Morphine modulates hippocampal neurogenesis and contextual memory extinction via miR-34c/Notch1 pathway in male ICR mice

JieWei Hu; FuHua Cui; XiaoDong Zhang

doi:10.1515/biol-2020-0006

Article Open Access

Morphine modulates hippocampal neurogenesis and contextual memory extinction via miR-34c/Notch1 pathway in male ICR mice

JieWei Hu , FuHua Cui and XiaoDong Zhang

Published/Copyright: February 28, 2020

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From the journal Open Life Sciences Volume 15 Issue 1

Abstract

Background

The opioid Morphine is known to affect neurogenesis in the hippocampus. Evidence has shown that several microRNAs modulate morphine-induced neurogenesis, and hence morphine-induced contextual memory. This complex network has yet to be elucidated. In this study, we screened for morphine addiction related microRNA and determined its effects on hippocampal neurogenesis and morphine-induced contextual memory using the conditioned place preference (CPP) model.

Methods

The previously established CPP model was utilized in this study. For differential expression of miRNA in the hippocampus, the GeneChip miRNA array was used. Lentivirus technology was used to overexpress or downregulate the miRNA, and changes in expression level was verified with qRT-PCR. Protein expression levels were measured with western blot. Immunofluorescence was used to observe the protein expression during the differentiation of NSCs.

Results

The results showed that morphine administration upregulated microRNA-34c (miR-34c) and Notch1. Downregulating miR-34c in vivo decreased Notch1 expression and partially rescued the morphine-induced inhibition of the differentiation of neural stem cells (NSCs). This did not affect the morphine-induced proliferation of cells. Furthermore, downregulating miR-34c in vivo prolonged the extinction of morphine-induced contextual memory without affecting acquired CPP response.

Conclusion

The miR-34c regulates the hippocampal neurogenesis in addicted mice by up-regulating Notch1 expression, by inhibiting differentiation of neural precursor cells. The miR-34c/Notch1 pathway may be a new potential target for the prevention and treatment of opioid psychotic dependence.

Keywords: miR-34c; morphine; hippocampal neurogenesis; neural differentiation; contextual memory

1 Introduction

Lifelong neurogenesis in hippocampal dentate gyrus (DG) has been substantially reported in various species [1, 2, 3]. Neural stem cells (NSC) in the subgranular zone (SGZ) constantly give rise to functional mature granular cells in adult animals [4, 5]. Besides other extrinsic factors [6, 7], several drugs of abuse [8, 9, 10, 11], have been shown to regulate hippocampal neurogenesis in adult animals. A decrease of cycling cells was demonstrated in the SGZ of opiate-addicted mice and suggested an inhibitory effect of opiates on the renewal of neural precursor cells [12]. Furthermore, opiates were found to increase the proliferation of hippocampal progenitors [13]. Morphine administration showed inhibitory effects on neural differentiation and a simultaneous decrease in spatial memory capacity [14]. Regulating extrinsic stimulus modulated drug-dependence memory, which is closely related to environmental stimulus-induced addiction relapse [8].

MircoRNAs (miRNAs) are non-coding short RNA molecules. They function as regulators of gene expression in both normal [15, 16] and pathologic nervous system [17]. The participation of miRNAs in morphine-induced neurogenesis has previously been identified. Morphine administration enhanced hippocampal neural progenitor cells differentiation via miR132 [18] and miR-181a/Prox1/Notch1 pathway [19, 20]. Although fentanyl, another u-opioid agonist, was shown to modulate neurogenic differentiation 1 (NeuroD) via miR-190 without influencing DG neurogenesis [21], NeuroD was proved to be critical in neural development and neurogenesis [22].

The miR-34 family is closely related to learning and memory functions and is involved in the regulation of proliferation and differentiation of neural stem cells [23, 24, 25]. Among them, miRNA-34c is related to neuronal function [26, 27], which suggest that morphine may affect hippocampal neurogenesis through the miR-34c pathway. In our study, we used a mouse CPP model to investigate how miR-34c participates in morphine-induced neurogenesis in mentally dependent mice and explore the neurogenesis mechanisms of morphine-dependence formation and retention.

