Article Figures & Data
Figures
- Figure 1.
MAS impairs hippocampus-dependent spatial memory. A, During an object location test, mice exposed to simultaneous stresses lasting 60–90 min failed to spend more time exploring the novel location compared with the familiar, as indicated by the discrimination ratio (novel vs familiar; n = 5 mice/group; t(12) = 3.23, *p < 0.01). B, Plasma CORT levels at the time of the training session, 2 h after the end of the stresses, were comparable between controls and stressed mice (n = 4 mice/group, p = 0.15). C, Comparable exploration durations between control and stressed mice during the training (10 min) and testing (5 min) phases of a novel location task (n = 8 mice/group, p > 0.05). D–F, Memory deficits provoked by multiple acute stresses persisted 1 d and 1 week later. Another cohort of stressed mice was allowed to recover for 24 h or 1 week, which was followed by training (10 min) and testing (5 min). Controls explored the object in a novel location more than the object left in the familiar location, whereas stressed mice trained 1 d or 1 week after the MAS and tested 6 h later spent almost equal time exploring either (D) (main effect of stress, F(1, 28) = 5.86, *p < 0.05). Exploration times during the training and testing phases did not distinguish between the controls and stressed animals that recovered for 24 h (E) or 1 week (F) (n = 8 mice per group; p > 0.05).
- Figure 2.
Multiple concurrent acute stresses impair LTP at area CA1 Schaffer–commissural synapses. A, Graph comparing the fEPSP and fiber-volley amplitudes in response to increasing intense stimulation in slices prepared from control (brown) and stressed (green) animals. Note that no difference was detected in slices prepared from the two groups (n = 5–6 mice per group). B, Representative fEPSPs recorded from slices from control (brown) and stressed (green) mice in response to increasing stimulation intensity. Scale bars: y = 1 mV, x = 20 ms. C, Graph illustrating PPF associated with different interpulse intervals in control (brown) and stressed (green) mouse slices demonstrates no group differences. D, Bar graph summarizing the effect of burst number on response size in control (brown) and stressed (green) mouse slices. E, LTP after delivery of TBS was attenuated in slices from stressed (green) mice compared with controls (brown) within 10 min of induction. Representative traces from baseline (black lines) and 30 min after induction (colored lines) are shown. All slices in the stress group were prepared from animals <1 h after the concurrent acute stresses (*p < 0.01 for minutes 30–80). Results are shown as means ± SEM. Values in parentheses are the numbers of slices from n = 5–6 mice.
- Figure 3.
Concurrent acute stresses (MAS) reduce the density of thin dendritic spines and increase the size of mushroom-type spines. A, Representative CA1 apical dendritic segments from stress-free controls versus mice experiencing MAS. Arrows indicate thin spines; arrowheads, mushroom-type spines; note augmented size of spine heads with stress. Scale bar, 5 μm. B, With stress, spine density is reduced on apical dendritic trees in CA1 stratum radiatum, corresponding to the Schaffer–commissural terminal field. (t(57) = 3.78, *p < 0.01) and this is primarily a result of reduced numbers of thin spines in stressed versus control mice (t(57) = 3.15, *p < 0.01). C, Mean diameter of mushroom-type spine heads is larger in stressed versus control mice (t(8) = 5.21, *p < 0.01) and the size–frequency distribution of mushroom-type spines is “shifted to the right” in stressed mice. D, Representative wide-field microscopy followed by FDT demonstrates fewer and larger PSD-95-positive synapses in stressed mice. Scale bar, 10 μm. E, Reduced PSD-95 positive synapse density (t(58) = 1.92, *p = 0.05). F, Shift in the size–frequency distributions of PSD-95 puncta are apparent using the independent method (n = 20 slices and 5 mice per group). The number of large synapses is augmented in MAS mice. (t(29) = 2.30, *p < 0.05).
- Figure 4.
Coordinate actions of CORT and CRH on synaptic physiology recapitulate the effects of multiple concurrent acute stresses. CRH infusion (100 nm) into hippocampal slices depressed the size (slope) of EPSPs but only after a delay of 1 h (green). CORT (100 nm) had no detectable effect on synaptic responses (blue). Infusion of CORT+CRH resulted in rapid, profound depression of synaptic responses (orange). The difference between the three groups at 50–55 min of the infusion was highly significant (p < 0.01, one-way ANOVA), with the combination treatment distinct from either of the CORT or CRH alone groups. The response size versus time of infusion interaction for the three groups from minutes 10 through 40 was significantly different (p < 0.01, two-way ANOVA; n = 4 animals per group, 2–3 slices per animal).
- Figure 5.
