Somatostatin Modulates Neurotransmitter Release From Cultured Rat Retinal GABAergic Amacrine Cells

Retina is not only a part of central nervous system, but also has a relatively simple structure. By studying it, we can have a better understanding of the function of neruronal circuit and network. Amacrine cells(ACs) is one of the major kinds of interneurons in the retina. It can form synapse with other neurons, such as bipolar cell and/or ganglion cell, to modulate the formation and processing of visual information. There are many kinds of amacrine cells and GABAergic AC is one of the two major inhibitory interneurons. Spontaneous postsynaptic current is a reflection of spontaneous neurotransmitter release that plays multiple roles in a variety of neurobiological activities, especially in the formation, maintaining and transformation of information of synapse. Many studies demonstrate that the spontaneous transmitter release needs calcium, including the calcium influx from the presynaptic voltage-gated calcium channel and release from the intracellular calcium stores. But there are also many evidences suggest that, in some places, the transmitter release is independent from calcium. Yet, the mechanisms of the transmitter release of the cultured rat retina GABAergic amacrine cell are still not well elucidated.Somatostatin(SRIF) is wide spread in the central nervous system as a neurotransmitter or neuromodulator. SRIF plays a neurotransmitter or neuromodulator role by activating the specific G protein couple receptors (sstl-sst5). In the rat retina, SRIF is localized in the ACs and a number of ganglion cells, but the sst receptors are expressed across the whole retina, but their function is not clarified. Previous study demonstrated that sst5 receptors were expressed in rat retinal GABAergic amacrine cells and its terminal, but its function is not clear.In the present study, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) by patch-clamp techniques in cultured rat retinal GABAergic ACs and examined if and how Ca2+ was involved in the induction of spontaneous GABA release from the terminals of these cells. In our experiment condition, sIPSCs were completely blocked by application of 10 μM Gabazine, and the reversal potential of sIPSCs was close to EC1-, indicating that these events were exclusively mediated by GABAA receptors. In order to examine the role of Ca2+ in sIPSCs. First, co-applicaion of L type voltage-gated calcium channe blocker nimodipine (10μM) and N type voltage-gated calcium channe blocker co-conotoxin GVIA (0.5μM) suppressed the sIPSCs, indicating that the calcium influx from the L and N type voltage-gated calcium channel are involved in the presynaptic transmitter release. Furthermore, the ryanodine receptor (RyR) antagonist dantrolene (10μM) failed to affect sIPSCs, suggest that the calcium from ryanodine receptor mediated intracellular calcium store is not involved in the presynaptic transmitter release. While the inositol 1,4,5-trisphosphate (IP3) receptor antagonists 2-aminoethyl diphenylborinate (2-APB,20μM) and xestospongin C (XeC,1μM) significantly decreased the frequency of sIPSCs. While 2-APB can blocker IP3 receptor, it also have effect on transient receptor potential channel(TRP). In the presence of SKF96365 (10μM), a non-specific TRP blocker,2-APB persisted to show its effect on sIPSCs. Here, we also investigated modulation of SRIF on GABA release in cultured ACs. The frequency of spontaneous IPSCs (sIPSCs) in the ACs was reduced significantly by SRIF (1μM), which was reversed by an sst5 receptor antagonist BIM 23056 (1μM). As we mentioned above, the presynaptic voltage-gated calcium channel is involved in the spontaneous GABA release in the cultured rat retinal AC. So, co-application of nimodipine (10μM) and co-conotoxin GVIA (0.5μM) suppressed the sIPSCs, and SRIF failed to further inhibit the currents, while 1 uM SRIF can directly suppress voltage-gated calcium currents. SRIF can modulate calcium channel through multiple intracellular signaling pathway. So we examined if cGMP-PKG pathway is involved in. Extracellular application of 8-Br-cGMP (1mM), a membrane permeable cGMP analog and it can constantly activate cGMP signal pathway, decreased significantly the frequency of sIPSCs, and SRIF shows further inhibition in the sIPSCs. An application of protein kinase G (PKG) inhibitor KT5823 (200 nM) had no remarkable effect on the sIPSCs, whereas SRIF inhibits the sIPSCs in the presence of KT5823.In conclusion, spontaneous GABA release from the terminals of GAB Aergic ACs is Ca2+ -dependent, and both extracellular calcium influx through presynaptic calcium channels and Ca2+-release through activation of the IP3-sensitive pathway, but not the ryanodine-sensitive one, from intracellular stores are responsible for the generation of sIPSCs under our experimental conditions. We also conclude that SRIF inhibits Ca2+ influx via the presynaptic Ca2+ channels by activating the sst5 receptors, which results in a decrease of GABA release from the ACs through activating the cGMP-PKG signaling pathways.