2 Materials & Methods

2.1 Animals

Eight-week-old male ICR mice, initially weighing 18.0-20.2 g, were obtained from the Second Military Medical University (SMMU) Laboratory Animal Center (Shanghai, China). All mice were group-housed eight to a cage with free access to food and water and maintained on a 12 h light/dark cycle, temperature within 20-25°C and humidity within 40%‒70%. The mice were divided randomly into groups of two with at least a week of acclimatization.

Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experimental protocols were approved by the Ethics Committee of the Second Military Medical University.

2.2 CPP training and extinction protocol

1. Apparatus

The apparatus consisted of two compartments (black and white) divided by a guillotine door. Time spent in each compartment was monitored by a surveillance camera and a tracking program (Jiliang, Shanghai, China).

2. Procedure

Mice were habituated with free access to both compartments for 15 min for 3 days. Drug-paired compartment was determined as the less preferred one on day 3. Training was conducted for two sessions a day for seven days. Mice were i.p. injected with morphine (dilute with saline, injection dose is 0.25 ml per mouse) 0.32 mg/kg, with an increment of 0.04 mg/kg per day and were immediately placed in drug-paired compartment for 50 min in the first session, and were injected with saline and placed in the other compartment in the second session. In the control experiment, mice were injected with saline in both sessions. CPP test was conducted 24 h after training: mice were given free access to both compartments for 15 min, and the time spent in drug-paired compartment was evaluated as CPP score. In extinction experiments, tests were conducted every 5 days after the first test to evaluate CPP scores.

2.3 MicroRNA high-throughput sequencing

Hippocampal tissue obtained from saline- and morphine-treated mice was kept at -20°C. The changes in the miRNA profiling were measured with GeneChip™ miRNA Array (Thermo Fisher Scientific, US) according to manufacturer’s instructions.

2.4 Total RNA isolation and quantification of miRNA by real-time qRT-PCR

The harvested hippocampal tissues from morphine- or saline-treated mice were homogenized in TRIzol Reagent (Invitrogen, USA). Total RNA was extracted according to the manufacturer’s instructions. RNA was quantified by spectrophotometry and dilution was performed to keep the RNA samples at 200 ng/μL.

2 μg of RNA from each sample was reverse transcribed at 42°C for 60 min with 1 μg of primer, 5× Reaction Buffer, 10mM dNTP Mix, RiboLock RNAsin (Thermo Fisher Scientific, US) and M-MLV. PCR reaction mixtures included cDNAs in optimum dilution, FastStart Universal SYBR Green Master Mix (Thermo Fisher Scientific, USA), and 7.5 μM primers in a total reaction volume of 25 μL. Cycling parameters were 95°C for 10 min followed by 40 cycles of 60°C/s temperature transition rate up to 95°C (15 s), 62°C (60 s), followed by melting curve analysis. All reactions were performed in triplicates with reference dye normalization and the median Ct (Cycle threshold) value was used for analysis.

2.5 qRT-PCR was performed with a set of primers for amplification of the miRNA

RT primer: 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG GCA ATC AG -3′;

q-RT PCR primer: 5′- ACA CTC CAG CTG GGA GGC AGT GTA GTT AGC T -3′.

2.6 Western blot analysis

After the establishment of CPP, mice were decapitated and hippocampal tissues were isolated and kept frozen at -80°C. Harvested tissue was homogenized with ice-cold RIPA lysis buffer (Beyotime Biotechnology) and phosphatase inhibitors. The mixture was then centrifuged at 14000 r/min for 5 min at -4°C. The supernatant was collected and stored at -80°C. Protein concentration was determined using a BCA assay kit (Beyotime Biotechnology). Proteins of 50 μL were loaded per lane and resolved using 10% SDS-polyacrylamide gel electrophoresis at 120 V prior to being transferred to PVDF membranes using wet tank electro transfer. After blocked with 5% BSA for 1 h at room temperature, membranes were incubated with primary antibodies against Notch1 (1: 1,000; Ab52627; Abcam) in blocking buffer overnight at 4°C. These membranes were subsequently incubated with secondary antibody (Horeseradish peroxidase conjugated-goat anti-Rabbit immunoglobulin G; 1:5,000; Beyotime Biotechnology) in blocking buffer for 1 h at room temperature. The blots were developed using DAB substrate kit (Beyotime Biotechnology). The immunoblots were imaged with Amersham Imager 600 (GE Healthcare Life Sciences) and analyzed ImageJ software. The Notch1 levels were normalized to β- actin in the same membranes.

