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Introduction: Light responses of rod photoreceptor cells traverse the retina through three pathways. The primary pathway involves synapses from rods to ON-type rod bipolar cells with OFF signals reaching retinal ganglion cells (RGCs) via sign-inverting glycinergic synapses. Secondly, rod signals can enter cones through gap junctions. Finally, rods can synapse directly onto cone OFF bipolar cells. Methods: To analyze these pathways, we obtained whole cell recordings from OFF-type α RGCs in mouse retinas while expressing channelrhodopsin-2 in rods and/or cones. Results: Optogenetic stimulation of rods or cones evoked large fast currents in OFF RGCs. Blocking the primary rod pathway with L-AP4 and/or strychnine reduced rod-driven optogenetic currents in OFF RGCs by ~1/3. Blocking kainate receptors of OFF cone bipolar cells suppressed both rod- and cone-driven optogenetic currents in OFF RGCs. Inhibiting gap junctions between rods and cones with mecloflenamic acid or quinpirole reduced rod-driven responses in OFF RGCs. Eliminating the exocytotic Ca2+ sensor, synaptotagmin 1 (Syt1), from cones abolished cone-driven optogenetic responses in RGCs. Rod-driven currents were not significantly reduced after isolating the secondary pathway by eliminating Syt1 and synaptotagmin 7 (Syt7) to block synaptic release from rods. Eliminating Syt1 from both rods and cones abolished responses to optogenetic stimulation. In Cx36 KO retinas lacking rod-cone gap junctions, optogenetic activation of rods evoked small and slow responses in most OFF RGCs suggesting rod signals reached them through an indirect pathway. Two OFF cells showed faster responses consistent with more direct input from cone OFF bipolar cells. Discussion: These data show that the secondary rod pathway supports robust inputs into OFF α RGCs and suggests the tertiary pathway recruits both direct and indirect inputs.

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The authors wish to make the following corrections to this paper [...].

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Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. The mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones measured using glutamate transporter anion currents (IA(glu)) was also halved. We also studied the release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of the release from paired pulse depression was twofold slower in the rods and cones lacking ribbons. The release from rods at -40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in the rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed up vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity.

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Células Fotorreceptoras Retinianas Conos , Sinapsis , Animales , Ácido Glutámico/metabolismo , Ratones , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología

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Vision under starlight requires rod photoreceptors to transduce and transmit single-photon responses to the visual system. Small single-photon voltage changes must therefore cause detectable reductions in glutamate release. We found that rods achieve this by employing mechanisms that enhance release regularity and its sensitivity to small voltage changes. At the resting membrane potential in darkness, mouse rods exhibit coordinated and regularly timed multivesicular release events, each consisting of ~17 vesicles and occurring two to three times more regularly than predicted by Poisson statistics. Hyperpolarizing rods to mimic the voltage change produced by a single photon abruptly reduced the probability of multivesicular release nearly to zero with a rebound increase at stimulus offset. Simulations of these release dynamics indicate that this regularly timed, multivesicular release promotes transmission of single-photon responses to post-synaptic rod-bipolar cells. Furthermore, the mechanism is efficient, requiring lower overall release rates than uniquantal release governed by Poisson statistics.

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Fotones , Células Fotorreceptoras Retinianas Bastones/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Femenino , Masculino , Potenciales de la Membrana , Ratones

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The vertebrate visual system can detect and transmit signals from single photons. To understand how single-photon responses are transmitted, we characterized voltage-dependent properties of glutamate release in mouse rods. We measured presynaptic glutamate transporter anion current and found that rates of synaptic vesicle release increased with voltage-dependent Ca2+ current. Ca2+ influx and release rate also rose with temperature, attaining a rate of ∼11 vesicles/s/ribbon at -40 mV (35°C). By contrast, spontaneous release events at hyperpolarized potentials (-60 to -70 mV) were univesicular and occurred at random intervals. However, when rods were voltage clamped at -40 mV for many seconds to simulate maintained darkness, release occurred in coordinated bursts of 17 ± 7 quanta (mean ± SD; n = 22). Like fast release evoked by brief depolarizing stimuli, these bursts involved vesicles in the readily releasable pool of vesicles and were triggered by the opening of nearby ribbon-associated Ca2+ channels. Spontaneous release rates were elevated and bursts were absent after genetic elimination of the Ca2+ sensor synaptotagmin 1 (Syt1). This study shows that at the resting potential in darkness, rods release glutamate-filled vesicles from a pool at the base of synaptic ribbons at low rates but in Syt1-dependent bursts. The absence of bursting in cones suggests that this behavior may have a role in transmitting scotopic responses.

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Células Fotorreceptoras Retinianas Bastones , Sinapsis , Vesículas Sinápticas , Animales , Calcio/metabolismo , Femenino , Masculino , Potenciales de la Membrana , Ratones , Células Fotorreceptoras Retinianas Conos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I

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AMPA-type glutamate receptors in the CNS are normally impermeable to Ca2+, but the aberrant expression of Ca2+-permeable AMPA receptors (CP-AMPARs) occurs in pathological conditions such as ischemia or epilepsy, or degenerative diseases such as ALS. Here, we show that select populations of retinal ganglion cells (RGCs) similarly express high levels of CP-AMPARs in a mouse model of glaucoma. CP-AMPAR expression increased dramatically in both On sustained alpha and Off transient alpha RGCs, and this increase was prevented by genomic editing of the GluA2 subunit. On sustained alpha RGCs with elevated CP-AMPAR levels displayed profound synaptic depression, which was reduced by selectively blocking CP-AMPARs, buffering Ca2+ with BAPTA, or with the CB1 antagonist AM251, suggesting that depression was mediated by a retrograde transmitter which might be triggered by the influx of Ca2+ through CP-AMPARs. Thus, glaucoma may alter the composition of AMPARs and depress excitatory synaptic input in select populations of RGCs.