Concerted actions of CORT and CRH on dendritic spines recapitulate the effects of multiple acute stresses. A, Combined CORT+CRH at levels presumed to obtain in hippocampus during stress (Chen et al., 2012; Maras and Baram, 2012) recapitulate the effects of concurrent short stresses on dendritic spine integrity in hippocampal CA1. CORT has little effect on thin spines (arrows) and tends to increase the size of mushroom-type spines (arrowheads). CRH reduces the numbers of thin spines, whereas the combined application of both hormones diminishes thin spine density and promotes aberrantly large mushroom-type spine heads (empty arrowheads). Scale bar, 6 μm. B, Reduced density of thin spines on apical dendrites of CA1 pyramidal neurons exposed to both hormones (repeated-measures one-way ANOVA, F(3, 39) = 24.87, p < 0.0001; post hoc, p < 0.05). C, Exposure to CORT+CRH promotes a shift in the size–frequency distributions of YFP-labeled mushroom-type spines in CA1 (repeated-measures one-way ANOVA, F(3, 33) = 25.28, p < 0.0001; post hoc, p < 0.01). D, An independent method (wide-field microscopy and FDT) demonstrates reduction of PSD-95-positive synapses by combined application of CORT+CRH. Scale bar, 8 μm. E, Combined application of CORT+CRH reduced total synapse number compared with vehicle and with each hormone applied alone (one-way ANOVA, F(3, 116) = 54.03, p < 0.0001; Bonferroni's post hoc test, *p < 0.01). The number of PSD-95 synapses was reduced by CRH alone compared with vehicle (*p < 0.05). F, CORT+CRH exposure reduced the number of small (0.15 μm3) synapses (one-way ANOVA, F(3, 60) = 14.66; p < 0.0001; post hoc, p < 0.001), increased the proportion of larger (>0.30 μm3) synapses (one-way ANOVA, F(3, 196) = 30.32, p < 0.0001; post hoc, p < 0.01), and shifted the size–frequency distribution of PSD-95-positive synapses toward larger diameters.
- Figure 6.
CORT and CRH effects converge on mechanisms regulating spine stability and specifically on the actin-regulating RhoA. A, Putative convergent mechanisms by which CORT and CRH influence spine integrity. CORT, via GR, prevents activation of RhoA in dendritic spine heads (Jafari et al., 2012). Activation of CRFR1 by CRH engages calpain in the spine through an NMDA-mediated mechanism (Andres et al., 2013), which destroys the Rho-like GTPase RhoA. This leads to pCofilin dephosphorylation, generating the active cofilin, which can cleave F-actin. Together, these hormone actions promote depolymerization of F-actin and spine collapse. B, The ratio of RhoA-positive+PSD-95-positive puncta to those immunolabeled for PSD-95 alone was reduced by coadministration of CORT and CRH (one-way ANOVA, F(3, 13) = 7.45, p < 0.005; Newman–Keuls multiple-comparisons test, p < 0.01), as well as by CRH alone, but not by CORT alone (post hoc, p < 0.01), as predicted by the model (A). C, In contrast, active RhoA at synapses, measured as a ratio of dually labeled active RhoA+PSD-95/single label PSD-95 puncta was reduced significantly only when both hormones acted in concert (one-way ANOVA, F(3, 31) = 5.27, p < 0.005; post hoc, p < 0.05). D, Representative raw images for colocalizion of total RhoA/PSD-95 (left) and active RhoA/PSD-95(right). PSD-95 is labeled in green and RhoA, active or total, is labeled in red. Groups are arrayed vertically: control, CORT, CRH, or both. An effect of treatment group on the proportion of RhoA-labeled PSD-95 puncta (appearing yellow) within the whole PSD-95-positive population is apparent and is quantified in B and C. CRH at 100 nm, CORT at 100 nm, and CORT+CRH at 100 nm each were applied for 15–30 min. Scale bars, 5 μm.
- Figure 7.
Blocking the actions of both CORT and CRH, but not each individually, abrogates memory impairments induced by acute concurrent stresses (MAS). A, Stressed mice explored the object in the novel location not more than expected by chance (one-sample t test, t(7) = 1.161, p = 0.238) and less than controls (F(1, 21) = 8.94; p < 0.01). The GR blocker RU38486 had little effect (F(1, 21) = 0.17, p = 0.69) and object exploration did not differ from chance (t(3) = 1.25, p = 0.303). B, After selective blocking of CRFR1 receptors using NBI 30775, stressed mice explored the object in the novel location less than controls (F(1, 16) = 6.86, p < 0.05) and the antagonist had little effect (F(1, 16) = 0.48, p = 0.50). C, Blocking both GR and CRFR1 by a mixture of RU38486+NBI 30775 prevented stress-induced deficits in spatial memory (stress × drug interaction, F(1, 15) = 6.22, p < 0.05; post hoc, p < 0.05, for MAS + vehicle < MAS + both antagonists). Stressed mice explored the object in the novel location less than controls, yet mixture-treated stressed mice spent significantly more time exploring the novel-located object than chance (one-sample t test, t(4) = 12.61, p < 0.01). In all cases, mice were trained after they recovered from the stress and receptor blockers were administered centrally before the stresses. Asterisks in A–C denote values that are different from those expected by chance. D, Blocking brain GR, CRFR1, or both did not interfere with activation of the hypothalamic–pituitary adrenal axis, as apparent from robust increase of plasma levels of CORT in all MAS groups (one-way ANOVA, F(4, 19) = 38.02, p < 0.001; post hoc, p < 0.0001, for control vehicle being lower than all stress groups; post hoc tests did not distinguish the stress vehicle from the stress antagonist groups). CORT levels were measured at 1 h from the onset of the ongoing stresses (Maras et al., 2014; n = 3–6 per group; *p < 0.05 vs stress groups).
- Figure 8.
The total duration of exploring the novel and familiar objects are comparable in stress-free and stressed mice with/without antagonist-treatment. Cannulas were implanted 5–6 d before the experiments. Blockers including RU38486 (750 ng in 1 μl), a specific blocker of glucocorticoid receptors (A); NBI 30775 (5 μg in 1 μl), a specific antagonist of CRH receptor type 1, CRFR1 (B); and a RU38486+NBI 30775 mixture (750 ng + 5 μg in 1 μl, respectively) (C) were delivered via the cannulas 30 min before a 2 h MAS. After a recovery period of 1.5–2 h, animals were trained (10 min) for exploring 2 objects (training session), followed by a 5 min testing (testing session) 6 h later the training (n = 8 mice per group, p > 0.05).
