Mechanisms underlying the development of reciprocal gabaergic feedback onto rod bipolar cells in mouse retina



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Universidad de Valparaíso



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Programa de Magister en Ciencias Biologicas Mencion Neurocienciass




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In the mammalian retina, GABAergic A17 amacrine cells are known to provide reciprocal inhibitory feedback to rod bipolar cell (RBC) terminals to regulate glutamate release and shape the time course of visual signaling in vivo. Remarkably, preliminary results from our laboratory indicate that this reciprocal feedback is completely absent prior to eye opening (postnatal <P15), suggesting that A17-RBC reciprocal synapses could be developmentally regulated to modify visual processing in the inner retina. However, how A17-RBC synapses are established to shape visual response remains poorly understood. Here, using electrophysiological and pharmacological approaches in acute mouse retinal slices, we evaluate the functional properties of A17-RBC synapses before and after eye opening in order to determine the mechanism(s) underlying the formation and establishment of this particular type of synapse in the mammalian retina. After eye opening, depolarization of voltage-clamped RBCs elicited sustained inward Ca2+ current, upon which was superimposed a fast and transient, feedback inhibitory postsynaptic current (IPSC). In contrast, the application of the same depolarization protocol before eye opening did not evoked reciprocal IPSCs and a substantial decrease in the magnitude of the sustained Ca2+ current was found. While this change in the Ca2+ current could explain the absence of feedback IPSC early in the development, we found that glutamate release from RBC terminals before and after the eye opening was sufficient to evoke excitatory postsynaptic currents (EPSCs) in A17 amacrine cells, suggesting that other mechanism rather than changes in glutamate release could be involved. For instance, RBC terminals are known to express type 1 cannabinoid receptor (CB1R), whose tonic activation might be involved in reciprocal synapse formation during development as has been shownfor other type of synapses in the brain. To evaluate this possibility, feedback IPSC was recorded from slices pre-incubated with AM251, a CB1R inverse agonist, and contrary to our thoughts, inhibition of CB1R did not recover feedback IPSC neither increase the magnitude of Ca2+ current recorded from RBC, suggesting that tonic eCB release does not contribute to the lack of A17-RBC reciprocal synapse in early mouse retina. Given that feedback IPSCs require GABA receptor (GABAR) activation at the RBC terminal, next we evaluate whether GABARs are functionally expressed in RBC terminals before eye opening. To test this possibility, first we elicited GABAR-mediated IPSC by applying GABA directly onto RBC terminal and found that GABA-evoked IPSCs are mediated by both GABAA and GABAC receptors. Similarly, GABA release triggered by a brief puff of AMPA onto A17s dendrites, elicited feedback IPSC in RBCs mediated by both GABAA and GABAC receptors. Together these results indicate that GABA release and GABAR subtypes are functional prior eye opening at RBC terminals, and that the absence of the reciprocal feedback IPSC elicited by brief depolarization is not due to an immature function of GABA receptors. As in adult retina, GABA release from A17 amacrine cells is triggered by activation of Ca2+-permeable AMPA receptors (CP-AMPARs), next we test the possibility that early in development A17 might not express CP-AMPARs necessary to trigger GABA release onto RBC terminals. To test this, we took advantage of the polyamine voltage-dependent block of CP- AMPARs and constructed current-voltage relationship (I-V) of AMPARs-mediated EPSCs, from both AII and A17 amacrine cells that are postsynaptic to RBC terminals. While AMPARs- mediated EPSC in AII amacrine cells showed an inward rectifying I-V relationship at positive potentials in both developmental stages, in A17 amacrine cells, AMPARs-mediated EPSC display an inward rectification after, but not before eye opening, suggesting a cell-specific switch of CP-AMPARs expression and/or function during retinal development in the inner retina. Our results demonstrate that RBC-A17 synapses are established early in development (prior to eye opening) and strongly suggest a change in subunit composition and/or function of AMPA receptors from Ca2+-impermeable to Ca2+-permeable -which are required for GABA release from A17s- occur after the eye opening. This observation ultimately might explain the absence of the reciprocal GABAergic feedback between A17 and RBC.


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• Programa de Magíster en Ciencias Biológicas mención Neurociencia, Facultad de Ciencias, Universidad de Valparaíso. • Proyecto FONDECYT 1151091. • Centro Interdisciplinario de Neurociencia (CINV): Proyecto código P09-022F • Núcleo Milenio de Biología de las Enfermedades Neuropsiquiátricas (NuMIND): Proyecto código NC 130011

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