(A, B) In situ hybridization of TRPC5-mRNA in the amygdala, hippocampus, somatosensory cortex, and auditory cortex. LA, lateral nucleus of the amygdala; BLA, basolateral nucleus of the amygdala; CE central nucleus of the amygdala; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; AuD, secondary auditory cortex, dorsal; Au1, primary auditory cortex; AuV, secondary auditory cortex, ventral; Ect, ectorhinal cortex; PRh, perirhinal cortex. Scale bar, 1mm. (C), TRPC5 (left) and CaMKIIα (middle, a marker of pyramidal neurons) colocalize in the lateral nucleus of the amygdala. Scale bar, 25 μm. (D) TRPC5 (left, in situ hybridization) and anti-CaMKIIα (middle) co-localize in the majority of pyramidal cells of the auditory cortex. Arrows indicate cells expressing both TRPC5 and CaMKIIα. Scale bar, 50 μm.

(A) Position of the stimulation (S_thalamic, S_cortical) and recording (R) electrodes. EC, external capsule. (B) Responses of LA neurons to current injection (150 pA) recorded in current-clamp mode. (C) Summary plots of the number of spikes in LA neurons evoked by current injection of increasing intensity in slices from control (open symbols) and TRPC5^−/− (filled symbols) mice, recorded as in (B). From 4 wt mice, n=32 neurons, and 5 null mice, n=39 neurons. ANOVA, P=0.75 (D) Synaptic input-output curves obtained in the cortical input to the LA. Cortico-amygdala EPSCs were recorded under voltage-clamp conditions (V_H=−70 mV). EPSC amplitude is plotted vs. stimulation intensity (wt mice, n=14 neurons; null mice, n = 10 neurons. ANOVA, P=0.7 (E) Action potential-EPSP pairing-induced LTP of the cortico-amygdala EPSP recorded in the LA neuron in slices. Insets show the average of 10 EPSPs recorded before, and 35min after, the induction (arrow). (F) Summary of LTP experiments at cortico-amygdala synapses (from 7 wt mice, n=20 neurons, and 7 null mice, n=19 neurons) t test, P=0.8. (G) Synaptic input-output curves obtained in the thalamic input to the LA (from 4 wt mice, n=22 neurons, and 5 null mice, n=24 neurons; ANOVA, P=0.8. (H) LTP of the thalamo-amygdala EPSP recorded in the LA neuron. Insets show the average of 10 EPSPs recorded before, and 35min after, the induction (arrow). (I) Summary of LTP experiments at the thalamo-amygdala synapses (from 5 wt mice, n=8 neurons, and 5 null mice, n=10 neurons; t test, P=0.87. Error bars indicate SEM.

(A) Cortico-amygdala synaptic responses in brain slices from a control mouse evoked by trains of high frequency stimulation before and after addition of CNQX (AMPAR blocker; 20μM), NMDAR blockers D-APV (50μM) and MK-801 (10μm) and CGP 35348 (GABA_BR blocker; 300μM). Stimulation trains consisted of 10 pulses at 100 Hz delivered once every 30s. Inset shows synaptic responses recorded in current-clamp mode before (1) and after (2) the addition of blockers to the external solution. The dashed line indicates the point where the EPSP amplitude was measured. (B) Experiment as in A), from a TRPC5^−/− mouse. (C) Summary plot of the experiments shown in A) and B), performed in cortical and thalamic inputs to the LA. The amplitude of the residual component of the EPSP (measured at dashed line in A, B) in the presence of blockers was significantly smaller in both pathways in slices from TRPC5^−/− mice (cortical input: from 7 wt mice, n=12 neurons, and 4 null mice, n=7 neurons. thalamic input: from 4 wt mice, n=8 neurons, and 5 null mice, n=11 neurons). (D, E) Short-train stimulation-induced cortico-amygdala EPSCs recorded at V_H ranging from −100 mV to +40 mV in slices from control (D) and TRPC5^−/− (E) mice in the presence CNQX, D-APV, MK-801, CGP 35348 and picrotoxin (100μM). (F) Summary current-voltage (I/V) plots of the peak current in the cortical input (as in D and E), as well as from control mice in the presence of MPEP (10μM, filled black symbols). From 7 wt mice, n=11 neurons, and 3 null mice, n=7 neurons; from 4 wt mice in the presence of MPEP, n=4 neurons. (G) Short-train stimulation-induced cortico-amygdala EPSCs (V_H = +40 mV) are sensitive to the mGluR5 antagonist MPEP (10 μM). The inset shows the time course of MPEP block in slices from control mice. (H) A specific antagonist of mGluR1, CPCCOEt (40μM), also reduced the size of the slow EPSC. (I) Summary plot of the effects of MPEP (10μM, n = 4 cells from 3 wt mice, P < 0.01 versus baseline) and CPCCOEt (40μM, n=5 cells from 3 wt mice, P<0.01 versus baseline), and MPEP and CPCCOEt applied simultaneously (n=10 neurons from 7 wt mice); percent of EPSC reduction induced by antagonists. P=0.34 for the effect of MPEP versus MPEP+CPCCOEt; P=0.16 for the effect of CPCCOEt versus MPEP. Error bars indicate SEM.

