Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 1. TRPC4 expression in adult mouse brain. A, B, In situ hybridization of TRPC4-mRNA in amygdala, hippocampus, somatosensory cortex, auditory thalamus, and auditory cortex. BLA, amygdala basolateral nucleus; S1, primary somatosensory cortex;S2,secondarysomatosensorycortex;AuT,auditorythalamus;AuD,secondaryauditorycortex,dorsal;Au1,primaryauditory cortex; AuV, secondary auditory cortex, ventral; Ect, ectorhinal cortex; PRh, perirhinal cortex. Scale bar, 1 mm. C, TRPC4 (left) and CaMKII(middle;amarkerofpyramidalneurons)colocalizeintheLA(right).D,CellsexpressingTRPC4(red)andGFAP(green;amarker ofglialcells)donotcolocalizeintheLA.E,CellsexpressingTRPC5(red)andGAD67(green;amarkerofinterneurons)donotcolocalizeinthe LA.Scalebars:C–E,10m.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Expressing, In Situ Hybridization

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure2. GenerationandconfirmationofTRPC4/mouse.A,TargetingstrategyforthedisruptionoftheTRPC4gene.Afterhomologousrecombination,deletionofexon4regionwascatalyzed by Cre-recombinase in ES cells. B, Targeting of the TRPC4 locus is confirmed by PCR analysis of tail genomic DNA. C, RT-PCR analysis of whole-brain mRNA from control and TRPC4/ littermates confirms the absence of exon 4. D, IP of TRPC4 protein in brain microsomes from control and TRPC4/ mice reveals loss of TRPC4 protein in TRPC4/ mice (top). Western blotting of NKA- confirmsequalproteinloadingincontrolandTRPC4/mice(bottom).E,ImmunohistochemicalstainingofbrainsectionsfromcontrolandTRPC4/littermatesrevealsselectivelossofTRPC4 expression in mutant mice. Scale bar, 1 mm.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 9. mGluR-EPSCs in LA neurons are suppressed in TRPC4/ mice. A, Synaptic responses in cortical input to the LA neuroninaslicefromacontrolmouseevokedbytrainsofhigh-frequencystimulationbeforeandduringadditionofCNQX(AMPA receptorantagonist;20M),NMDAreceptorantagonists,D-APV(50M),andMK-801(10m)andGABABRantagonistCGP35348 (300 M) recorded as described previously (Riccio et al., 2009). Stimulation trains consisted of 10 pulses at 100 Hz which were delivered once every 30 s. Inset shows synaptic responses recorded in current-clamp mode before (1) and after (2) the addition of antagonists to the external medium. The dashed line indicates the time point where the EPSP amplitude was measured. B, The experiment was identical to A, but the recording was obtained from a TRPC4/ mouse. C, Summary data for the experiments as in A and B, performed in both cortical and thalamic inputs to the LA. The amplitude of the residual component of the EPSP in the presence of antagonists was smaller in both pathways in slices from TRPC4/mice comparedwithcontrollittermates.D,EPSCsincorticalinputrecordedinvoltage-clampmodeatholdingpotentialsranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). E, Current–voltage (I–V) plots of the peak current in cortical input (as in D) in slices from control and TRPC4/ mice. F, EPSCs in thalamic input recorded in voltage-clamp mode at holding potentials ranging from –100 mV to 40 mV in slices from control (left) and TRPC4/ (right) mice in the presence of the antagonists (as in A). G, Current–voltage (I–V) plots of the peak current in thalamic input (as in F) in slices from control and TRPC4/ mice. H, Left, Representative immunoblotshowssimilaramountsofTRPC5proteininbrainmicrosomesextractedfromcontrolorTRPC4/littermates. Western blotting of Na -K -ATPase indicates equal protein loading (bottom). Right, Quantification of three different blots. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Control, Western Blot

Journal: Journal of Neuroscience

Article Title: Decreased Anxiety-Like Behavior and G q/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

doi: 10.1523/jneurosci.2274-13.2014

Figure Lengend Snippet: Figure 10. CCK-mediated increase in spike firing in LA neurons is diminished in TRPC4/ mice. A, Spikes evoked in LA neurons by current injection (150 pA) recorded in current-clamp mode under baseline conditions and in the presence of 3 M CCK4 in a slice from a control mouse. B, Summary plot showing CCK4-induced depolarization in LA neurons in slices from control and mutant mice. C, Spikes evoked in LA neuron under baseline conditions and in the presence of 3 M CCK4 in a slice from a TRPC4/ mouse. D, The percentage increase in spike frequency in the presence of CCK4 relative to the baseline frequency (taken as 100%) in slices from control and null mice. E, Left, Fluorescence double labeling for CCKergic fibers (green) and CaMKII (red) reveals no differences in innervation of the LA by CCK-containing fibers in brain sections from control and TRPC4/ littermates. Right, Mean CCK fluorescence after normalization to CaMKII fluorescence from six images per group taken from different stainings (two images from each of 3 control and 3 KO mice). Scale bar, 10 m. Results are shown as mean SEM.

Article Snippet: Primary antibody mixtures consisted of either rabbit monoclonal antibody to CaMKII (1:1000; clone EP1829Y, Abcam), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP; 1:2000; Abcam), or rabbit polyclonal antibody to Gad67 (1:100; AnaSpec) combined with mouse monoclonal to TRPC4 (1:500; clone: N77/15, NeuroMab, UC Davis) and diluted in DaVinci Green diluent (Biocare Medical) applied for 1 h. Mouse monoclonal antibody to CaMKII (1: 1000; Abcam) was combined with rabbit polyclonal antibody to CCK8 (1:200; ImmunoStar).

Techniques: Injection, Control, Mutagenesis, Fluorescence, Labeling