Long-term potentiation (LTP) of synaptic transmission is thought to be an important cellular mechanism underlying memory formation. A widely accepted model posits that LTP requires the cytoplasmic carboxyl tail (C-tail) of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GluA1. To find the minimum necessary requirement of the GluA1 C-tail for LTP in mouse CA1 hippocampal pyramidal neurons, we used a single-cell molecular replacement strategy to replace all endogenous AMPA receptors with transfected subunits. In contrast to the prevailing model, we found no requirement of the GluA1 C-tail for LTP. In fact, replacement with the GluA2 subunit showed normal LTP, as did an artificially expressed kainate receptor not normally found at these synapses. The only conditions under which LTP was impaired were those with markedly decreased AMPA receptor surface expression, indicating a requirement for a reserve pool of receptors. These results demonstrate the synapse's remarkable flexibility to potentiate with a variety of glutamate receptor subtypes, requiring a fundamental change in our thinking with regard to the core molecular events underlying synaptic plasticity.

In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

The amino-terminal domain (ATD) of AMPA receptors (AMPARs) accounts for approximately 50% of the protein, yet its functional role, if any, remains a mystery. We have discovered that the translocation of surface GluA1, but not GluA2, AMPAR subunits to the synapse requires the ATD. GluA1A2 heteromers in which the ATD of GluA1 is absent fail to translocate, establishing a critical role of the ATD of GluA1. Inserting GFP into the ATD interferes with the constitutive synaptic trafficking of GluA1, but not GluA2, mimicking the deletion of the ATD. Remarkably, long-term potentiation (LTP) can override the masking effect of the GFP tag. GluA1, but not GluA2, lacking the ATD fails to show LTP. These findings uncover a role for the ATD in subunit-specific synaptic trafficking of AMPARs, both constitutively and during plasticity. How LTP, induced postsynaptically, engages these extracellular trafficking motifs and what specific cleft proteins participate in the process remain to be elucidated.

Proper brain function depends on neurovascular coupling: neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand1. Neurovascular coupling is the basis for functional brain imaging2, and impaired neurovascular coupling is implicated in neurodegeneration1. The underlying molecular and cellular mechanisms of neurovascular coupling remain poorly understood. The conventional view is that neurons or astrocytes release vasodilatory factors that act directly on smooth muscle cells (SMCs) to induce arterial dilation and increase local blood flow1. Here, using two-photon microscopy to image neural activity and vascular dynamics simultaneously in the barrel cortex of awake mice under whisker stimulation, we found that arteriolar endothelial cells (aECs) have an active role in mediating neurovascular coupling. We found that aECs, unlike other vascular segments of endothelial cells in the central nervous system, have abundant caveolae. Acute genetic perturbations that eliminated caveolae in aECs, but not in neighbouring SMCs, impaired neurovascular coupling. Notably, caveolae function in aECs is independent of the endothelial NO synthase (eNOS)-mediated NO pathway. Ablation of both caveolae and eNOS completely abolished neurovascular coupling, whereas the single mutants exhibited partial impairment, revealing that the caveolae-mediated pathway in aECs is a major contributor to neurovascular coupling. Our findings indicate that vasodilation is largely mediated by endothelial cells that actively relay signals from the central nervous system to SMCs via a caveolae-dependent pathway.

Protein kinase A (PKA) integrates inputs from G-protein-coupled neuromodulator receptors to modulate synaptic and cellular function. Gαs signaling stimulates PKA activity, whereas Gαi inhibits PKA activity. Gαq, on the other hand, signals through phospholipase C, and it remains unclear whether Gαq-coupled receptors signal to PKA in their native context. Here, using two independent optical reporters of PKA activity in acute mouse hippocampus slices, we show that endogenous Gαq-coupled muscarinic acetylcholine receptors activate PKA. Mechanistically, this effect is mediated by parallel signaling via either calcium or protein kinase C. Furthermore, multiple Gαq-coupled receptors modulate phosphorylation by PKA, a classical Gαs/Gαi effector. Thus, these results highlight PKA as a biochemical integrator of three major types of GPCRs and necessitate reconsideration of classic models used to predict neuronal signaling in response to the large family of Gαq-coupled receptors.

RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in vivo delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed from pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline.

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.