Homeostatic switch in hebbian plasticity and fear learning after sustained loss of Cav1.2 calcium channels

Abstract

Ca(2+) influx through postsynaptic Ca(v)1.x L-type voltage-gated channels (LTCCs) is particularly effective in activating neuronal biochemical signaling pathways that might be involved in Hebbian synaptic plasticity (i.e., long-term potentiation and depression) and learning and memory. Here, we demonstrate that Ca(v)1.2 is the functionally relevant LTCC isoform in the thalamus-amygdala pathway of mice. We further show that acute pharmacological block of LTCCs abolishes Hebbian plasticity in the thalamus-amygdala pathway and impairs the acquisition of conditioned fear. On the other hand, chronic genetic loss of Ca(v)1.2 triggers a homeostatic change of the synapse, leading to a fundamental alteration of the mechanism of Hebbian plasticity by synaptic incorporation of Ca(2+)-permeable, GluA2-lacking AMPA receptors. Our results demonstrate for the first time the importance of the Ca(v)1.2 LTCC subtype in synaptic plasticity and fear memory acquisition.

Figure 1.

Hebbian LTP in the thalamo-amygdala pathway is LTCC dependent. a, Expression of Cav1.x mRNA in the mouse brain. Frontal sections (panels 1 and 3) and sagittal sections (panels 2 and 4) were labeled with a Ca_v1.3-specific (panels 1 and 2) and Ca_v1.2-specific (panels 3 and 4) probes, as shown. CTX, Cerebral cortex; HIP, hippocampus; THL, thalamus; CRB, cerebellum; OB, olfactory bulb. b, Response of a LA cell to current injection of −20 to 50 pA. Neural spiking thresholds, resting potentials, spiking latencies, frequency adaptations, and input resistances for each pharmacological or genetic alteration were derived from these traces and are shown in supplemental Table S1, available at www.jneurosci.org as supplemental material. c, Schematic drawing of stimulating and recording electrode placement. Afferent fibers from the thalamus enter the LA medially (red arrow). CE, Central nucleus of the amygdala; ec, external capsule; oc, optic tract. d, The LTCC blocker isradipine (10 μm; red) or the NR2B-blocker ifenprodil (10 μm, green) abolished the induction of Hebbian LTP (control, black) at thalamic inputs. Time course of normalized EPSP slope over 30 min after Hebbian LTP induction. Inset: Traces from individual experiments before and 30 min after induction are shown. Scale bars: Vertical, 5 mV; horizontal, 50 ms. e, Normalized EPSP slope levels averaged over 30 min after LTP induction. *p < 0.05 relative to vehicle controls; #, no significant difference to baseline. Ctr, Vehicle control, Ifen, ifenprodil, Isra, isradipine. Sample sizes are indicated inside the bars.

Figure 2.

Morphological, biochemical, and cellular electrophysiological characterization of brain-specific Ca_v1.2^NesCre mice. a, Schematic representation of the wild type (WT), knock-out (L1), and conditional CACNA1C alleles (L2). The numbers indicate the exon number. Brain-specific activation of Cre recombinase (Nestin-Cre) results in the deletion of CACNA1C exons 14 and 15. Restriction sites are as follows: A, Acc65I; B, BamHI; C, ClaI; EI, EcoRI; Red triangles, loxP sites. b, Golgi-Cox stain demonstrating unchanged dendritic length, branching, and spine density of Ca_v1.2^NesCre neurons. Ctr, Control; KO, knock-out. c, Analysis of dendritic length and spine number. Panels 1 and 2, Sholl analysis was used to investigate the total dendritic length of hippocampal pyramidal neurons and revealed no differences between the litter-matched controls (Ctr) and Ca_v1.2^NesCre (KO) groups (n = 10). Panels 3 and 4, Analysis of dendrite order and spine number. No differences were found between the two groups in terms of dendritic order (data not shown) or spine number (n = 10). d, e, Western blot analysis of proteins from mouse brain (d) or amygdala (e) punches using an anti-Ca_v1.2 antibody demonstrates lack of Ca_v1.2 protein. ERK1/2 was used as loading control. CTX, Cerebral cortex; AMY, amygdale; HIP, hippocampus; CRB, cerebellum; OB, olfactory bulb. kD, kilodalton. f, Isradipine (20 μm, solid bar) blocks the DHP-sensitive component of barium currents in Ctr pyramidal neurons of the LA (black; n = 5) but has no effect in Ca_v1.2^NesCre neurons (red; n = 5). *p < 0.05 relative to baseline.

