The co-expression of the depolarizing and hyperpolarizing mechanosensitive ion channels in mammalian retinal neurons

Abstract

Introduction: The elevation of the intraocular and extraocular pressures is associated with various visual conditions, including glaucoma and traumatic retinal injury. The retina expresses mechanosensitive channels (MSCs), but the role of MSCs in retinal physiology and pathologies has been unclear.

Methods: Using immunocytochemistry, confocal microscopy, and patch-clamp recording techniques, we studied the co-expression of K⁺-permeable (K-MSCs) TRAAK and big potassium channel BK with the epithelial sodium channel ENaC and transient receptor potential channel vanilloid TPRV4 and TRPV2 favorably permeable to Ca²⁺ than Na⁺ (together named N-MSCs), and TRPV4 activity in the mouse retina.

Results: TRAAK immunoreactivity (IR) was mainly located in Müller cells. Photoreceptor outer segments (OSs) expressed BK and ENaCα intensively and TRAAK, TRPV2, and TRPV4 weakly. Somas and axons of retinal ganglion cells (RGCs) retrograde-identified clearly expressed ENaCα, TRPV4, and TRPV2 but lacked TRAAK and BK. Rod bipolar cells (RBCs) showed TRPV4-IR in somas and BK-IR in axonal globules. Horizontal cells were BK-negative, and some cone BCs lacked TRPV4-IR. TRPV4 agonist depolarized RGCs, enhanced spontaneous spikes and excitatory postsynaptic currents, reduced the visual signal reliability (VSR = 1-noise/signal) by ~50%, and resulted in ATP crisis, which could inactivate voltage-gated sodium channels in RGCs.

Conclusion: Individual neurons co-express hyperpolarizing K-MSCs with depolarizing N-MSCs to counterbalance the pressure-induced excitation, and the level of K-MSCs relative to N-MSCs (R_K/N ratio) is balanced in the outer retina but low in RGCs, bringing out novel determinants for the pressure vulnerability of retinal neurons and new targets for clinical interventions.

Keywords: BK; ENaC; TRAAK; TRPV2; TRPV4; confocal microscopy; immunocytochemistry; whole-cell patch-clamp.

Figure 1

BK expression in the mouse retina. (A,E) Retinal slices labeled for BK (green), GABA, and Calbindin. (B) The retina was retrogradely labeled for RGCs with Lucifer yellow (LY, blue) and neurobiotin (NB, red) and stained for PKCα (green), showing the size and location of the characteristic globules of axon terminals of rod bipolar cells (RBCs). (C) Labeled for BK (green) and TO-PRO-3. (D) The fitting curves and functions of pixels of BK immunoreactivity (IR) in seven retinal layers in (C). The BK-IR is primarily present in the OSL and IPL. Weak BK-IR is present in some somas in the INL and GCL and large globules of RBCs (arrows, inset of A), which contact BK-and GABA-positive profiles of putative A17 ACs. (E) The two plots show the linear profile of pixels along the two straight lines in the image. HCs identified by calbindin were nearly negative for BK. BCs, ACs, HCs, and RGCs: bipolar, amacrine, horizontal, and ganglion cells, respectively. OSL: photoreceptor layer. ONL and INL: inner and outer nuclear layer, respectively. OPL and IPL: inner and outer plexiform layer, respectively. Scale bars are 20 μm.

Figure 2

The expression of TRAAK and TRPV4 in the mouse retina. (A,B) Retinal slices were retrogradely labeled for ganglion cells (GCs) by neurobiotin (NB) and stained for TRPV4 (green) and TRAAK (A, pink) or glutamine synthetase (GS, blue, B). TRPV4 signals are heavier in the GCL. (A) The image was inverted and displaced on a white background. TRAAK is heavily expressed in Müller cells and weakly expressed in OSL and OPL. (B) Axons and somas of GCs are brightly positive for TRPV4. TRPV4 is present in Müller cells (MC). Some somas of putative cone BCs in the second soma row of the INL contain no TRPV4 signals (open arrow, inset b). (C,D) Retinal slices from wild-type (w.t.) and TRPV4 transgenic mice (TRPV4−/−) were labeled for TRPV4 (green) and PKCa (red). (C) Some TRPV4 puncta are present in rod BCs (asterisks) and cone BCs (triangles). (D) TRPV4 signal is nearly absent in TRPV4 transgenic mice. OSL: outer segment layer; ISL: inner segment layer; OPL: outer plexiform layer; INL: inner nuclear layer; BCL-bipolar cell layer; ACL-amacrine cell layer; IPL-inner plexiform layer; GCL-ganglion cell layer; NFL-nerve fiber layer. The scale bar is 5 μm for C and 20 μm for others.

Figure 3

TRPV2 expression in mouse retina. RGCs were retrogradely labeled by neurobiotin (NB, red). (A,B) TRPV2 (green) is expressed in the OSL, OPL, IPL, and GCL (A). Some puncta are present in RBCs identified by PKCα immunoreactivity (B, blue). The expression in the ACL and GCL is heavier in DBA/2J mice (D2, a congenital glaucoma model, in C), including amacrine cells labeled for GABA (C, blue). B6: C57BL/6J mice. Scale bars are 20 μm.

Figure 4

The expression of the mechanosensitive ENaC in mouse retina. Retinas were retrogradely labeled for RGCs by NB (red) and stained for TRAAK (blue) and ENaCα (A,B) or ENaCβ (C) (green). (A,B) ENaCα is intensively expressed in photoreceptors, OPL, IPL, and RGC somas and dendrites, and the INL is weakly labeled. TRAAK is primarily expressed in the soma, process, and end foot of Müller cells and OSL (A–C). (C) ENaCβ identifies blood vessels and weakly labels the OSL, OPL, and IPL. Scale bars are 20 μm.

Figure 5

TRPV4 mediates excitatory visual noises and dysfunction of mouse RGCs. RGCs were recorded under loose patch mode (A,B) and whole-cell voltage-clamp mode at various holding potentials (D–G). (A,B,F,G) TRPV4 agonists enhance spontaneous action potentials (A,B, arrow) and the spontaneous postsynaptic currents (arrow) and light-evoked excitatory currents (F,G), reducing the visual signal reliability [VSR = 1-noise (N)/signal (S)] by ~50% (C). (D,E) Confocal images of a recorded RGC filled with Lucifer yellow (D: The x-y view; E: The y-z view revealing the dendritic tree and axon of the cell).

Figure 6

The activation of MSCs induces membrane depolarization, ATP depletion, and dysfunction of mouse RGCs. RGCs are recorded under current-clamp (A,B) and voltage-clamp (C) modes. (A,B) Activating MSCs by low osmolarity (Osm) and TRPV4 agonists 4aPDD and GSK101 (GSK) depolarizes MP of RGCs to −40 to −50 mV and elicits spontaneous firing of action potentials (A), which raises the ATP consume close to the peak level and predicts ATP depletion. (C) The threshold of NaVs in mouse RGCs is close to −50 mV and sensitive to TTX. The duration of the sodium current is ~2 ms. (D) Blue curve: the standard ATP consumption for RGCs to maintain RP was plotted per Equation 2. TRPV4 agonists raised the ATP consumption (green dot) near the inactivation level of half NaVs (purple dot). Vh: holding potential. MP: membrane potential. RP: resting potential. NaV: voltage-gated sodium channel. RR: ruthenium red, nonspecific blocker of TRPs.