Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Regulation of postsynaptic gephyrin cluster size by protein phosphatase 1

The scaffolding protein gephyrin is essential for the clustering of glycine and GABA(A) receptors (GABA(A)Rs) at inhibitory synapses. Here, we provide evidence that the size of the postsynaptic gephyrin scaffold is controlled by dephosphorylation reactions. Treatment of cultured hippocampal neurons with the protein phosphatase inhibitors calyculin A and okadaic acid reduced the size of postsynaptic gephyrin clusters and increased cytoplasmic gephyrin staining. Protein phosphatase 1 (PP1) was found to colocalize with gephyrin at selected postsynaptic sites and to interact with gephyrin in transfected cells and brain extracts. Alanine or glutamate substitution of the two established serine/threonine phosphorylation sites in gephyrin failed to affect its clustering at inhibitory synapses and its ability to recruit gamma 2 subunit containing GABA(A)Rs. Our data are consistent with the postsynaptic gephyrin scaffold acting as a platform for PP1, which regulates gephyrin cluster size by dephosphorylation of gephyrin- or cytoskeleton-associated proteins. (C) 2010 Elsevier Inc. All rights reserved

NSC164599

Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brain‘s responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type–specific expression. Fluorescent Ca2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo. Although suboptimal Ca2+-binding dynamics and Ca2+-induced fluorescence changes in FCIPs are important factors, low levels of expression also seem to play a role. Here we report that delivering D3cpv, an improved fluorescent resonance energy transfer–based FCIP, using a recombinant adeno-associated virus results in expression sufficient to detect the Ca2+ transients that accompany single action potentials. In upper-layer cortical neurons, we were able to detect transients associated with single action potentials firing at rates o

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca₂₊ sensors

Measurement of population activity with single-neuron resolution is pivotal for understanding how information is represented and processed in the brain and how the brain's responses are altered by experience. Because neuronal activity in the neocortex is sparse and different neuron types perform different tasks, such measurements need to resolve single action potentials in single neurons, and need to be targeted to neuronal sub-classes. This is greatly facilitated by the use of genetically-encoded fluorescent calcium indicator proteins (FCIPs) of neuronal activity. We have employed recombinant adeno-associated viruses to deliver different FCIPs to neurons at a sufficiently high levels to detect the Ca2+ transients that accompany single action potentials. Based on these transients we were able to detect action potentials with high reliability not only in cultured brain slices but also in cortical layer 2/3 pyramidal cells in living animals. Cell-type targeting and long-term recording thus make FCIPs highly suitable to follow the activity of identified cells over the periods of weeks to months. This allows the study of the development and plasticity of neural maps. Preliminary results suggest that with FCIPs functional imaging of the same cells is possible over periods o

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca₂₊ sensors

Measurement of population activity with single-neuron resolution is pivotal for understanding how information is represented and processed in the brain and how the brain's responses are altered by experience. Because neuronal activity in the neocortex is sparse and different neuron types perform different tasks, such measurements need to resolve single action potentials in single neurons, and need to be targeted to neuronal sub-classes. This is greatly facilitated by the use of genetically-encoded fluorescent calcium indicator proteins (FCIPs) of neuronal activity. We have employed recombinant adeno-associated viruses to deliver different FCIPs to neurons at a sufficiently high levels to detect the Ca2+ transients that accompany single action potentials. Based on these transients we were able to detect action potentials with high reliability not only in cultured brain slices but also in cortical layer 2/3 pyramidal cells in living animals. Cell-type targeting and long-term recording thus make FCIPs highly suitable to follow the activity of identified cells over the periods of weeks to months. This allows the study of the development and plasticity of neural maps. Preliminary results suggest that with FCIPs functional imaging of the same cells is possible over periods o