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Link to original content: http://pubmed.ncbi.nlm.nih.gov/24155300/
NMDA receptor activation and calpain contribute to disruption of dendritic spines by the stress neuropeptide CRH - PubMed Skip to main page content
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. 2013 Oct 23;33(43):16945-60.
doi: 10.1523/JNEUROSCI.1445-13.2013.

NMDA receptor activation and calpain contribute to disruption of dendritic spines by the stress neuropeptide CRH

Adrienne L Andres  1 , Limor Regev, Lucas Phi, Ronald R Seese, Yuncai Chen, Christine M Gall, Tallie Z Baram
Affiliations

NMDA receptor activation and calpain contribute to disruption of dendritic spines by the stress neuropeptide CRH

Adrienne L Andres et al. J Neurosci. .

Abstract

The complex effects of stress on learning and memory are mediated, in part, by stress-induced changes in the composition and structure of excitatory synapses. In the hippocampus, the effects of stress involve several factors including glucocorticoids and the stress-released neuropeptide corticotropin-releasing hormone (CRH), which influence the integrity of dendritic spines and the structure and function of the excitatory synapses they carry. CRH, at nanomolar, presumed-stress levels, rapidly abolishes short-term synaptic plasticity and destroys dendritic spines, yet the mechanisms for these effects are not fully understood. Here we tested the hypothesis that glutamate receptor-mediated processes, which shape synaptic structure and function, are engaged by CRH and contribute to spine destabilization. In cultured rat hippocampal neurons, CRH application reduced dendritic spine density in a time- and dose-dependent manner, and this action depended on the CRH receptor type 1. CRH-mediated spine loss required network activity and the activation of NMDA, but not of AMPA receptors; indeed GluR1-containing dendritic spines were resistant to CRH. Downstream of NMDA receptors, the calcium-dependent enzyme, calpain, was recruited, resulting in the breakdown of spine actin-interacting proteins including spectrin. Pharmacological approaches demonstrated that calpain recruitment contributed critically to CRH-induced spine loss. In conclusion, the stress hormone CRH co-opts mechanisms that contribute to the plasticity and integrity of excitatory synapses, leading to selective loss of dendritic spines. This spine loss might function as an adaptive mechanism preventing the consequences of adverse memories associated with severe stress.