Figure 3.

Acute, but not sustained loss of Ca²⁺ signaling via Ca_v1.2 impairs acquisition of fear memories. a, b, Auditory-cued fear memory 24 h after conditioning in control mice given intracerebroventricular bolus infusions (2 μl, arrows) of vehicle of isradipine 30 min before (a) or right after conditioning (b). *p < 0.0005 relative to vehicle controls. c, Auditory-cued fear memory 24 after conditioning in litter-matched Ctr and Ca_v1.2^NesCre mice. Sample sizes are indicated inside the bars. US, Unconditioned stimulus.

Figure 4.

Lack of DHP effect on Hebbian LTP in the thalamo-amygdala pathway in Ca_v1.2^NesCre mice. a, Time course of normalized EPSP slope over 30 min after Hebbian LTP induction in Ctr (black) and Ca_v1.2^NesCre (red) LA slices. b, Isradipine (10 μm) abolished the induction of Hebbian LTP at thalamic inputs in control (black) but not in Ca_v1.2^NesCre (red) LA slices. Insets (a, b): Traces from individual experiments before and 30 min after induction are shown. Scale bars: Vertical, 5 mV; horizontal, 50 ms. c, Normalized EPSP slope levels averaged over 30 min after LTP induction. Control (Ctr) (black), Ctr plus 10 μm isradipine (Isra) (gray), knockout (KO) (Ca_v1.2^NesCre, red), KO plus 10 μm Isra (pink), *p < 0.05 relative to control slices; #, no significant difference to baseline. n.s., no significant difference between KO and KO plus Isra groups. Sample sizes are indicated inside the bars.

Figure 5.

A switch of the mechanism of Hebbian LTP to calcium-permeable AMPA receptors homeostatically compensates for the loss of CACNA1C in Ca_v1.2^NesCre mice. a, Time course of normalized EPSP slope over 30 min after Hebbian LTP induction in Ca_v1.2^NesCre (red) and philanthotoxin-treated Ca_v1.2^NesCre (10 μm, blue) LA slices. Inset: Traces from individual experiments before and 30 min after induction are shown. Scale bars: Vertical, 5 mV; horizontal 50 ms. b, The specific blocker of cp-AMPARs PhTX-433 (10 μm) abolished (blue) the induction of Hebbian LTP at thalamic inputs in Ca_v1.2^NesCre slices (red) but had no effect on control (Ctr) (black, gray) LA slices. *p < 0.05 relative to Ca_v1.2^NesCre (KO, knockout) slices; #, no significant difference to baseline. c, Loss of Ca_v1.2 drives GluA1 into synapses. Averaged examples of evoked AMPA receptor-mediated synaptic responses recorded at −70 mV and +40 mV from control LA pyramidal neurons (black) and Ca_v1.2^NesCre cells (red). d, Average rectification index values (I_{−70 mV}/I_{+40 mV}) for control and Ca_v1.2^NesCre neurons. (*p < 0.05, t test). e, Left: Representative Western blots of GluA1 and GluA2 in control and Ca_v1.2^NesCre amygdala punches (top). ERK1/2 was used as a loading control. Right: Quantitative analysis of Western blot signal intensities (normalized to controls) shows that GluA1 levels were increased in Ca_v1.2^NesCre mice (*p < 0.001, t test), while GluA2 levels were unchanged. Sample sizes are indicated inside the bars.