Abstract
Purpose :
To identify the exocytotic Ca2+ sensor used by rods and cones of mouse retina. While mouse photoreceptors show evidence for Synaptotagmin-1 (Syt1), salamander photoreceptors lack Syt1 and show low Ca2+ cooperativity unlike other Syt1-mediated synapses.
Methods :
To measure Ca2+ cooperativity, we varied extracellular Ca2+ and recorded synaptic currents evoked in horizontal cells by extracellular stimulation of cones. To eliminate Syt1 from rods or cones, we generated mice with Cre-sensitive loxP sites flanking exon 6 in Syt1 and and then crossed these mice with Rho-iCre or HRGP-Cre mice that express Cre only in rods or cones, respectively. After confirming Syt1 elimination and evaluating retinal anatomy by immunohistochemistry, we tested neurotransmission from rods and cones with ex vivo electroretinogram (ERG) recordings. Anion currents activated by glutamate transporter activity were used to measure glutamate release in individual rods and cones. In cones, we also assessed release by the inhibition of Ca2+ currents (ICa) from vesicular protons.
Results :
Similar to amphibians, mouse cones showed a low Ca2+ cooperativity for release of N=2.7. Elimination of Syt1 from rods suppressed ERG b-waves evoked by dim flashes, while elimination of Syt1 from cones reduced responses to bright flashes and to high frequency flicker. Neither ERG a-waves nor presynaptic ICa were altered. Eliminating Syt1 suppressed glutamate release from rods and cones evoked by brief depolarizing steps, but spontaneous release and release evoked by longer 500 ms steps were not reduced. Unlike other presynaptic mutations that impair release from rods, removal of Syt1 did not promote ectopic sprouting of bipolar and horizontal cell dendrites.
Conclusions :
Syt1 is the Ca2+ sensor that regulates fast synaptic release by mouse rods and cones; other sensors contribute to spontaneous and slower forms of release. Loss of the b-wave shows that Syt1 also mediates tonic release in darkness. Salamander photoreceptors do not use Syt1, but both species show similar low Ca2+ cooperativity suggesting this property is not due to a particular Syt. The undisturbed synaptic anatomy seen after deletion of Syt1 suggests a limited role for neurotransmission in maintaining photoreceptor synapse integrity, emphasizing the importance of proteins involved in active zone organization.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.