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Figures

Figure 1.
Figure 1.
The stress neuropeptide CRH causes a loss of PSD95-ir puncta and dendritic spines in cultured rat hippocampal neurons. Exposure to 100 nm CRH leads to a significant reduction in PSD95-ir puncta, an indication of dendritic spine loss. A, B, Control neuron and (B) neuron exposed to 100 nm CRH at 36°C for 30 min and processed for ICC for PSD95 (green) and for F-actin (red). C, Confocal images were used to quantify PSD95-ir puncta and dendritic spines from GFP-expressing neurons. Neurons used for quantification were clearly demarcated and devoid of dendritic crossings from other neurons that could confound counts. D, Example of a confocal image processed for quantification with 20 μm segments measured out from the soma. E, Exposure to CRH reduced the density of PSD95-ir puncta along dendritic branches (F(1,66) = 3.81, p = 0.006), and this effect became more apparent in third-order (p = 0.004) and fourth-order (p < 0.001) branches (n = 12). Similar results were obtained by quantifying PSD95 by distance from the soma. F, Graph quantifying PSD95-ir puncta per 20 μm segment in cultures incubated in the presence or absence of CRH (F(1,88) = 7.09, p = 0.014; n = 12). G, CRH reduced PSD95 puncta in a time-dependent manner (F(3,80) = 17.66, p < 0.001; n = 6). H, Lentiviral infection of neurons did not change the size or shape of the soma, and enabled direct visualization of spines. I, J, An example of a control GFP-filled dendrite and (J) a dendrite after exposure to 100 nm CRH at 36°C for 60 min. K, Graph quantifying GFP-filled spines per 20 μm segment with and without exposure to CRH (F(1,48) = 7.55, p = 0.017; n = 12). The values for spine density were approximately half of those found using PSD95-ir puncta because the GFP analysis is limited to spines perpendicular to the dendrite. Scale bars: A–D, H, 20 μm; I, J, 5 μm.
Figure 2.
Figure 2.
CRH-induced spine loss involves CRHR1. Cultured hippocampal neurons express CRHR1. Nonpermeable and permeabilized ICC conditions discriminate surface versus internal pools of CRHR1. A, Surface CRHR1 (green) was immunolabeled under nonpermeable conditions. After permeabilization, the neuron was processed for ICC for internal CRHR1 (red). The dashed lines approximate the contour of the dendrite. Green arrows point to surface CRHR1, the majority of which are located away from the dendrite and likely on dendritic spines. Red arrows point to external CRHR1 away from the dendritic shaft that colocalize with internal pools of CRHR1. These colocalized puncta are likely spines carrying the external receptor within the postsynaptic density as well as internal pools of CRHR1. B, Graph depicting that the CRHR1-selective antagonist (NBI30775; 100 nm) prevents CRH-induced loss of PSD95 puncta (p > 0.05; n = 12). C, D, CRH-induced spine loss requires neuronal activity. In the presence of TTX, CRH no longer reduces PSD95 puncta (F(3,368) = 20.31, p < 0.001; n = 12; C) or GFP-filled spines (F(3,176) = 6.29, p = 0.001; n = 12; D). Scale bars: A, C, D, 5 μm.
Figure 3.
Figure 3.
CRHR1 colocalizes with ionotropic glutamate receptors on dendritic spines. A, GFP-expressing neurons were immunostained for CRHR1 (red) and the GluR1 subunit of AMPA receptors (blue). White arrows point to spines that contain both CRHR1 and GluR1, red arrows point to spines that have CRHR1 without GluR1, and blue arrows point to spines that have GluR1 but lack CRHR1. B, GFP-expressing neurons immunostained for CRHR1 (red) and the NR2A subunit of NMDA receptors (blue). White arrows point to spines that contain both CRHR1 and NR2A. Scale bar, 5 μm.
Figure 4.
Figure 4.
CRH selectively eliminates GluR1-lacking dendritic spines. A, Neurons were exposed to CRH or a control medium for 30 min. ICC for surface GluR1 was performed under nonpermeabilized conditions (see Materials and Methods). In the control condition, ∼40% of PSD95-ir puncta (red) colocalized with sGluR1 (green). CRH reduced the number of PSD95-ir puncta, and of those remaining, the majority colocalized with surface GluR1. These findings indicate that GluR1-negative spines are more vulnerable to CRH. B, Graph showing the effects of CRH on sGluR1-positive PSD95-ir puncta (dotted lines; F(1,88) = 1.43, p = 0.244; n = 12) compared with total, PSD95-ir puncta (solid lines). These differed among groups (F(1,88) = 20.11, p = 0.0002; n = 12): CRH led to a marked reduction in total PSD95-ir puncta, but did not influence significantly the density of dual-labeled puncta. C, Upon exposure to CRH, the ratio of sGluR1-positive PSD95-ir puncta over the total PSD95-ir puncta was increased throughout the dendrite (F(1,88) = 35.27, p < 0.0001; n = 12), supporting the preferential loss of sGluR1-lacking dendritic spines. Scale bars: A, 5 μm.
Figure 5.
Figure 5.
CRH-induced spine loss requires NMDA receptor activation, but not AMPA receptor activation. To distinguish the roles of NMDA- and AMPA-type receptors, neurons were exposed to CRH in the presence of selective blockers, APV and CNQX, respectively. A, Example of dendrites exposed to CRH in the presence of APV + CNQX. The combined antagonists prevented CRH-induced spine loss. B, Graph quantifying CRH spine loss in the presence of both glutamate receptor antagonists (F(2,180) = 64.22, p < 0.0001; n = 12). C, Exposing neurons to CRH in the presence of APV or of CNQX revealed that NMDA receptor activation was required for CRH-induced spine loss. D, PSD95 quantification demonstrates that the AMPA receptor blocker CNQX (purple) partially ameliorated the effects of CRH on PSD95-ir puncta (F(2,180) = 38.79, p < 0.0001; post hoc CRH vs CNQX + CRH p > 0.05 at all distance points; n = 12), but did not protect from the effects of CRH on GFP-filled spines (F(2,240) = 43.05, p < 0.0001 n = 12). E, In contrast, NMDA receptor blockade abolished CRH-induced reduction in PSD95 puncta (F(2,180) = 62.78, p < 0.0001; n = 12) and GFP-filled spines (F(2,240) = 34.65, p < 0.0001; n = 12). Scale bars: A, C, 5 μm.
Figure 6.
Figure 6.
Exposure to CRH increases NMDA receptor-dependent calpain activity. A, Cultured hippocampal neurons express calpain throughout the soma and dendrites. The punctate appearance of calpain-1 is consistent with its presence in dendritic spines. B, To distinguish CRH-induced calpain activation from constitutively active calpain, all of the groups were exposed to 500 nm calpain inhibitor III for 3 h before the onset of the experiment. The amount of SBDP (140 kDa) increased with exposure to CRH (pink column). The increase in the SBDP was abolished by calpain inhibitor III (purple and green columns). C, Graph showing optical density analysis of the ratio of SBDP/full-length spectrin for each treatment group (F(4,13) = 5.592, p = 0.001) and of the ratio of the SBDP/actin loading control (F(4,13) = 9.356, p = 0.001); results were from two to four experiments. D, Representative gel, showing that the NMDA receptor blocker, APV, prevented CRH-induced increase in calpain activation. E, Quantitative graph derived from two experiments. Optical densities of SBDP/full-length spectrin and of SBDP/actin loading control were increased by CRH, and this effect was blocked by APV. Scale bar, A, 7 μm.
Figure 7.
Figure 7.
The calpain inhibitor prevents dendritic spine loss induced by CRH. A, The 100 nm calpain inhibitor prevented CRH-induced reduction of PSD95-ir puncta. B, The calpain inhibitor abolished the loss of GFP-filled spines. C, Graph showing quantification of PSD95-ir puncta (F(5,264) = 37.21, p < 0.0001; n = 12) and GFP-filled spines (F(5,264) = 65.56, p < 0.0001; n = 12). Scale bars: A, B, 5 μm.
Figure 8.
Figure 8.
Schematic of the proposed molecular signaling involved in CRH-induced dendritic spine loss. The CRH receptor, CRHR1, is located on dendritic spine heads, within the postsynaptic density and in close proximity to NMDA- and AMPA-type ionotropic glutamate receptors. When CRH (released during stress from hippocampal interneurons), binds CRHR1 in the presence of network activity, this triggers an NMDA receptor-dependent signaling cascade that culminates in spine loss. Specifically, the influx of calcium ions through NMDA receptors activates calpain. Calpain cleaves actin-associated scaffolding proteins, such as spectrin, leading to the breakdown of the spine cytoskeleton and spine loss. The presence of GluR1 may protect subsets of mature spines from the actions of CRH.
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