2.7 Lentivirus construction

Lentiviruses expressing miR-34c-5p-sponge (34c-vir: 5′-GCAATCAGCTAACTACACTGCCTTATACGCAATCAG-CTAACTACACTGCCTACATCGCAATC-3′) or an empty vector as control were constructed in pHB-U6-MCS-zsgreen-puro using the Lentivirus-siRNA system (Hanbio Biotechnology) according to the manufacturer’s instructions. Thus, con-vir and 34c-vir also express GFP. The lentivirus expressing 34c-vir was generated by inserting miR-34c-5p-sponge cDNA between the U6 and MCS sites. Viruses were produced by transfecting 293T cells with pHB-U6-MCS-zsgreen-puro constructs together with pSPAX2^、pMD2G (Hanbio Biotechnology). Viral titers (~2 × 108 transducing units/mL) were determined in 293T cells. Infection efficiency was >60%.

2.8 Stereotaxic surgery and drug injection

Mice were anesthetized with 1% sodium pentobarbital (40 mg/kg) and secured in a stereotaxic frame. Virus was injected into DG (AP -2.6 mm, ML 1.8 mm, DV 2.0 mm) at a rate of 0.8 μL/min for total 6 μL by a microsyringe pump. Mice were allowed to recover at home cage for 7 days after injection for further experiments.

2.9 Antibodies and immunohistochemistry (IHC)

Mice were anesthetized with pentobarbital sodium (40 mg/kg) and perfused with 4% paraformaldehyde. Brains were post-fixed for 24 h with 4% paraformaldehyde. BrdU (5-Bromo-2´-Deoxyuridine) (5 mg/kg; Invitrogen) was injected intraperitoneally into mice in the last 2 days of CPP training. Tissues were sectioned coronally at 40 μm on a freezing microtome.

Immunohistochemical analyses were performed as described previously with modifications [28]. Briefly, sections were boiled in EDTA buffer, pH 8.0, and blocked in 3% BSA for 0.5 h at room temperature. Incubation with primary antibodies was performed at 4°C overnight. Secondary antibodies were applied to sections for 50 min at room temperature. The following primary antibodies were used: rat anti-nestin (1:300; Ab6142; Abcam); rabbit anti-DCX (1:500; Ab18725; Abcam); rabbit anti-NeuN (1:3000; Ab177487; Abcam). Secondary antibodies were conjugates of Alexa Fluor 488 (1:200; Abcam). The labelling of BrdU was preformed according to the instructions of EdU staining kit (RiboBio Co.), and DAPI (4′,6′-diamidino-2-phenylindole) (0.01%; Invitrogen) was used as nuclear counterstain.

2.10 Microscopic analysis and quantification

To quantitatively examine the double-labelled cells of interest, a typical hippocampal section of each mouse was selected for imaging using a fluorescent inverted microscope (BM optical instruments, Shanghai, China) with 40× objective. Image-Pro Plus5.0 was used for image analyses.

2.11 Statistical analysis

All the data were provided in the form of mean ± S.E. Two sets of data that met the normal distribution and homogeneity of variance were analyzed by Student’s t-test. Multigroup comparisons of the means were carried out by ANOVA with post hoc contrasts by Tukey test. Differences between treatments and controls were considered significant at p < 0.05. Results were analyzed with SPSS 21.0 statistical software.

3 Results

3.1 Morphine-induced CPP Upregulated miR-34c and Notch1

3.1.1 Chronic morphine administration induces conditioned place preference

Mice treated with morphine or saline were trained with conditioned place preference (CPP) for 7 days after habituation. Morphine dependence was evaluated as time spent in drug-paired compartment in the CPP test 24 h after training. As shown in Figure 1, morphine-treated WT mice exhibited significant preference for drug-paired compartment after training either compared to pre-training test within group or to after-training test of saline-treated WT group (p < 0.01, ANOVA (F (3,8) =65.67) and the post hoc tests (Tukey method) was applied to investigate the differences one by one). Saline-treated WT mice exhibited no place preference after training compared to pre-training test within group (p>0.05). CPP training successfully established morphine-related contextual memory.

Figure. 1

Chronic morphine induces CPP of adult mice. The CPP scores of wildtype mice treated with saline or morphine. Error bars represent SEM. N=8. Significant differences between two groups were determined using ANOVA (F (3,8) =65.67) and the post hoc tests (Tukey method) was applied to investigate the differences one by one. **, p < 0.01 compared to pre-training of morphine wildtype group; ##, p < 0.01 compared to post-training of saline wildtype group. WT, wildtype; S, saline; M, morphine.

