Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

Three-dimensional reconstruction of the axon extending from the dermal photoreceptor cell in the extraocular photoreception system of a marine gastropod, onchidium.

The marine gastropod Onchidium has a multiple photoreceptive system consisting of stalk eyes, dorsal eyes, photosensitive neurons, and extraocular dermal photoreceptor cells (DPCs). The DPCs were widespread all over the dorsal mantle and distributed singly or in groups in the dermis, but were not discernible by the naked eye. The DPC was oval in shape and large in size, and characterized by features specific to gastropod photoreceptor cells such as massive microvilli, photic vesicles, and a depolarized response. DPC-17, one of a group of 19 DPCs, was examined on serial semi-thin sections of 0.4 µm in thickness with a high-voltage transmission electron microscope (HVTEM). The axon emerged specifically from the lateral side between the distal microvillous portion and proximal cytoplasm, travelled through the connective tissue, and joined a small nerve bundle (NB). Two types of supportive cells were found along the length of the axon. The first type was a covering cell (CC) surrounding the surface of the DPC body and continuing onward to the axon sheath. DPC-17 was covered by 11 CCs, while the larger DPC-6 was only covered by four CCs. The second type was a sheath cell (ShC) wrapping the surface of the small NB where the axon of the DPC merged with undefined nerve fibers. The axon extending directly from DPC-17 was reconstructed three-dimensionally (3D) using DeltaViewer software. The 3D-reconstructed image of the sheath of the axon and the CC demonstrated the continuity between the two structures, especially when the image was rotated using DeltaViewer.

The OFF-pathway dominance of P2X(2)-purinoceptors is formed without visual experience.

Visual input in the critical period is an important determinant of the functions of the visual system, affecting for example the formation of the ocular dominance column in the visual cortex. The final map of columnar organization is usually determined by plastic changes in the critical period, but organization is distorted without adequate visual input. Here, we examined whether formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling in the mouse retina is the result of visual experience. The P2X(2)-purinoceptor signaling pathway developed during the critical period. However, visual experience in this period produced no plastic change in the formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling. Our findings suggest that the OFF-pathway dominance of P2X(2)-signaling in the mouse retina is intrinsically programmed.

Distribution of immunoreactivity for P2X3, P2X5, and P2X6-purinoceptors in mouse retina.

Previous findings have shown that P2X-purinoceptor-mediated signaling pathways regulate the release of ACh in the retina. We previously reported the existence of immunoreactivity for P2X1-, P2X2-, P2X4-, and P2X7-purinoceptors in mouse retina and speculated that P2X2 and P2X7-purinoceptors may modulate the activity of cholinergic amacrine cells. In the present study, we used an immunohistochemical technique to examine whether P2X3-, P2X5, and P2X6-purinoceptors are also important for the modulation of cholinergic amacrine cells in mouse retina. Immunoreactivity for P2X3-, P2X5-, and P2X6-purinoceptors was observed in mouse retina. Immunoreactivity for P2X3- purinoceptors was observed in the dendrites of cholinergic amacrine cells. Immunoreactivity for P2X5-purinoceptors existed in the soma of cholinergic amacrine cells. P2X6-purinoceptor immunoreactivity was not colocalized with the cholinergic amacrine cells. We concluded that, among the three P2X-purinoceptors that were examined, P2X3-purinoceptors seem to affect the function of cholinergic amacrine cells in the mouse retina.

Characterization of voltage-gated ionic channels in cholinergic amacrine cells in the mouse retina.

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.