Upregulation of Netrin-1 in the hippocampus mediates the formation of visceral hypersensitivity induced by maternal separation

Abstract

Early adverse life events (EALs), such as maternal separation (MS), can cause visceral hypersensitivity, which is thought to be a key pathophysiological mechanism of irritable bowel syndrome (IBS). Previous studies mainly focused on EALs-induced visceral hypersensitivity in adulthood but did not consider that it may have occurred in the preadult period. We previously found that rats who experienced MS suffered from visceral hypersensitivity starting from the post-weaning period. Moreover, the hippocampus is considered to be critical in regulating the formation of visceral hypersensitivity induced by MS. But the underlying mechanisms throughout different life periods are unclear. In this study, behavioral tests, RNA-seq, lentiviral interference, and molecular biology techniques were applied to investigate the molecular mechanism in the hippocampus underlying MS-induced long-lasting visceral hypersensitivity. It was found that both visceral sensitivity and anxiety-like behaviors were significantly increased in MS rats in post-weaning, prepubertal, and adult periods, especially in the prepubertal period. Subsequently, RNA-seq targeting the hippocampus identified that the expression level of Netrin-1 was significantly increased in all periods, which was further confirmed by quantitative real-time PCR and Western blot. Knocking-down hippocampal Netrin-1 in the post-weaning period by lentivirus interference alleviated visceral hypersensitivity and anxiety-like behaviors of MS rats in the later phase of life. In addition, deleted in colorectal cancer (DCC), instead of neogenin-1(Neo-1) or uncoordinated (UNC5), was proved to be the specific functional receptor of Netrin-1 in regulating visceral hypersensitivity, whose upregulation may result in the most severe symptoms in the prepubertal period. Furthermore, the activation of the Netrin-1/DCC pathway could enhance long-term potentiation (LTP) in the hippocampus, probably via recruitment of the AMPA receptor subunit GluA1, which finally resulted in the formation of visceral hypersensitivity. These novel findings suggest that long-lasting over-expression of Netrin-1 can mediate visceral hypersensitivity and anxiety disorder from the post-weaning period to adulthood by activating DCC/GluA1 pathway in the hippocampus. Moreover, early intervention of Netrin-1 in the post-weaning period could lead to significant symptom relief afterward, which provides evidence that the Netrin-1/DCC/GluA1 signaling pathway may be a potential therapeutic target for the treatment of visceral hypersensitivity in clinics.

Keywords: DCC; GluA1; Netrin-1; hippocampus; maternal separation; visceral hypersensitivity.

FIGURE 1

The schematic of experimental procedures. (A) Maternal separation (MS) procedures were established on postnatal day (PND) 1–14 (n = 60), and the normal control (NC) rats remained undisturbed (n = 58). MS and NC rats were randomly subdivided into PND21, PND35, and PND70 groups. The open-field test (OFT), elevated plus maze (EPM) test, and surgery were performed a day before electromyogram (EMG) recording. The visceromotor response (VMR) to colorectal distension (CRD) was recorded by EMG on PND21, PND35, and PND70. After visceral sensitivity was measured, the rats were sacrificed and the hippocampus were dissected for RNA-Seq, quantitative real-time PCR (qRT-PCR), and Western blot. (B) MS procedures were established on PND 1–14 (n = 24). On PND 21, rats were randomly assigned into two groups and were subjected to anti-Netrin-1 shRNA (Lenti-shNTN-1) or scrambled control shRNA (Lenti-Scramble) microinjection into the CA1 regions of the hippocampus. EMG was recorded on PND35, and the OFT, EPM, and surgery were performed a day in advance. After visceral sensitivity was measured, rats were sacrificed and the hippocampus were dissected for qRT-PCR and Western blot. The accuracy of injection sites was confirmed by fluorescence imaging.

FIGURE 2

Visceral hypersensitivity occurred in MS rats from the post-weaning period to adulthood and was more pronounced on PND35. (A–C) VMR amplitudes to 40 and 60 mmHg CRD on PND21 (A), PND35 (B), and PND70 (C) were significantly higher than those of NC rats. On PND21: NC, n = 12; MS, n = 12. On PND35: NC, n = 12; MS, n = 12. On PND70: NC, n = 12; MS, n = 12. All data were given as mean ± SEM. ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test. (D) The relative VMR ratios of MS to NC rats in three different age periods. The relative VMR ratios of MS rats to NC rats on PND35 were significantly higher than that of PND21 and PND70. All data were given as mean ± SEM. ^**P < 0.01, ^***P < 0.001, one-way ANOVA test followed by least-significant difference test. AUC/s, the area under the curve per second. (E) The representative external abdominal oblique muscle EMG recordings of NC and MS rats.

FIGURE 3

Anxiety-like behaviors were increased in rats of MS groups. Before EMG recordings, all rats were subjected to the OFT test (A–C) and EPM test (D–F) to evaluate anxiety-like behaviors. (A) The distance traveled by MS rats in the open field was significantly less than that of NC rats. (B) On PND35 and PND70, MS rats showed less entries into the center zone (CZ). (C) There was no significant difference in the number of rearing in the three age periods. (D) MS rats on PND21, PND35, and PND70 showed a significantly lower probability of open arms (OA) entries than NC rats. (E) MS rats in three groups spent significantly less percentage of time in the OA than NC rats. (F) MS rats in three groups showed a significantly higher anxiety index than NC rats. The anxiety index integrated with the EPM behavioral measures was calculated as follows: anxiety index = 1 − [(time spent in OA/total time on the maze) + (number of entries to the OA/total exploration on the maze)/2]. On PND21: NC, n = 12; MS, n = 12. On PND35: NC, n = 12; MS, n = 12. On PND70: NC, n = 12; MS, n = 12. All data were given as mean ± SEM. *P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test. (G) Representative animal track in the OFT and (H) EPM.

