The TRPM1 Channel Is Required for Development of the Rod ON Bipolar Cell-AII Amacrine Cell Pathway in the Retinal Circuit.

Neurotransmission plays an essential role in neural circuit formation in the central nervous system (CNS). Although neurotransmission has been recently clarified as a key modulator of retinal circuit development, the roles of individual synaptic transmissions are not yet fully understood. In the current study, we investigated the role of neurotransmission from photoreceptor cells to ON bipolar cells in development using mutant mouse lines of both sexes in which this transmission is abrogated. We found that deletion of the ON bipolar cation channel TRPM1 results in the abnormal contraction of rod bipolar terminals and a decreased number of their synaptic connections with amacrine cells. In contrast, these histological alterations were not caused by a disruption of total glutamate transmission due to loss of the ON bipolar glutamate receptor mGluR6 or the photoreceptor glutamate transporter VGluT1. In addition, TRPM1 deficiency led to the reduction of total dendritic length, branch numbers, and cell body size in AII amacrine cells. Activated Goα, known to close the TRPM1 channel, interacted with TRPM1 and induced the contraction of rod bipolar terminals. Furthermore, overexpression of Channelrhodopsin-2 partially rescued rod bipolar cell development in the TRPM1-/- retina, whereas the rescue effect by a constitutively closed form of TRPM1 was lower than that by the native form. Our results suggest that TRPM1 channel opening is essential for rod bipolar pathway establishment in development.SIGNIFICANCE STATEMENT Neurotransmission has been recognized recently as a key modulator of retinal circuit development in the CNS. However, the roles of individual synaptic transmissions are not yet fully understood. In the current study, we focused on neurotransmission between rod photoreceptor cells and rod bipolar cells in the retina. We used genetically modified mouse models which abrogate each step of neurotransmission: presynaptic glutamate release, postsynaptic glutamate reception, or transduction channel function. We found that the TRPM1 transduction channel is required for the development of rod bipolar cells and their synaptic formation with subsequent neurons, independently of glutamate transmission. This study advances our understanding of neurotransmission-mediated retinal circuit refinement.

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

Larval bullfrog skin lacks amiloride-blockable epithelial transport because α-ENaC is located within intracellular vesicles in epidermal apical cells and not in the apical plasma membrane.

The epithelial Na channel (ENaC) plays an essential role in sodium transport across epithelia such as adult frog skin. Transport across the skin, measured as short-circuit current (SCC), is blocked by amiloride. Bullfrog alpha-ENaC (α-fENaC) is expressed in adult bullfrog skin, and the SCC across this skin is blocked by amiloride. In contrast, an amiloride-blockable SCC is not detected in larval bullfrog skin, even though it expresses α-fENaC. We examined the subcellular localization of α-ENaC in such larval and adult skins. Immunofluorescent and immunoelectron microscopy of apical cells in the larval epidermis revealed α-fENaC localization within intracellular vesicles, but not in the plasma membrane. In contrast, in adult skin α-fENaC was localized to the apical-side membrane and to intracellular vesicles in Stratum granulosum cells. This may support the view that amiloride-blockable SCC is absent from larval skin, but is present in adult skin.

Localization of ENaC subunit mRNAs in adult bullfrog skin.

Adult amphibian skin has served as a model for the investigation of Na(+)-transporting epithelia, such as mammalian renal tubules. The amiloride-blockable epithelial Na(+) channel (ENaC), which is located in the apical membrane of the outer living cell layer, regulates Na(+) transport across the epithelium. ENaC is thought to develop during the terminal differentiation of epidermal cells, but the details are unclear. Here, we used in situ hybridization to examine the localization of the ENaC subunit mRNAs in skin of adult bullfrogs, to clarify the development of ENaC. We found that α-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while ß-ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of γ-ENaC mRNA, possibly for technical reasons. α- and ß-ENaC mRNAs, at least, were present in the sub-apical cells, in which ENaC protein is not necessary for amphibian skin to possess its Na(+)-transport function. Our results may mean that the sub-apical cells are already producing the ENaC subunit mRNAs prior to the final step in their differentiation.

Vasotocin- and mesotocin-induced increases in short-circuit current across tree frog skin.

In adult amphibian skin, Na(+) crosses from outside to inside. This Na(+) transport can be measured as the amiloride-blockable short-circuit current (SCC) across the skin. We investigated the effects of arginine vasotocin (AVT) and mesotocin (MT), and those of antagonists of the vasopressin and oxytocin receptors, on the SCC across Hyla japonica skin. (1) Both AVT (100 pmol/L or more) and MT (1 nmol/L or more) increased the SCC. (2) The AVT- and MT-induced increases in SCC recovered with time (downregulation). (3) These AVT/MT-induced effects were blocked by application of OPC-31260 (vasopressin V(2)-receptor antagonist). (4) The OPC-31260 concentration needed to block the AVT-induced response was lower upon post-application (after application of agonist) than upon pre-application (before application of agonist), suggesting the number of receptors may have decreased after AVT application. (5) Upon repeated application of AVT (100 pmol/L), the induced SCC increase did not differ significantly between the 1st and 2nd applications. (6) The time to reach the half-maximum value of the AVT-induced or MT-induced increase in SCC was not significantly different between washout and post-application of OPC-31260, suggesting that post-application of OPC-31260 cleared AVT and MT from their receptors. The effects of AVT, MT, and their antagonists in H. japonica, which is adapted to a terrestrial habitat, are compared with our previously published data on Rana catesbeiana (=Lithobates catesbeianus), which is adapted to a semiaquatic habitat.

Effects of arginine vasotocin and mesotocin on the activation and development of amiloride-blockable short-circuit current across larval, adult, and cultured larval bullfrog skins.

Amphibian skin has osmoregulatory functions, with Na(+) crossing from outside to inside. Na(+) transport can be measured as the short-circuit current (SCC). We investigated the short-term and long-term effects of arginine vasotocin (AVT) and mesotocin (MT) (which modulate Na(+) transport) on the activation and development of an amiloride-blockable SCC (adult-type feature) in larval, adult, and corticoid-cultured larval bullfrog skins. We found: (1) AVT-receptor (AVT-R) and MT-receptor (MT-R) mRNAs could be detected in both larval and adult skins, (2) in the short term (within 60 min), the larval SCC (amiloride-stimulated SCC) was increased by AVT, forskolin, and MT, suggesting that AVT and MT did not activate the inactive ENaC (epithelial sodium channel) protein thought to be expressed in larval skin, (3) in the short term (within 90 min), AVT, forskolin, and MT stimulated the adult SCC (amiloride-blockable SCC), (4) AVT and MT increased both the larval and adult SCC via receptors insensitive to OPC-21268 (an antagonist of the V(1)-type receptor), OPC-31260 (an antagonist of the V(2)-type receptor), and ([d(CH(2))(5),Tyr(Me)(2),Thr(4),Orn(8),des-Gly-NH (2) (9) ]VT) (an antagonist of the oxytocin receptor), (5) culturing EDTA-treated larval skin with corticoids supplemented with AVT (1 microM) or MT (1 microM) for 2 weeks (long-term effects of AVT and MT) did not alter the corticoid-induced development of an amiloride-blockable SCC (adult-type feature). AVT and MT thus have the potential to stimulate SCC though channels that are already expressed, but they may not influence the development of the amiloride-blockable SCC (an adult-type feature) in larval skin.