Calcium-permeable AMPA receptors in layer 5 interneurons of the mouse visual cortex

Cortical sensory processing is widely studied in the visual cortex. Such processing depends on specific neuronal connectivity, and the reliable activation of inhibitory interneurons. Excitatory connections onto both excitatory and inhibitory neurons exhibit synaptic plasticity thought to underlie circuit refinement and function. The mechanisms of induction and expression of short- and long-term plasticity can differ between neurons and depend on synapse-specific differences in molecular machinery. Compared to synapses onto principal cells, less is known about plasticity at synapses onto inhibitory neurons. In part, this reflects difficulties of classifying these diverse cells. Mechanistically, postsynaptic N-methyl-D-aspartate-type glutamate receptors are necessary for most forms of plasticity. However, in the hippocampus, postsynaptic amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs) of the calcium-permeable (CP-) subtype are thought to underlie a form of non-Hebbian plasticity. I sought to examine the expression of CP-AMPARs at synapses onto the two major classes of inhibitory interneuron in layer 5 of the mouse visual cortex, Basket cells (BCs) and Martinotti cells (MCs). I made patch-clamp recordings from cells in acute brain slices from wild-type and transgenic animals. Interneurons were distinguished using laser scanning two-photon microscopy to characterize their defining patterns of axonal arborisation, and voltage recording to determine their characteristic firing properties. To examine synaptic AMPARs, I recorded miniature excitatory postsynaptic currents, determined their kinetic properties, their rectification due to voltage-dependent block by intracellular polyamines, and their sensitivity to the selective blocker of CP-AMPARs, 1-naphthyl acetyl spermine. I also used antibody labelling to examine the presence of the GluA2 subunit in the two interneuron classes. Although MCs proved less amenable to voltage-clamp recording than did BCs, my findings indicate the presence of CP-AMPARs in BCs but not in MCs, suggesting distinct activation of these two inhibitory inputs to Pyramidal cells. I conclude by discussing the likely influence of differential CP-AMPAR expression on the dynamics of the cortical network

Synapse Type-Dependent Expression of Calcium-Permeable AMPA Receptors

Calcium-permeable (CP) AMPA-type glutamate receptors (AMPARs) are known to mediate synaptic plasticity in several different interneuron (IN) types. Recent evidence suggests that CP-AMPARs are synapse-specifically expressed at excitatory connections onto a subset of IN types in hippocampus and neocortex. For example, CP-AMPARs are found at connections from pyramidal cells (PCs) to basket cells (BCs), but not to Martinotti cells (MCs). This synapse type-specific expression of CP-AMPARs suggests that synaptic dynamics as well as learning rules are differentially implemented in local circuits and has important implications not just in health but also in disease states such as epilepsy

Synapse-specific expression of calcium-permeable AMPA receptors in neocortical layer 5

In the hippocampus, calcium‐permeable AMPA receptors have been found in a restricted subset of neuronal types that inhibit other neurons, although their localization in the neocortex is less well understood. In the present study, we looked for calcium‐permeable AMPA receptors in two distinct populations of neocortical inhibitory neurons: basket cells and Martinotti cells. We found them in the former but not in the latter. Furthermore, in basket cells, these receptors were associated with particularly fast responses. Computer modelling predicted (and experiments verified) that fast calcium‐permeable AMPA receptors enable basket cells to respond rapidly, such that they promptly inhibit neighbouring cells and shut down activity. The results obtained in the present study help our understanding of pathologies such as stroke and epilepsy that have been associated with disordered regulation of calcium‐permeable AMPA receptors

Neurons Specifically Activated by Fear Learning in Lateral Amygdala Display Increased Synaptic Strength

The lateral amygdala (LA) plays a critical role in the formation of fear-conditioned associative memories. Previous studies have used c-fos regulated expression to identify a spatially restricted population of neurons within the LA that is specifically activated by fear learning. These neurons are likely to be a part of a memory engram, but, to date, functional evidence for this has been lacking. We show that neurons within a spatially restricted region of the LA had an increase in both the frequency and amplitude of spontaneous postsynaptic currents (sPSC) when compared to neurons recorded from home cage control mice. We then more specifically addressed if this increased synaptic activity was limited to learning-activated neurons. Using a fos-tau-LacZ (FTL) transgenic mouse line, we developed a fluorescence-based method of identifying and recording from neurons activated by fear learning (FTL+ ) in acute brain slices. An increase in frequency and amplitude of sPSCs was observed in FTL+ neurons when compared to nonactivated FTL- neurons in fear-conditioned mice. No learning-induced changes were observed in the action potential (AP) input-output relationships. These findings support the idea that a discrete LA neuron population forms part of a memory engram through changes in synaptic connectivity

Using a Multiplex Nucleic Acid <i>in situ</i> Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I h , which is important for modulating neuron excitability. Four genes (HCN1-4) encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid in situ hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula (medial and lateral) and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of I h -mediated "sag" in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels