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The transient receptor potential vanilloid isoform 4 (TRPV4) functions as polymodal transducer of swelling, heat, stretch, and lipid metabolites, is widely expressed across sensory tissues, and has been implicated in pressure sensing in vertebrate retinas. Although TRPV4 knockout mice exhibit a variety of mechanosensory, nociceptive, and thermo- and osmoregulatory phenotypes, it is not known whether the transmission of light-induced signals in the eye is affected by the loss of TRPV4. We utilized field potentials, a measure of rod and cone signaling, to determine whether TRPV4 impacts on the generation and/or transmission of the photoreceptor light response and neurotransmission. Luminance intensity-response relationships were acquired in anesthetized wild-type and TRPV4 -/- mice and evaluated for peak amplitude and implicit time under scotopic and photopic conditions. We found that the morphology of the outer retina is unaffected by the ablation of the Trpv4 gene. Calcium imaging of dissociated Müller glia showed that selective TRPV4 stimulation induces oscillatory calcium signals in adjacent rods. However, no differences in scotopic or photopic light-evoked signaling in the distal retina were observed in TRPV4-/- eyes, suggesting that TRPV4 signaling in healthy Müller cells does not modulate the transmission of light-evoked signals at rod and cone synapses.

Glutamate induced cell death mechanisms gained considerable attention lately as excessive release of extracellular glutamate was reported to cause neurodegeneration in brain areas including the retina. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to provide neuroprotection through anti-apoptotic effects in the glutamate-model and also in other degeneration assays. Although PACAP is known to orchestrate complex intracellular signaling primarily through cAMP production, the mechanism that mediates the anti-apoptotic effect in glutamate excitotoxicity remains to be clarified. To study this mechanism we induced retinal neurodegeneration in newborn Wistar rats by subcutaneous monosodium-glutamate injection. 100 pmol PACAP and enzyme inhibitors were administered intravitreally. Levels of caspase 3, 9, and phospho-protein kinase A were assessed by Western blots. Changes in cAMP levels were detected employing a competitive immunoassay. We found that cAMP blockade by an adenylyl-cyclase inhibitor (2',4'-dideoxy-adenosine) did not abrogate the neuroprotective effect of PACAP1-38. We show that following intravitreal PACAP1-38 treatment cAMP was unaltered, consistent with the inhibitor results and phospho-protein kinase A, an effector of the cAMP pathway was also unaffected. On the other hand, blockade of the alternative phosphatidylcholine-specific PLC pathway using an inhibitor (D609CAS) abrogated the neuroprotective effects of PACAP1-38. Our results highlight PACAP1-38 ability in protecting retinal cells against apoptosis through diverse signaling cascades. It seems that at picomolar concentrations, PACAP does not trigger cAMP production, but nonetheless, exerts a significant anti-apoptotic effect through PLC activation. In conclusion, PACAP1-38 may signal via both AC and PLC activation producing the same protective outcome.

Although L-glutamate is the main excitatory neurotransmitter in the retina, excess glutamate level triggers severe neuronal damages. Therefore, monosodium glutamate has been used to probe neurodegenerative mechanisms but precise toxicity schedule is not available in vivo. We report, for the first time, a temporal analysis of apoptotic processes induced by subcutaneously applied monosodium glutamate. We investigated the glutamate triggered subcellular processes over a time scale of 48 h in neonatal retina. We employed immunoblots to measure the level of activated apoptotic factors and immunocytochemistry to reveal the dying cells. Upregulation of active caspase-9 started at 3 h and peaked at 6 h post-injection. Activations of caspase-3, caspase-6 and caspase-7 consistent with their late-phase roles increased at 6 h post-injection. The apoptotic processes were terminated by 24 h post-injection. Caspase 12 and calpain-2 seemed unaffected by subcutaneous monosodium glutamate administration. Uniquely, we found that the ubiquitous calpain-1 is not expressed in newborn rat retina.