Arginine vasopressin ameliorates spatial learning impairments in chronic cerebral hypoperfusion via V1a receptor and autophagy signaling partially

Abstract

Chronic cerebral hypoperfusion (CCH) is a major factor contributing to neurological disorders and cognitive decline. Autophagy activation is believed to provide both beneficial and detrimental roles during hypoxic/ischemic cellular injury. Although arginine vasopressin (AVP) has been strongly involved in many behaviors, especially in learning and memory, the effects of AVP on CCH and their molecular mechanisms remain unclear. Here, to investigate whether there was neuroprotective effects of AVP on CCH through V1a receptor (an AVP receptor) signaling, permanent bilateral carotid arteries occlusion (two vessel occlusion, 2VO) was used to establish a rat model of CCH, and hypertonic saline (5.3%) was injected intraperitoneally to induce the secretion of AVP. Results showed that hypertonic saline effectively alleviated spatial learning and memory deficit, enhanced synaptic plasticity of CA3-CA1 hippocampal synapses, upregulated N-methyl-d-aspartate receptor subunit 2B (NR2B) and postsynaptic density protein 95 (PSD-95) surface expressions, reduced oxidative stress and increased Nissl bodies in 2VO model rats. These phenomena were significantly decreased by V1a receptor antagonist SR49059. Interestingly, hypertonic saline also upregulated autophagy in the hippocampus of 2VO rats partly through V1a receptor. These findings imply that AVP has a beneficial role for the treatment of cognitive impairments partly through V1a receptor signaling in CCH, which is possibly related to improving synaptic plasticity by promoting NR2B and PSD-95 externalization and by enhancing autophagy.

Figure 1

The expressions of AVP and V1aR in hippocampus of sham, 2VO, 2VO + HTS and 2VO + HTS + SR49059 groups. (a) Representative immunoreactive bands of AVP (34 kDa), V1aR (43 kDa) and β-actin (43 kDa). Proteins lysed in RIPA were loaded at 30 μg per well. (b) Quantitative analysis of the optical density ratio of AVP/β-actin. (c) Quantitative analysis of the optical density ratio of V1aR/β-actin. Data are shown as mean ± s.e.m. *P<0.05, **P<0.01 versus sham group; ^#P<0.05, ^##P<0.01 versus 2VO group; ^&&P<0.01 versus 2VO + HTS group (one-way ANOVA analysis followed by LSD test). n=4 per group. (d) Representative immunofluorescence photographs of AVP in the hippocampal CA1 region. Yellow arrows denote AVP expression. (e) Representative immunofluorescence photographs of V1aR in the hippocampal CA1 region. Yellow arrows denote V1a receptor expression. Scale bar=50 μm. ANOVA, analysis of variance; AVP, arginine vasopressin; HTS, hypertonic saline; LSD, least significant difference; 2VO, two vessel occlusion; VIaR, V1a receptor.

Figure 2

The performance of rats in IT and SET stages of MWM experiment. (a) Calculated escape latency on each day in IT stage of sham, 2VO, 2VO + HTS and 2VO + HTS + SR49059 groups (two-way repeated measures ANOVA with LSD or Dunnett’s T3 post hoc test). (b) Swimming speeds on each day in the IT stage of four groups (two-way repeated measures ANOVA). (c) Representative swim traces of all four groups in SET stage. (d) The number of platform crossings in the SET stage (Mann–Whitney U-test). (e) Percentage of time spent in target quadrant (quadrant III) in SET stage (one-way ANOVA analysis followed by LSD test). Data are shown as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 versus sham group; ^#P<0.05, ^##P<0.01 versus 2VO group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 versus 2VO + HTS group. n=8 per group. ANOVA, analysis of variance; HTS, hypertonic saline; IT, initial training; LSD, least significant difference; MWM, Morris water maze; SET, space exploring test; 2VO, two vessel occlusion.

Figure 3

Long-term potentiation (LTP) from Schaffer collaterals to the hippocampal CA1 and synaptic protein expressions in hippocampus of sham, 2VO, 2VO + HTS and 2VO + HTS + SR49059 groups. (a) The changes of time coursing in normalized fEPSP slopes in sham, 2VO, 2VO + HTS and 2VO + HTS + SR49059 groups. The first 30 min of evoked responses were normalised and used as the baseline responses of LTP. Arrow represents application of a theta burst stimulation (TBS). (b) The mean normalized fEPSP slopes between 45 and 60 min after the TBS. n=8 per group. (c) Representative immunoreactive bands of NR2B (180 kDa), PSD-95 (95 kDa), SYP (38 kDa) and β-actin (43 kDa) in different subcellular fractions. Proteins were loaded at 5 μg per well for the LS1, 10 μg per well for the H, P2, LP1, TxS and TxP and 30 μg per well for the S2. (d–f) Quantitative analysis of the optical density ratio of NR2B/β-actin (d), PSD-95/β-actin (e) and SYP/β-actin (f) in different subcellular fractions. Data are shown as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 versus sham group; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus 2VO group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 versus 2VO + HTS group (one-way ANOVA analysis followed by LSD test). n=4 per group. ANOVA, analysis of variance; fEPSP, field excitatory postsynaptic potential; HTS, hypertonic saline; LSD, least significant difference; NR2B, N-methyl-D-aspartate receptor subunit 2B; PSD-95, postsynaptic density protein 95; 2VO, two vessel occlusion.

Figure 4

The changes of oxidative parameters and autophagy levels in the hippocampus of sham, 2VO, 2VO + HTS and 2VO + HTS + SR49059 groups. (a) The MDA level in hippocampus of the four groups (Mann–Whitney U-test). (b) The T-SOD activity in hippocampus of the four groups (Mann–Whitney U-test). (c) The CAT activity in hippocampus of the four groups (one-way ANOVA analysis followed by LSD test). (d) Representative immunoreactive bands of SQSTM1/p62 (75 kDa), Beclin-1 (60 kDa), LC3-I (16 kDa), LC3-II (14 kDa) and β-actin (43 kDa). Proteins lysed in RIPA were loaded at 30 μg per well. (e–g) Quantitative analysis of the optical density ratio of p62/β-actin (e), Beclin-1/β-actin (f), and LC3-II/LC3-I (g; one-way ANOVA analysis followed by LSD test). Data are shown as mean±s.e.m. *P<0.05, **P<0.01 versus sham group; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus 2VO group; ^&P<0.05, ^&&P<0.01, ^&&&P<0.001 versus 2VO + HTS group. n=4 per group. (h) Representative immunofluorescence photographs showed the co-localization of LC3 and LAMP1 in CA1 regions as indicated by yellow punctiform staining. Higher magnification views of the areas marked with ‘*’ were shown as insets. White arrows denote LC3, LAMP1 and merged punctas. Scale bar=50 μm. ANOVA, analysis of variance; CAT, catalase; HTS, hypertonic saline; LSD, least significant difference; MDA, malondialdehyde; T-SOD, total superoxide dismutase; 2VO, two vessel occlusion.

Figure 5

The underlying mechanisms that AVP ameliorates spatial learning impairments. The red ‘+’ represented the enhancement effect; the red ‘−’ represented the inhibiting effect; the dashed arrow represented the process, which was not verified in our study. AVP, arginine vasopressin.