3.1.2 Chronic morphine administration upregulates miR-34c and protein Notch1

After the establishment of CPP, microRNA microarray analysis was performed with harvested hippocampal tissues to screen for microRNAs of morphine-induced differential transcription. Among those microRNAs of transcription difference over 2 folds, miR-34c topped with a 4.8-fold transcription difference.

To validate the morphine-induced upregulation of miR-34c, quantification by means of q-RT PCR was conducted with both morphine- and saline-treated WT mice. Consistent with results from microarray analysis, miR-34c level increased 50% in morphine-treated WT mice compared to saline-treated group (Figure 2A).

Figure. 2

Chronic morphine upregulates miR-34c and Notch1. (A) The expression of miR-34c in the hippocampi of wildtype mice treated with saline or morphine was determined by qRT-PCR after the CPP training. WT+S is considered to be 1. All data represent the mean ± SEM of three independent experiments. Significant differences between groups were determined using unpaired Student’s t test. **, p < 0.01 compared to the saline wildtype group. (B) The protein level of Notch1 in the hippocampi of wildtype mice treated with saline or morphine was determined by western-blot. The results were normalized against the level of β-actin. (C) Notch1 protein level. All data represent the mean ± SEM of three independent experiments. WT+S is considered to be 1. Significant differences between groups were determined using unpaired Student’s t test. *, p < 0.05 compared to the saline wildtype group. WT, wildtype; S, saline; M, morphine.

Three common databases, TargetScan, EIMMo and DIANA, were consulted to predict the target genes of miR-34c. Notch1 overlapped and topped the prediction. Western Blot analysis was performed after CPP establishment. Notch1 protein level in morphine-treated WT mice was approximately 40% higher compared to saline-treated group (Figure 2B and C).

We show that chronic morphine significantly up-regulates the levels of miR-34c and the protein Notch1.

3.2 Downregulating miR-34c modulated morphine-induced neurogenesis

While the modulating effects of morphine on hippocampal neurogenesis in adult mice are well-established, how microRNAs are involved in the process is not. We next investigated how miR-34c participates in morphine-induced neurogenesis.

3.2.1 Down-regulating miR-34c inhibited Notch1 expression

Lentivirus construction expressing miR-34c-5p-sponge was utilized to downregulate miR-34c. Hippocampal tissue was obtained from mice of morphine-treated WT, empty vector (EV) and sponge group for qRT-PCR after CPP training. Compared to morphine-treated WT mice, miR-34c level of sponge group but not EV group decreased (Figure 3A, p < 0.01, ANOVA (F (2,6) =24.05) and the post hoc tests (Tukey method) was applied to investigate the differences one by one). miR-34c level was down-regulated by lentivirus.

Figure. 3

Modulating miR-34c level downregulates Notch1 expression. (A) The expression of miR-34c in the hippocampi of wildtype, EV and sponge mice treated with morphine was determined by qRT-PCR after the CPP training All data represent the mean ± SEM of three independent experiments. WT+S is considered to be 1. Significant differences between each two groups were determined using ANOVA (F (2,6) =24.05) and the post hoc tests (Tukey method) was applied to investigate the differences one by one. **, p < 0.01 compared to the wildtype group. ##, p < 0.01 compared to the EV group. (B) The protein level of Notch1 in the hippocampi of EV or sponge mice treated with morphine was determined by western-blot. The results were normalized against the level of β-actin. (C) Notch1 protein level. All data represent the mean ± SEM of three independent experiments. WT+S is considered to be 1. Significant differences between groups were determined using upaired Student’s t test. *, p < 0.05 compared to the EV group. WT, wildtype; EV, empty vector; S, saline; M, morphine.

Next, western blot analysis was conducted to examine Notch1 expression after miR-34c downregulation. As shown in Figure 3B and CC, Notch1 protein level of sponge group was significantly decreased compared to EV group after CPP establishment. Thus, Notch1 expression is positively modulated as a down-stream factor of miR-34c.