(A) Membrane currents induced in LA neurons (3.5s ramps from −100 to +40mV) under baseline conditions (black line), and after CCK4 agonism of CCK2 receptors (3μM, green line) in a control mouse. External solution also contained 0.5μM TTX, 200μM CdCl₂, and 50μM picrotoxin. ([Ca2+]_i was buffered to 100nM; see Methods). (B) CCK4-induced current at V_H=−70mV, with voltage ramps applied every 60s; 66.9±12.9pA, n=9 cells from 4 wt mice. (C) 2-APB (100μM) abrogated CC4-induced membrane currents in LA neurons (2.5±1.4pA, n=4 cells from 3 mice, significantly different from CCK4 effects without the blocker, t-test, P =0.0079). (D) CCK4-induced currents in slices from wt (left) and TRPC5^−/− (right) mice. (E) Baseline-subtracted CCK4-mediated currents in LA neurons during ramps from −100 to +40mV (from 4 wt mice; n=9 neurons, and 6 null mice; n=8 neurons). (F) Averaged amplitudes of the CCK4-induced currents at −70 mV (from 4 wt mice, n=9 neurons, and 6 null mice, n=8 neurons, P<0.05). (G) Spikes evoked in LA neurons by current injection (150pA) recorded in current-clamp mode under baseline conditions and in the presence of 3μM CCK4. (H) CCK4-induced depolarization in LA neurons (from 2 wt mice, n=8 neurons, and 3 null mice, n=14 neurons). P=0.005 for depolarization in control mice versus depolarization in TRPC5^−/− mice (I) Summary plot of the experiments as in (G), showing the percent increase in spike frequency in the presence of CCK4 relative to the baseline frequency (taken as 100%; n=8 neurons from 2 wt mice and n=14 neurons from 3 TRPC5 null mice; t test, P=0.004). Error bars indicate SEM.

(A) The acoustic startle response to auditory stimuli at 95, 100 and 105dB in control (n=10; white) and null (n=9; red) mice. ANOVA, F(1,17)=2.9, P=0.1. (B) Activity levels in control and null mice sampled immediately preceding the onset of the startle stimuli for the experiments shown in (A). (C) Locomotor activity of TRPC5^−/− and wt mice were indistinguishable (8 mice per group; P=0.7). (D) Conditioned ‘freezing’ following single-trial fear conditioning in control mice (n=10) and TRPC5^−/− mice (n=10) at 30min (ANOVA, P=0.054) and 24h (ANOVA, F(1,18) = 0.02, P=0.9) post-training. (E) Elevated plus-maze experiments: TRPC5^−/− mice entered the open arm of the maze more commonly (10 mice per group; open arms: t₍₁₈₎=2.75, P<0.05; closed arms t₍₁₈₎=1.44, P=0.17, not shown). (F–H) In open field experiments, TRPC5^−/− mice spent more time in the center of the arena [F(1,14)=5.16, P=0.04] (F) and (G) entered it more frequently [F(1,14)=5.6, P=0.03]; ≤5 min versus >5 min [F(1,14)=7.0, P=0.02]. Total path length did not differ between groups (H) Total path lengths; [F(1,14)=2.7, P=0.1]; 8 mice per group). (I, J) Social interaction test; (I) Duration of time spent by mice of both genotypes in each of the 3 areas of the testing apparatus during the preference for social novelty phase (7 mice per group). Mutant mice spent more time with the novel mouse (t test, t=2.2, P=0.04); (J) Average difference between the number of nose contacts with the novel and familiar mouse. Error bars indicate SEM.

