(2R,6R)-hydroxynorketamine acts through GluA1-induced synaptic plasticity to alleviate PTSD-like effects in rat models

Abstract

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with high morbidity and great social and economic relevance. However, extant pharmacotherapies of PTSD require long-term use to maintain effectiveness and have enormous side effects. The glutamatergic system, especially the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), is an important target of current research on the mechanism of PTSD. Postsynaptic AMPAR function and expression are known to be increased by (2R, 6R)-hydronorketamine (HNK), the primary metabolite of ketamine. However, whether (2R,6R)-HNK alleviates PTSD-like effects via AMPAR upregulation is yet to be known. In the present study, rats were exposed to single prolonged stress and electric foot shock (SPS&S). Afterwards, gradient concentrations of (2R,6R)-HNK (20, 50, and 100 μM) were administered by intracerebroventricular (i.c.v.) injection. Open field, elevated plus maze, freezing behavior, and forced swimming tests were used to examine PTSD-like symptoms. In addition, the protein levels of GluA1, BDNF and PSD-95 were analyzed using western blotting and immunofluorescence, and the synaptic ultrastructure of the prefrontal cortex (PFC) was observed by transmission electron microscopy. We found that (2R,6R)-HNK changed SPS&S-induced behavioral expression, such as increasing autonomous activity and residence time in the open arm and decreasing immobility time. Likewise, (2R,6R)-HNK (50 μM) increased GluA1, BDNF, and PSD-95 protein expression in the PFC. Changes in synaptic ultrastructure induced by SPS&S were reversed by administration of (2R,6R)-HNK. Overall, we find that (2R,6R)-HNK can ameliorate SPS&S-induced fear avoidance in rats, as well as rat cognates of anxiety and depression. This may be related to GluA1-mediated synaptic plasticity in the PFC.

Keywords: (2R,6R)-Hydroxynorketamine ((2R,6R)-HNK); GluA1; Glutamatergic nervous system; Post-traumatic stress disorder (PTSD); Synaptic plasticity.

Fig. 1

Experimental schedule of the SPS&S procedure Before the experiment, all rats were allowed to acclimate to their new environment for 7 days. Then, 14 days after SPS&S, the designated groups were treated with drugs injection in their lateral ventricles.

Fig. 2

Effect of SPS&S procedure on behaviors. A) Distance traveled by group in the center square (Mann-Whitney U test: z = −2.948, P = 0.002) in the OFT. B) Numbers of crossings (t = 3.781, P = 0.001) C, D) Number of entries into the open arms (t = 3.005, P = 0.008) and time spent in the open arms (t = 5.158, P = 0.000). E) The time spent in center area (t = 3.496, P = 0.003). F) Up-right numbers (t = 2.640, P = 0.017). G) Passive immobility time in FST (t = 2.944, P = 0.009). H) Freezing time in FBT (t = 2.829, P < 0.011). I, J, K, L) Representative video tracking images of OFT and EPMT. I, K) control group, J, L) SPS&S group. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001compared with control group.

Fig. 3

Effect of SPS&S procedure on synaptic ultrastructure and expression of GluA1 and BDNF in the PFC. A, B, C) Relative expression of GluA1 (t = 3.486, P = 0.025), BDNF (t = 3.091, P = 0.037), and PSD-95 (t = 3.108, P = 0.036). D, E, F) Localization and distribution of GluA1, BDNF, and PSD-95. Bar = 20 μm. G) GluA1 in PFC (t = 7.073, P = 0.002). H) BDNF in PFC (t = 5.431, P = 0.007). I) PSD-95 in the PFC (t = 11.79, P = 0.000). J, K) PFC synaptic ultrastructure. L, M, N) Synaptic cleft width (L,t = −3.563, P = 0.012), postsynaptic density (M, z = -2.309, P = 0.021), and synaptic interface curvature (N,t = 2.708, P = 0.035). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

Fig. 4

Effect of (2R,6R)-HNK on PTSD-like behaviors under the SPS&S procedure. A) Time spent in the center area in the OFT (P = 0.024, Kruskal-Wallis test). B) Up-right numbers (F (3, 36) = 4.753, P = 0.007) C, D) Time spent in the open arms (F (3, 36) = 6.233, P = 0.002) and number of entries into the open arms (P = 0.057 at Kruskal-Wallis test). E) Distance traveled in the center square by group (F (3, 36) = 0.5714, P = 0.638). F) Number of crossings (F (3, 36) = 3.109, P = 0.038). G) Passive immobility time in FST (F (3, 36) = 4.647, P = 0.008). H) Freezing time in FBT (F (3, 36) = 7.795, P = 0.000). I–P) Representative video tracking images of OFT and EPMT. I, M) SPS&S-Sal group; J, N) SPS&S-20 group; K, O) SPS&S-50 group; L, P) SPS&S-100 group. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

Fig. 5

Polynomial fitting between dose of medicine and behavior.

Fig. 6

Effect of (2R,6R)-HNK on the expression of GluA1 and BDNF in the PFC under the SPS&S procedure and results of correlation analysis. A, B, C) Relative expression of GluA1 (F (3, 8) = 7.780, P = 0.009), BDNF (F (3, 8) = 4.886, P = 0.033) and PSD-95 (F (3, 8) = 4.448, P = 0.041). D, E, F) Correlations between PSD-95 and BDNF (D), GluA1 and BDNF €, and GluA1 and PSD-95 (F) across all rats (n = 12). Correlations were computed using Pearson's R. F (3, 8) = 7.253, P = 0.011.

Fig. 7

Effect of (2R,6R)-HNK on the expression of GluA1, BDNF in the PFC under the SPS&S procedure. A) PFC image capture location diagram. B) Low magnification microscopic image of PFC. Bar = 200 μm. C, D, E) Localization and distribution of GluA1, BDNF, and PSD-95. Bar = 20 μm. F) GluA1 in PFC (P = 0.033, Kruskal-Wallis test). G) BDNF in PFC (F (3, 8) = 12.941, P = 0.002). H) PSD-95 in PFC (F (3, 8) = 16.1, P = 0.001). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

Fig. 8

Effect of (2R,6R)-HNK on the synaptic ultrastructure in the PFC under the SPS&S procedure. A) Synaptic ultrastructure. B) Curvature of synaptic interface (F (3, 12) = 5.633, P = 0.012) C) Postsynaptic density (F (3, 12) = 6.021 P = 0.010) D) Synaptic cleft width (F (3, 12) = 3.705, P = 0.043). Data were represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.