Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

Abstract

Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation.

Fig. 1.

BDNF stimulates RyR2, RyR3, and PKMζ mRNA and protein expression. Relative mRNA levels for RyR2 (A), RyR3 (B), and PKMζ (C) were determined by qRT-PCR; protein contents for RyR2 (D), RyR3 (E), and PKMζ (F) were quantified from immunoblots and normalized relative to β-actin. Preincubation for 1 h with 50 μM ryanodine (Rya) or TrkB-Fc (20 nM) prevented BDNF-induced changes. Data (Mean ± SE, n ≥ 3) represent ratios between experimental cultures and controls. Statistical significance was analyzed by one-way ANOVA followed by Newman-Keuls post test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

BDNF enhances the expression of functional RyR proteins. (A) Confocal images of control (I) or BDNF-treated (II) pyramidal neurons immunostained for RyR2 (green); the respective differential interference contrast (DIC) confocal images are shown at right. (B) Confocal images of neurons immunostained for RyR3 (red) and β-tubulin (green). I: Controls; II: Neurons incubated with BDNF (50 ng/mL, 6 h). (Scale bars in A and B, 10 μm.) (C) Representative Ca²⁺ signals (mean ± SE, n = 3–5) elicited by 4-CMC (0.5 mM, added at arrow) displayed by control and BDNF-treated (50 ng/mL, 6 h) neurons, some of which were preincubated with ryanodine (Rya, 50 μM, 1 h).

Fig. 3.

BDNF-induced spine remodeling requires RyR-mediated Ca²⁺ release. (A) (Upper) Time-lapsed confocal images of a neurite, visualized with calcein fluorescence, after BDNF addition (50 ng/mL); spine elongation and formation of a new spine can be appreciated. (Lower) Preincubation with ryanodine (Rya, 50 μM for 6 h) prevented BDNF-induced spine formation. (Scale bars, 2 μm.) (B) Average changes (mean ± SE) in spine length with time; controls (n = 8); exposed to BDNF (n = 10); preincubated with ryanodine (Rya 50 μM, 6 h) before BDNF addition (n = 10). *P < 0.05 and ***P < 0.001, significant differences at a given time between controls and BDNF-incubated cells; ≠, significant differences (P < 0.05) between cells incubated with BDNF and BDNF plus ryanodine. (C) Average changes (mean ± SE) in spine number with time; controls (n = 5), exposed to BDNF (n = 6), preincubated with ryanodine before BDNF addition (n = 5). *P < 0.05. In B and C, results were analyzed by two-way ANOVA plus Newman-Keuls posttest. (D) Representative neurite images obtained from a control culture, after BDNF addition (50 ng/mL, 6 h), after addition of ryanodine (Rya, 50 μM, 1 h) followed by BDNF addition for 6 h, or after incubation with ryanodine (Rya) for 7 h. (Scale bars, 5 μm.) (E) Quantification of spine density (mean ± SE, n = 4). Results were analyzed by one-way ANOVA followed by Newman-Keuls post test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

BDNF injection and spatial memory training increase RyR2, RyR3, and PKMζ mRNA and protein levels. (A) Western blot images and densitometry analysis (n ≥ 3) showing RyR2, RyR3, InsP3R1, PKMζ, BDNF, and β-actin content in rat hippocampal tissues extracted 6 h after bilateral intrahippocampus injection with BDNF (0.25 μg per hemisphere). I, injected with BDNF; C, injected with saline. (B) Levels of RyR1, RyR2, RyR3, and PKMζ mRNA, normalized with β-actin, determined by qRT-PCR analysis of hippocampal tissue extracts (mean ± SE, n ≥ 5) from control or trained rats. (C) Western blots showing RyR2, RyR3, PKMζ, and β-actin content in hippocampal tissue from control (C) or trained (T) rats. All values (mean ± SE; n ≥ 3) were analyzed by Mann-Whitney test. *P < 0.05, **P < 0.01.

Fig. 5.

Hippocampal ryanodine injection improves spatial memory formation. (A) [³H]-Ryanodine (10 nmol per hemisphere) was administered bilaterally intrahippocampus. Radioactivity was determined in different brain regions 24 h later, and ryanodine concentrations (mean ± SE, n = 3) were calculated from tissue weight, assuming a density of 1.0. (Inset) Photomicrograph of a representative coronal brain section showing the injection track ending in the dorsal hippocampus CA1 region; M.O.: Medulla Oblongata. (B) Ryanodine (10 nmol per hemisphere) or sham was injected as above 24 h before training. Escape latency values (mean ± SE) were measured daily during training; *P < 0.05, determined by two-way ANOVA. (C) Spatial acuity, determined at training day 6. (D) Time spent in Quadrant 4 and Zone B respect to shams, measured at day 9 (mean ± SE; n ≥ 6). *P < 0.05, determined by unpaired Student's t test.