(A) Progressive block by MK-801 (40μM) of the NMDA receptor EPSC recorded in cortical input to the LA in the presence of CNQX (20μM; V_H=−40 mV). MK-801 was applied to the slice in the absence of presynaptic stimulation for >10min and the external capsule was then stimulated at 0.1Hz to measure the rate of MK-801 block. Inset shows the baseline NMDAR EPSC (1) and its block at the end of presynaptic stimulation in the presence of MK-801 (2). (B) Summary graphs of the experiments with MK-801 protocol in cortical input (as in A). In each individual experiment, EPSC amplitudes were normalized by the first EPSC (from 3 wt mice, n=7 neurons and 4 TRPC5 null mice, n=10 neurons; ANOVA, P=0.9). (C) Representative traces of the asynchronous quantal EPSCs evoked by stimulation (at arrow) of the cortical input (V_H= −70 mV). In these experiments, Sr2+ was substituted for extracellular Ca²⁺. (D) Cumulative amplitude histograms of asynchronous quantal events recorded in the cortical input to the LA (from 3 wt mice, n=11 neurons and 4 TRPC5 null mice, n=17 neurons). (E) Progressive block by MK-801 (40μM) of the NMDA receptor EPSC recorded in the thalamic input to the LA at V_H=−40 mV (experimental conditions as in A). Inset shows the baseline NMDAR EPSC (1) and its block at the end of presynaptic stimulation in the presence of MK-801 (2). (F) Summary graphs of the experiments with MK-801 protocol in thalamic input (from 3 wt mice, n=7 neurons and 4 TRPC5 null mice, n=8 neurons; ANOVA, P=0.9). (G) Representative traces of the asynchronous quantal EPSCs evoked in the thalamic input at V_H =−70 mV. (H) Cumulative amplitude histograms of asynchronous quantal events recorded in the thalamic input to the LA (from 3 wt mice, n=11 neurons and 4 TRPC5 null mice, n=16 neurons). Error bars indicate SEM.

(A) Superimposed postsynaptic responses (all-or-none extracellular spikes) evoked in a LA neuron by stimulation of the cortical input and recorded in a cell-attached patch configuration (as in Otmakhov et al., 1993). The recordings were performed in the presence of 50μM PTX and 2μM CGP55845. (B) Recordings under current clamp conditions at −70 mV from the same neuron as in A) after establishing a whole-cell recording configuration. (C) Examples of responses in the LA neuron (recorded in a cell-attached patch configuration) to stimulation pulses of increasing intensity, delivered to the cortical input in a slice from a control mouse. The intensity of stimulation was increased from the threshold stimulus required to elicit the spike, determined in each individual experiment, with an increment of 25μA. (D) Identical experiment as in C) but in a slice from a TRPC5^−/− mouse. (E, F) The number of extracellular spikes evoked in LA neurons by presynaptic pulses of increasing intensity in cortical (E: from 4 wt mice, n=17 neurons and 4 TRPC5 null mice, n=12 neurons; ANOVA, P=0.02) and thalamic (F: from 4 wt mice, n=16 neurons and 4 TRPC5 null mice, n=15 neurons; ANOVA, P=0.02) pathways. First points represent responses evoked by the stimuli at the threshold +25 μA. Error bars indicate SEM.