Journal: The Journal of Neuroscience
Article Title: ATP Modulation of Excitatory Synapses onto Interneurons
Figure Lengend Snippet: P2X2 channels are the targets for ATP at interneuron synapses. A , Top left, Diagrammatic representation of the endogenous mouse P2X2 (mP2X2) locus with 11 exons. Top right, Representation of the targeted locus with exons 2-11 deleted and replaced with Neo. In both diagrams, the black bars indicate the approximate positions of the primers used for PCR analysis of tail genomic DNA, as shown in the gel to the left. Both the PCR and Southern blot (results not shown) approaches show that the mP2X2 gene is disrupted with Neo, but because we ran the PCR screen with all three primers, this approach additionally shows that exons downstream of exon 1 are deleted because no wild-type band was detected (left gel). Right gel, RT-PCR results from P2X2 + / + and P2X2 - / - mice with mRNA harvested from brain and testis. There was P2X2 mRNA in both brain and testis from P2X2 + / + mice but not from P2X2 - / - mice. mRNA for β-actin (β-act) was present in both genotypes and tissues. mw, Molecular weight markers. B, C , Nissl staining in P2X2 + / + ( B ) and P2X2 - / - mice ( C ). The representative voltage waveforms show responses of interneurons to depolarizing and hyperpolarizing current injections. D , EPSC frequency against time for 11 interneurons recorded from a P2X2 + / + mouse; the inset indicates that 64% of neurons responded to P2X activation in this mouse. E , mEPSC frequency against time for 13 interneurons recorded from a P2X2 - / - mouse; the inset indicates that only 15% of neurons responded to P2X activation in this mouse. F , Summary of recordings for all neurons that responded to ATPγS from seven P2X2 - / - and seven P2X2 + / + mice. G , In the P2X2 - / - mice, the proportion of neurons that receive P2X-modulated synapses is significantly reduced ( p
Article Snippet: We isolated mRNA from whole brain and testis with oligo-dT beads using the Quick Prep Micro mRNA purification kit (Amersham Biosciences, Arlington Heights, IL). cDNA was reverse-transcribed from mRNA using Moloney murine leukemia virus reverse transcriptase and oligo-dT primers (Advantage reverse transcriptase for PCR kit; Clontech, Palo Alto, CA), and PCR was performed using Taq polymerase ( Taq PCR core kit; Qiagen, Hilden, Germany).
Techniques: Polymerase Chain Reaction, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Activated Clotting Time Assay, Molecular Weight, Staining, Activation Assay