Preparation of Rat Organotypic Hippocampal Slice Cultures Using the Membrane-Interface Method

Cultured hippocampal slices from rodents, in which the architecture and functional properties of the hippocampal network are largely preserved, have proved to be a powerful substrate for studying healthy and pathological neuronal mechanisms. Here, we delineate the membrane-interface method for maintaining organotypic slices in culture for several weeks. The protocol includes procedures for dissecting hippocampus from rat brain, and collecting slices using a vibratome. This method provides the experimenter with easy access to both the brain tissue and culture medium, which facilitates genetic and pharmacological manipulations and enables experiments that incorporate imaging and electrophysiology. The method is generally applicable to rats of different ages, and to different brain regions, and can be modified for culture of slices from other species including mice

Protein kinase A (PKA) phosphorylation of Shp2 inhibits its phosphatase activity and modulates ligand specificity.

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function as mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We have identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the A-kinase anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates protein kinase A (PKA) phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We have identified two key amino acids in Shp2 that are phosphorylated by PKA: Thr73 contributes a helix-cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phospho-deficient (T73A/S189A) and phospho-mimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response

Mechanisms for localising calcineurin and CaMKII in dendritic spines

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca2+ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca2+ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca2+ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behaviour. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca2+ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes

Homomeric GluA2(R) AMPA receptors can conduct when desensitized

Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors (AMPARs), which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we present evidence that homomeric Q/R-edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large fractional steady-state currents and anomalous current-variance relationships. Our results from fluctuation analysis, single-channel recording, and kinetic modeling, suggest that the steady-state current is mediated predominantly by conducting desensitized receptors. When combined with crystallography this unique functional readout of a hitherto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane

The intracellular domain of homomeric glycine receptors modulates agonist efficacy

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for ICD in pentameric ligand-gated channels

AKAP79 enables calcineurin to directly suppress protein kinase A activity

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes towards LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes

Sound-Induced Flash Illusion is Resistant to Feedback Training

A single flash accompanied by two auditory beeps tends to be perceived as two flashes (Shams et al. Nature 408:788, 2000, Cogn Brain Res 14:147–152, 2002). This phenomenon is known as ‘sound-induced flash illusion.’ Previous neuroimaging studies have shown that this illusion is correlated with modulation of activity in early visual cortical areas (Arden et al. Vision Res 43(23):2469–2478, 2003; Bhattacharya et al. NeuroReport 13:1727–1730, 2002; Shams et al. NeuroReport 12(17):3849–3852, 2001, Neurosci Lett 378(2):76–81, 2005; Watkins et al. Neuroimage 31:1247–1256, 2006, Neuroimage 37:572–578, 2007; Mishra et al. J Neurosci 27(15):4120–4131, 2007). We examined how robust the illusion is by testing whether the frequency of the illusion can be reduced by providing feedback. We found that the sound-induced flash illusion was resistant to feedback training, except when the amount of monetary reward was made dependent on accuracy in performance. However, even in the latter case the participants reported that they still perceived illusory two flashes even though they correctly reported single flash. Moreover, the feedback training effect seemed to disappear once the participants were no longer provided with feedback suggesting a short-lived refinement of discrimination between illusory and physical double flashes rather than vanishing of the illusory percept. These findings indicate that the effect of sound on the perceptual representation of visual stimuli is strong and robust to feedback training, and provide further evidence against decision factors accounting for the sound-induced flash illusion

Parametric study of EEG sensitivity to phase noise during face processing

<b>Background: </b> The present paper examines the visual processing speed of complex objects, here faces, by mapping the relationship between object physical properties and single-trial brain responses. Measuring visual processing speed is challenging because uncontrolled physical differences that co-vary with object categories might affect brain measurements, thus biasing our speed estimates. Recently, we demonstrated that early event-related potential (ERP) differences between faces and objects are preserved even when images differ only in phase information, and amplitude spectra are equated across image categories. Here, we use a parametric design to study how early ERP to faces are shaped by phase information. Subjects performed a two-alternative force choice discrimination between two faces (Experiment 1) or textures (two control experiments). All stimuli had the same amplitude spectrum and were presented at 11 phase noise levels, varying from 0% to 100% in 10% increments, using a linear phase interpolation technique. Single-trial ERP data from each subject were analysed using a multiple linear regression model. <b>Results: </b> Our results show that sensitivity to phase noise in faces emerges progressively in a short time window between the P1 and the N170 ERP visual components. The sensitivity to phase noise starts at about 120–130 ms after stimulus onset and continues for another 25–40 ms. This result was robust both within and across subjects. A control experiment using pink noise textures, which had the same second-order statistics as the faces used in Experiment 1, demonstrated that the sensitivity to phase noise observed for faces cannot be explained by the presence of global image structure alone. A second control experiment used wavelet textures that were matched to the face stimuli in terms of second- and higher-order image statistics. Results from this experiment suggest that higher-order statistics of faces are necessary but not sufficient to obtain the sensitivity to phase noise function observed in response to faces. <b>Conclusion: </b> Our results constitute the first quantitative assessment of the time course of phase information processing by the human visual brain. We interpret our results in a framework that focuses on image statistics and single-trial analyses

Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.

γ-Aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABAAR function requires high-resolution structures. Here we present the first atomic structures of a GABAAR chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABAARs in neurological disorders