SummaryDynamic regulation of calcium-permeable (CP-) AMPARs is important in normal synaptic transmission, plasticity and pathological changes. While the involvement of TARPs in trafficking of calcium-impermeable (CI-) AMPARs has been extensively studied, their role in surface expression and function of CP-AMPARs remains unclear. Here we examined AMPAR-mediated currents in cerebellar stellate cells from stargazer mice, lacking the prototypical TARP stargazin (γ-2). We found a marked increase in the contribution of CP-AMPARs to synaptic responses, indicating that, unlike CI-AMPARs, these can localize at synapses in the absence of γ-2. In contrast with CP-AMPARs in extrasynaptic regions, synaptic CP-AMPARs displayed an unexpectedly low channel conductance and strong block by intracellular spermine, suggesting they were ‘TARPless’. As proof of principle that TARP-association is not an absolute requirement for AMPAR clustering at synapses, mEPSCs mediated by TARPless AMPARs were readily detected in stargazer granule cells following knock down of their only other TARP, γ-7.

SummaryRegulation of calcium-permeable AMPA receptors (CP-AMPARs) is critical in normal synaptic function and neurological disease states. While transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (γ-2) modulate the properties of calcium-impermeable (CI-) AMPARs and promote their synaptic targeting, the TARP-specific rules governing CP-AMPAR synaptic trafficking remain unclear. We used RNA interference to manipulate AMPAR subunit- and TARP expression in γ-2-lacking stargazer cerebellar granule cells – the classic model of TARP deficiency. We found that TARP γ-7 selectively enhanced synaptic expression of CP-AMPARs and suppressed CI-AMPARs, identifying a pivotal role of γ-7 in regulating the prevalence of CP-AMPARs. In the absence of associated TARPs, although their properties were altered, both CP- and CI-AMPARs were able to localize at synapses and mediate transmission. Finally, our results establish that TARPed synaptic receptors in granule cells require both γ-2 and γ-7, and reveal an unexpected basis for the loss of AMPAR-mediated transmission in stargazer mice.

Medical measures that bear no known danger for the adult brain may trigger active neuronal death in the developing brain. Pharmacological blockade of N-methyl-D-aspartate or activation of GABA(A) receptors, blockade of voltage-dependent sodium channels, and oxygen induce widespread apoptotic neurodegeneration during the period of rapid brain growth in rodents. Because such measures are often necessary in critically ill infants and toddlers, search for adjunctive neuroprotective strategies is warranted. We report that 17beta-estradiol ameliorates neurotoxicity of drugs that block N-methyl-D-aspartate receptors, activate GABA(A) receptors, or block voltage-gated sodium channels and reduces neurotoxicity of oxygen in the infant rat brain. This neuroprotective effect is reversed by tamoxifen and cannot be reproduced by 17alpha-estradiol. 17Beta-estradiol did not affect GABA(A) or N-methyl-D-aspartate currents in hippocampal neuronal cultures, indicating that direct modulation of neurotransmitter receptor/channel properties by this compound cannot explain neuroprotective effect. 17beta-Estradiol did, however, increase levels of phosphorylated extracellular signal-regulated kinase 1/2 and AKT, suggesting that activation of these prosurvival proteins may represent one mechanism for its neuroprotective action. 17Beta-estradiol and related compounds may be neuroprotective agents suitable for use in critically ill infants and toddlers. Its supplementation may particularly help to improve neurocognitive outcome in preterm infants who are prematurely deprived of maternal estrogen.

Juvenile Batten disease is the most common progressive neurodegenerative disorder of childhood. It is associated with mutations in the CLN3 gene, causing loss of function of CLN3 protein and degeneration of cerebellar and retinal neurons. It has been proposed that changes in granule cell AMPA-type glutamate receptors (AMPARs) contribute to the cerebellar dysfunction. In this study, we compared AMPAR properties and synaptic transmission in cerebellar granule cells from wild-type and Cln3 knock-out mice. In Cln3Δex1–6 cells, the amplitude of AMPA-evoked whole-cell currents was unchanged. Similarly, we found no change in the amplitude, kinetics, or rectification of synaptic currents evoked by individual quanta, or in their underlying single-channel conductance. We found no change in cerebellar expression of GluA2 or GluA4 protein. By contrast, we observed a reduced number of quantal events following mossy-fiber stimulation in Sr2+, altered short-term plasticity in conditions of reduced extracellular Ca2+, and reduced mossy fiber vesicle number. Thus, while our results suggest early presynaptic changes in the Cln3 Δex1–6 mouse model of juvenile Batten disease, they reveal no evidence for altered postsynaptic AMPARs.

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here we show that inclusion of the spider toxin analogue 1‑naphthylacetyl spermine (NASPM) in the intracellular solution results in complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.

No abstract