3.2.2 Down-regulating miR-34c promotes the differentiation of NSCs

To elucidate the role of miR-34c in the neurogenesis process, morphine- and saline-treated WT mice and morphine-treated sponge mice, which stereotaxic injections were performed in the GD of hippocampus, were examined after CPP training. In the last 2 days of training, BrdU was i.p. injected into mice to label proliferating cells. The hippocampal tissues were harvested 2 days, 7 days and 14 days after training to examined newborn NSCs, immature neurons and mature neurons, respectively (Figure 4A).

Figure. 4

The process of adult hippocampal neurogenesis after morphine administration in wildtype and miR-34c sponge mice. (A) Timeline of the experiment. Animals were habituated for 3 days and trained for 7 days. EdU (5 mg/kg) was i.p. injected in the last 2 days of training. The animals were sacrificed on day 2 (p2), day 7 (p7) or day 14 (p14) post training and analyzed by immunohistochemistry. (B) Fluorescent images showing the co-localization of EdU and the NSC marker Nestin, immature neuron marker DCX or mature neuron marker NeuN, in the DG of morphine- and saline-treated wildtype mice and morphine-treated sponge mice. Blue: DAPI; Red: EdU; Green: Nestin, DCX or NeuN, respectively; Images represent three individual animals with similar results. Scale bar: 50 μm. (C) Quantification of the co-localization between EdU and Nestin, DCX or NeuN. Total numbers of EdU-labeled cells co-stained with Nestin (EdU⁺Nestin⁺), DCX (EdU⁺DCX⁺) or NeuN (EdU⁺NeuN⁺) antibodies in adult mouse hippocampus sections were counted and compared. Error bars represent SEM (N = 8). Significant differences between two groups were determined using ANOVA (F (2,6) =25.00 for Nestin, F (2,6) =18.18 for DCX and F (2,6) =8.455 for NeuN) and the post hoc tests (Tukey method) was applied to investigate the differences one by one. *, p < 0.05 (**, p<0.01) compared to the saline-treated wildtype mice; &, p<0.05 compared to the morphine-treated wildtype group. WT, wildtype; S, saline; M, morphine.

The number of newborn neural stem cells, labeled as ErdU⁺/Nestin⁺ cells, was determined 2 days after training. As shown in the upper panels of Figure 4B and C, a quantitative increase of Edu+/Nestin+ cells was observed in both morphine-treated WT mice and sponge mice, compared to saline-treated WT mice, but no difference was observed between morphine-treated WT mice and miR-34c mice (p<0.05, ANOVA (F (2,6) =25.00 for Nestin, F (2,6) =18.18 for DCX, F (2,6) =8.455 for NeuN and the post hoc tests (Tukey method) was applied to investigate the differences one by one). Together, these results indicate chronic morphine promotes the proliferation of NSCs but not via miR-34c modulation.

Next, the number of newborn immature neurons was determined 7 days after training, shown as ErdU⁺/DCX⁺ cells. The number of ErdU⁺/DCX⁺ neurons in morphine- treated WT mice and sponge mice was significantly decreased compared to saline-treated WT mice, but significantly more ErdU+/NeuN+ cells was observed in sponge mice compared to morphine-treated WT mice (Figure 4B and C, middle panels). Together, these results indicate chronic morphine treatment inhibits the differentiation of NSCs into neurons, and at least partially via miR-34c pathways.

Similarly, the number of newborn mature neurons was determined 14 days after training, labeled as ErdU+/NeuN+ neurons. As shown in panels of Figure 4B and C, a decrease of ErdU+/NeuN neurons in morphine-treated WT mice was revealed compared to saline-treated WT mice. No differences were observed between morphine-treated WT mice and sponge mice, or between saline-treated WT mice and morphine-treated sponge mice. These results suggest that chronic morphine decreases the number maturation of newborn neurons, but not via miR-34c pathways.

3.3 Down-regulating miR-34c prolonged the morphine-induced CPP retention without affecting its acquisition

The differentiation of NSCs was reported to contribute to the formation of drug-related contextual formation. We next tested how miR-34c modulates CPP response. Morphine-treated WT mice and sponge mice were trained with CPP conditioning. To examine the acquisition and extinction of CPP response, CPP scores were evaluated 1 day after training and once every 5 days after. No difference between the two groups was observed in the CPP scores 1 day after training (Figure 5A), but time required for extinction of CPP response of morphine-treated sponge mice was prolonged compared to that of morphine-treated WT mice (Figure 5B). Together, we conclude that down-regulation of miR-34c prolongs the extinction without affecting the acquisition of morphine-induced CPP response.

