscious ocular sensations but is sufficient to increase the activity of corneal cold thermoreceptors, leading to an increased reflex lacrimation and blinking that may relieve dry eye condition thus reducing ocular discomfort and pain.

Visual Disfunction due to the Selective Effect of Glutamate Agonists on Retinal Cells.

One of the causes of nervous system degeneration is an excess of glutamate released upon several diseases. Glutamate analogs, like N-methyl-DL-aspartate (NMDA) and kainic acid (KA), have been shown to induce experimental retinal neurotoxicity. Previous results have shown that NMDA/KA neurotoxicity induces significant changes in the full field electroretinogram response, a thinning on the inner retinal layers, and retinal ganglion cell death. However, not all types of retinal neurons experience the same degree of injury in response to the excitotoxic stimulus. The goal of the present work is to address the effect of intraocular injection of different doses of NMDA/KA on the structure and function of several types of retinal cells and their functionality. To globally analyze the effect of glutamate receptor activation in the retina after the intraocular injection of excitotoxic agents, a combination of histological, electrophysiological, and functional tools has been employed to assess the changes in the retinal structure and function. Retinal excitotoxicity caused by the intraocular injection of a mixture of NMDA/KA causes a harmful effect characterized by a great loss of bipolar, amacrine, and retinal ganglion cells, as well as the degeneration of the inner retina. This process leads to a loss of retinal cell functionality characterized by an impairment of light sensitivity and visual acuity, with a strong effect on the retinal OFF pathway. The structural and functional injury suffered by the retina suggests the importance of the glutamate receptors expressed by different types of retinal cells. The effect of glutamate agonists on the OFF pathway represents one of the main findings of the study, as the evaluation of the retinal lesions caused by excitotoxicity could be specifically explored using tests that evaluate the OFF pathway.

Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches.

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.

Automatic Tuning of a Retina Model for a Cortical Visual Neuroprosthesis Using a Multi-Objective Optimization Genetic Algorithm.

The retina is a very complex neural structure, which contains many different types of neurons interconnected with great precision, enabling sophisticated conditioning and coding of the visual information before it is passed via the optic nerve to higher visual centers. The encoding of visual information is one of the basic questions in visual and computational neuroscience and is also of seminal importance in the field of visual prostheses. In this framework, it is essential to have artificial retina systems to be able to function in a way as similar as possible to the biological retinas. This paper proposes an automatic evolutionary multi-objective strategy based on the NSGA-II algorithm for tuning retina models. Four metrics were adopted for guiding the algorithm in the search of those parameters that best approximate a synthetic retinal model output with real electrophysiological recordings. Results show that this procedure exhibits a high flexibility when different trade-offs has to be considered during the design of customized neuro prostheses.

A novel formulation based on 2,3-di(tetradecyloxy)propan-1-amine cationic lipid combined with polysorbate 80 for efficient gene delivery to the retina.

PURPOSE: The aim of the present study was to evaluate the potential application of a novel formulation based on a synthesized cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 to deliver the pCMS-EGFP plasmid into the rat retina. METHODS: We elaborated lipoplexes by mixing the formulation containing the cationic lipid and the polysorbate 80 with the plasmid at different cationic lipid/DNA ratios (w/w). Resulted lipoplexes were characterized in terms of size, charge, and capacity to condense, protect and release the DNA. In vitro transfection studies were performed in HEK-293 and ARPE-19 cells. Formulations were also tested in vivo by monitoring the expression of the EGFP after intravitreal and subretinal injections in rat eyes. RESULTS: At 2/1 cationic lipid/DNA mass ratio, the resulted lipoplexes had 200 nm of hydrodynamic diameter; were positive charged, spherical, protected DNA against enzymatic digestion and transfected efficiently HEK-293 and ARPE-19 cultured cells exhibiting lower cytotoxicity than LipofectamineTM 2000. Subretinal administrations transfected mainly photoreceptors and retinal pigment epithelial cells; whereas intravitreal injections produced a more uniform distribution of transfection through the inner part of the retina. CONCLUSIONS: These results hold great expectations for other gene delivery formulations based on this cationic lipid for retinal gene therapy purposes.