FIGURE 4

RNA-seq results of the hippocampus in MS and NC rats of three different age periods (n = 4 per group). (A–C) The differentially expressed genes (DEGs) on PND21 (A), PND35 (B), and PND70 (C) were significantly enriched in various KEGG signaling pathways. The top-ranked pathways were visualized with bubble plots. (D) Venn diagram of enriched pathways showed that axon guidance pathway was the only shared enriched pathway which ranked among top five of all age periods. (E) Venn diagram of DEGs in the hippocampus showed that 54 DEGs were shared in different age periods. (F) A total of 54 shared DEGs were significantly enriched in various pathways, including the axon guidance pathway. (G) A simplified schematic of the axon guidance pathway. Netrin-1, its specific receptor deleted in colorectal cancer (DCC), as well as other two molecules marked in red were upregulated among MS rats of three periods. Netrin-1 and DCC were specific ligands or receptors for each other.

FIGURE 5

The mRNA expression of Netrin-1 and GluA1 were significantly increased in the hippocampus in MS rats of three different age periods. (A) On PND21, the mRNA expression of Netrin-1 was significantly increased, but its receptor uncoordinated (UNC5D) was significantly decreased. (B) On PND35, the mRNA expression of Netrin-1, and its receptor DCC, neogenin-1 (Neo-1) were significantly increased, but UNC5C was significantly decreased. (C) On PND70, the mRNA expression of Netrin-1 was significantly increased, but its relative receptors showed no significant upregulation. (D–F) GluA1, NR2A, NR2B, and postsynaptic density 95 (PSD95) were significantly increased in all three MS groups. Besides, NR1 was found upregulated in MS rats on PND35 (E) and PND70 (F). On PND21: NC, n = 8; MS, n = 8. On PND35: NC, n = 8; MS, n = 8. On PND70: NC, n = 6; MS, n = 8. All data were given as mean ± SEM. *P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test.

FIGURE 6

Netrin-1 and GluA1 protein were upregulated in the hippocampus in rats of three MS groups. Representative immunoblots showed the expression of Netrin-1, DCC, Neo-1, GluA1, NR1, NR2A, NR2B, PSD95, and β-Tubulin on PND21 (A), PND35 (C), and PND70 (E). (B) Western blot analysis showed increased expression of Netrin-1, GluA1, NR2A, NR2B, and PSD95 in the hippocampus in MS rats on PND21, (D) PND35, and (F) PND70. Furthermore, the protein expression level of DCC was upregulated on PND35. On PND21: NC, n = 8; MS, n = 8. On PND35: NC, n = 8; MS, n = 8. On PND70: NC, n = 8; MS, n = 8. All data were given as mean ± SEM. *P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test.

FIGURE 7

Knockdown of Netrin-1 (NTN-1) in hippocampal CA1 regions decreased not only visceral sensitivity but also the expression levels of mRNA and proteins of Netrin-1, DCC, GluA1, and PSD95. (A) The representative external abdominal oblique muscle EMG recordings. (B) VMR amplitudes to 40 and 60 mmHg CRD were significantly reduced in rats expressing Lenti-shNTN-1 (MS + shNTN-1) than in rats injected Lenti-Scramble (MS + Scramble). MS + Scramble: n = 12. MS + shNTN-1: n = 12. All data were given as mean ± SEM. ^***P < 0.001, two-tailed unpaired Student’s t-test. (C) The mRNA and (E) protein expressions of Netrin-1, DCC, GluA1, and PSD95 in the hippocampus were significantly reduced in the MS + shNTN-1 group. (D) Representative immunoblots showed the expression of Netrin-1, DCC, GluA1, NR2A, NR2B, PSD95, and β-Tubulin. (F) A representative fluorescence image confirmed the successful GFP expression in shNTN-1 infected cells in the hippocampus. Scale bar, 500 μm. MS + Scramble: n = 4. MS + shNTN1: n = 4. All data were given as mean ± SEM. *P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test.

FIGURE 8

Knockdown of Netrin-1 (NTN-1) in hippocampal CA1 regions reversed the increase of anxiety-like behaviors caused by MS. (A) In OFT, compared with rats injected Lenti-Scramble (MS + Scramble), rats expressing Lenti-shNTN-1 (MS + shNTN-1) showed longer distance traveled, (B) more entries into the center zone, and (C) more rearing. (D) In the EPM test, the percentage of numbers of entries into OA and (E) the percentage of time spent in OA in the MS + shNTN-1 group were significantly increased, and (F) the anxiety index was reduced significantly. (G) Representative animal track in the OFT and (H) EPM. MS + Scramble: n = 12. MS + shNTN1: n = 12. All data were given as mean ± SEM. *P < 0.05, ^**P < 0.01, ^***P < 0.001, two-tailed unpaired Student’s t-test.