Rigby, M; (2013) Presynaptic AMPA receptor-mediated modulation of GABA release from cerebellar molecular layer interneurons. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Presynaptic ionotropic glutamate receptors that modulate neurotransmitter release are widespread in the central nervous system, yet their regulation and mechanism of action are poorly understood. Indeed, their presumed dependence on transmembrane auxiliary proteins, which profoundly shape the behaviour of somatodendritic receptors, is an open question. The trafficking and function of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs) is regulated by transmembrane AMPAR regulatory proteins (TARPs). I examined the role of TARPs at presynaptic sites in cerebellar molecular layer interneurons (MLIs). The reduction in evoked GABA release triggered by glutamate spillover from climbing fibres, and the increased quantal release following AMPA application, were markedly attenuated in stargazer mice lacking the prototypical TARP stargazin (γ-2). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a partial agonist at TARP-associated AMPARs, had comparable effects on release that were also abolished in stargazer mice. These findings were replicated in dissociated Purkinje cells with adherent synaptic boutons, demonstrating the presynaptic locus of modulation. The absence of AMPAR-mediated effects in recordings from stargazer dissociated Purkinje cells, suggests that presynaptic AMPARs do not function without γ-2-association. This contrasts to postsynaptic and extrasynaptic AMPARs in MLIs that can function TARPless, or in association with TARP γ-7, respectively. Mechanistically, presynaptic AMPARs predominantly modulate release through regulation of voltage-gated Ca2+ channels (VGCCs). Pre-incubation of acutely dissociated Purkinje cells with either the specific VGCC blocker ω-Agatoxin-IVA, or the slow binding Ca2+ chelator, ethylene glycol-bis(2-aminoethylether)-N,N',N'-tetraacetic acid (EGTA), revealed that AMPARs mainly regulate the activity of P/Q- and/or N-type VGCCs coupled to the active zone in the microdomain. In addition, treatment with the Ca2+-permeable AMPAR blocker, philanthotoxin-433, suggested that direct Ca2+ influx through the AMPAR channel may further contribute to effects on release at MLI – MLI boutons. My findings identify γ-2 as a crucial subunit for presynaptic AMPAR-mediated modulation of GABA release that occurs via regulation of VGCCs remote from active zones.

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