Michael C. Ashby

The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity.

The AMPA receptor (AMPAR) GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity. Most neuronal AMPARs contain this critical subunit; however, in certain restricted neuronal populations and under certain physiological or pathological conditions, AMPARs that lack this subunit are expressed. There is a current surge of interest in such GluR2-lacking Ca2+-permeable AMPARs in how they affect the regulation of synaptic transmission. Here, we bring together recent data highlighting the novel and important roles of GluR2 in synaptic function and plasticity.

Removal of AMPA Receptors (AMPARs) from Synapses Is Preceded by Transient Endocytosis of Extrasynaptic AMPARs

AMPA receptors (AMPARs) are dynamically regulated at synapses, but the time course and location of their exocytosis and endocytosis are not known. Therefore, we have used ecliptic pHluorin-tagged glutamate receptor 2 to visualize changes in AMPAR surface expression in real time. We show that synaptic and extrasynaptic AMPARs respond very differently to NMDA receptor activation; there is a rapid internalization of extrasynaptic AMPARs that precedes the delayed removal of synaptic AMPARs.

Coordinated developmental recruitment of latent fast spiking interneurons in layer IV barrel cortex.

Feedforward inhibitory GABAergic transmission is critical for mature cortical circuit function; in the neonate, however, GABA is depolarizing and believed to have a different role. Here we show that the GABAA receptor–mediated conductance is depolarizing in excitatory (stellate) cells in neonatal (postnatal day [P]3–5) layer IV barrel cortex, but GABAergic transmission at this age is not engaged by thalamocortical input in the feedforward circuit and has no detectable circuit function. However, recruitment occurs at P6–7 as a result of coordinated increases in thalamic drive to fast-spiking interneurons, fast-spiking interneuron–stellate cell connectivity and hyperpolarization of the GABAA receptor–mediated response. Thus, GABAergic circuits are not engaged by thalamocortical input in the neonate, but are poised for a remarkably coordinated development of feedforward inhibition at the end of the first postnatal week, which has profound effects on circuit function at this critical time in development.

It's green outside: tracking cell surface proteins with pH-sensitive GFP

Green fluorescent protein (GFP) and mutated GFP variants have proved to be immensely powerful tools that have had a profound impact on research in biological sciences. This review considers the development, use and future implications of pH-dependent GFP variants (e.g. pHluorins). These proteins hold considerable promise for the relatively non-invasive monitoring of events such as exocytosis, endocytosis and protein surface expression in living neurons with high spatial and temporal resolution.

Maturation of a Recurrent Excitatory Neocortical Circuit by Experience-Dependent Unsilencing of Newly-Formed Dendritic Spines

Local recurrent excitatory circuits are ubiquitous in neocortex, yet little is known about their development or architecture. Here we introduce a quantitative technique for efficient single-cell resolution circuit mapping using 2-photon (2P) glutamate uncaging and analyze experience-dependent neonatal development of the layer 4 barrel cortex local excitatory circuit. We show that sensory experience specifically drives a 3-fold increase in connectivity at postnatal day (P) 9, producing a highly recurrent network. A profound dendritic spinogenesis occurs concurrent with the connectivity increase, but this is not experience dependent. However, in experience-deprived cortex, a much greater proportion of spines lack postsynaptic AMPA receptors (AMPARs) and synaptic connectivity via NMDA receptors (NMDARs) is the same as in normally developing cortex. Thus we describe a approach for quantitative circuit mapping and show that sensory experience sculpts an intrinsically developing template network, which is based on NMDAR-only synapses, by driving AMPARs into newly formed silent spines.

Coordinated activation of distinct Ca 2+ sources and metabotropic glutamate receptors encodes Hebbian synaptic plasticity

At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated presynaptic and postsynaptic spikes to activate postsynaptic NMDA receptors (NMDARs). The magnitudes of the ensuing Ca2+ transients within dendritic spines are thought to determine the amplitude and direction of synaptic change. In contrast, we show that at mature hippocampal Schaffer collateral synapses the magnitudes of Ca2+ transients during plasticity induction do not match this rule. Indeed, LTP induced by time-correlated pre- and postsynaptic spikes instead requires the sequential activation of NMDARs followed by voltage-sensitive Ca2+ channels within dendritic spines. Furthermore, LTP requires inhibition of SK channels by mGluR1, which removes a negative feedback loop that constitutively regulates NMDARs. Therefore, rather than being controlled simply by the magnitude of the postsynaptic calcium rise, LTP induction requires the coordinated activation of distinct sources of Ca2+ and mGluR1-dependent facilitation of NMDAR function.

Spatial characterisation of ryanodine-induced calcium release in mouse pancreatic acinar cells

In pancreatic acinar cells, agonists evoke intracellular Ca(2+) transients which are initiated in the apical region of these polarized cells. There are contradictory experimental data concerning Ca(2+) release from ryanodine receptors (RyRs) in the apical region. In the present study, we have used low doses of ryanodine to open RyRs leading to the release of Ca(2+) from intracellular stores. Ryanodine causes Ca(2+) release that is initiated in the apical region of the cell but is dependent upon functional inositol 1,4,5-trisphosphate receptors (IP(3)Rs). These results suggests that co-ordinated release from co-localized RyRs and IP(3)Rs underlies the increased sensitivity of the apical region to initiation of intracellular Ca(2+) transients.

Measuring membrane protein dynamics in neurons using fluorescence recovery after photobleach.

The use of genetically encoded fluorescent tags such as green fluorescent protein (GFP) as reporters to monitor processes in living cells has transformed cell biology. One major application for these tools has been to analyze protein dynamics in neurons. In particular, fluorescence recovery after photobleach (FRAP) of surface expressed fluorophore-tagged proteins has been instrumental to addressing outstanding questions about how neurons orchestrate the synaptic delivery of proteins. Here, we provide an overview of the methodology, equipment, and analysis required to perform, analyze, and interpret these experiments.

Altered Synapse Stability in the Early Stages of Tauopathy

Summary Synapse loss is a key feature of dementia, but it is unclear whether synaptic dysfunction precedes degenerative phases of the disease. Here, we show that even before any decrease in synapse density, there is abnormal turnover of cortical axonal boutons and dendritic spines in a mouse model of tauopathy-associated dementia. Strikingly, tauopathy drives a mismatch in synapse turnover; postsynaptic spines turn over more rapidly, whereas presynaptic boutons are stabilized. This imbalance between pre- and post-synaptic stability coincides with reduced synaptically driven neuronal activity in pre-degenerative stages of the disease.

Real-time imaging of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor) movements in neurons

The mechanisms that regulate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) synthesis, transport, targeting and surface expression are of fundamental importance for fast excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system. It has become apparent that these control processes involve complex sets of protein-protein interactions and many of the proteins responsible have been identified. We have been working to visualize AMPAR movement in living neurons in order to investigate the effects of blocking protein interactions. Here we outline the approaches used and the results obtained thus far.