Figure. 5

Downregulating miR-34c modulates extinction but not acquisition of CPP. (A) The CPP scores of wildtype and sponge mice treated with morphine during the acquisition session. Error bars represent SEM (N=8). Significant differences among groups were determined using unpaired Student’s t test. (B) The time required for the extinction of CPP of wildtype and sponge mice treated with morphine. Error bars represent SEM. N=8. Significant differences between groups were determined using unpaired Student’s t tests. *, p < 0.05 compared to the wildtype group.

4 Discussion

The neurogenesis in adult animals goes through a series processes, including the proliferation and differentiation of neural stem cells, the maturation of newborn neurons and their immigration and integration into existent neural networks [4]. Although morphine administration modulates adult animals’ neurogenesis, its mechanisms, in particular the involvement of miRNAs, have yet been substantially reported. Previous studies have demonstrated that the miR-34 family is closely involved in learning and memory [27] and modulates the differentiation of NSCs [23]. In our study, we demonstrated that by down-regulating miR-34c in morphine treated mice inhibits NSC differentiation into neurons. This effect was partially rescued via modulating Notch1 expression, and subsequently enhanced the retention of morphine-induced CPP by prolonging time required for extinction.

As demonstrated in this study, morphine promotes the proliferation of NSC and but inhibits its differentiation into and survival of neurons, which is consistent with previous reports [13, 19, 20]. Chi Xu et al. has substantially demonstrated that morphine attenuated the neural progenitor cell differentiation into mature neurons by promoting astrocyte-preferential differentiation via the miR181a/Prox1/Notch1 pathway [19, 20]. Since Notch1 functions in both pathways, the activation miR-34c/Notch1 is likely to decrease newborn immature neurons by promoting the NPC differentiation into astrocytes, which is yet to be investigated. Moreover, morphine was reported to upregulate NeuroD1 [21], and Notch1 was demonstrated to be one of its targets [29]. It is likely that a network of Notch1 signaling pathways, involving multiple miRNAs and other factors, participate in the morphine-induced neurogenesis.

Our further investigation on morphine-related contextual memory by downregulating miR-34c demonstrates a coincidental increase of neuronal differentiation and CPP retention without affecting its acquisition. Similar results have been reported in several studies by counter-regulating factors in several pathways. Reversing morphine-induced decreases in NeuroD activities and neurogenesis [30], and prolonged the time required for extinction of morphine-induced CPP without affecting its acquisition. Upregulating Prox1 exhibited similar effects [31]. These findings suggest morphine modulates contextual memory by regulating neurogenesis via a network of pathways. Studies have shown that low expression of miR-34c does not reverse the formation of mouse CPP, but whether the number and extent of mouse addiction change requires us to make a step forward. Mice with low expression of miR-34c have prolonged disappearance of addiction, which is inconsistent with our hypothesis that miR-34c alleviates mouse addiction behavior. We next need to establish a mouse model of mental dependence recurrence, detect miR-34c and Notch1 level during addiction, relapse and exogenous intervention in miR-34c level to observe whether it affects the behavior and neurogenesis during the various stages of addiction.

In summary, we have shown that morphine modulates neural differentiation and hence contextual memory via miR-34c/Notch1 pathway. This indicates a potential target for drug addiction treatment. A more detailed network that control adult animals’ neurogenesis and drug memory is yet to be elucidated.

5 Conclusions

This study determined that morphine modulates neural differentiation and hence contextual memory probably by inhibiting the differentiation of NSCs in the adult DG via miR-34c/Notch1 pathway, indicating a potential target for drug addiction treatment. These findings further consolidate the proposal of microRNAs functioning in morphine-induced hippocampal neurogenesis and identify one more factor in this complex network.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81176260).

Authors̕ contribution: H.J.W and C.F.H contributed equally to this work. H.J.W and C.F.H performed the experiments. Z.X.D and C.F.H designed the experiments. H.J.W and C.F.H analyzed the data. H.J.W wrote the paper. Z.X.D revised and edited the manuscript.
Conflict of interest: Authors state no conflict of interest.

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Received: 2019-08-17

Accepted: 2019-12-02

Published Online: 2020-02-28

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/biol-2020-0006

Keywords for this article

miR-34c; morphine; hippocampal neurogenesis; neural differentiation; contextual memory

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BY 4.0