Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors

<div><p>The regulation of adult neurogenesis by opiates has been implicated in modulating different addiction cycles. At which neurogenesis stage opiates exert their action remains unresolved. We attempt to define the temporal window of morphine’s inhibition effect on adult neurogenesis by using the POMC-EGFP mouse model, in which newborn granular cells (GCs) can be visualized between days 3–28 post-mitotic. The POMC-EGFP mice were trained under the 3-chambers conditioned place preference (CPP) paradigm with either saline or morphine. We observed after 4 days of CPP training with saline, the number of EGFP-labeled newborn GCs in sub-granular zone (SGZ) hippocampus significantly increased compared to mice injected with saline in their homecage. CPP training with morphine significantly decreased the number of EGFP-labeled GCs, whereas no significant difference in the number of EGFP-labeled GCs was observed with the homecage mice injected with the same dose of morphine. Using cell-type selective markers, we observed that morphine reduced the number of late stage progenitors and immature neurons such as Doublecortin (DCX) and βIII Tubulin (TuJ1) positive cells in the SGZ but did not reduce the number of early progenitors such as Nestin, SOX2, or neurogenic differentiation-1 (NeuroD1) positive cells. Analysis of co-localization between different cell markers shows that morphine reduced the number of adult-born GCs by interfering with differentiation of early progenitors, but not by inducing apoptosis. In addition, when NeuroD1 was over-expressed in DG by stereotaxic injection of lentivirus, it rescued the loss of immature neurons and prolonged the extinction of morphine-trained CPP. These results suggest that under the condition of CPP training paradigm, morphine affects the transition of neural progenitor/stem cells to immature neurons via a mechanism involving NeuroD1.</p></div

Morphine injections for four days decreased the number of later stage progenitors and immature neurons.

<p>(A-E) Fluorescent images of EGFP labeled cells and multiple neurogenesis markers: Nestin, Sox2, NeuroD, DCX, TUJ-1. There were no or very little Nestin or Sox2 positive neurons that were also EGFP positive (<5%), but there was a high percentage of NeuroD (20%-40%) or DCX (60%-80%) positive cells colocalized with EGFP neurons (colocalization marked by white arrow heads. Scale bar: 50 μm). (F-H) Measurement of the numbers of nestin, Sox 2 or NeuroD marked early neural precursors (N = 6/per group, no significant differences). (I-J) Measurement of the numbers of DCX or Tuj-1 labeled type III cells or immature neurons (N = 8/per group, *p<0.05). Statistical significance was determined by two-way ANOVA with a Bonferroni test for post hoc comparisons.</p

Overexpression of NeuroD by injection of lenti-virus in mouse hippocampus.

<p>(A) Fluorescent images of four groups of mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153628#pone.0153628.g005" target="_blank">Fig 5A</a>, sacrificed on day 9) that received different lenti-virus injections (control/ND) and CPP training (saline/morphine injection). Left panel: GFP-BrdU double staining; Right panel: DCX-Tuj-1 double staining (scale bar: 50 μm). (B-C) Measurement of the numbers of EGFP and BrdU labeled neural precursors (N = 8, *p<0.05). (D-E) Measurement of the numbers of DCX and Tuj-1 labeled immature neurons (N = 6, *p<0.05). Data represent mean ± SEM of 6–8 animals in separate experiments. Statistical significance was determined by two-way ANOVA with a Bonferroni test for post hoc comparisons.</p

Overexpression or knock down of NeuroD protein in the system.

<p>(A) Experiment design: Lenti-virus of control oligonucleotide (control), NeuroD cDNA (ND) or NeuroD siRNA (siND) was injected one week prior to the CPP study. A subset of mice from the control and ND groups was sacrificed on day 9 for the following staining analysis. (B) Western blot test of the NeuroD virus <i>in vitro</i>. (C) Immunoactivity test of the NeuroD and NeuroD siRNA virus <i>in vivo</i>. The NeuroD cDNA virus increases the total number of NeuroD expressing cells in the DG by 1.8-fold compared to the no-injection control, while NeuroD siRNA decreases the number of NeuroD expressing cells in the DG to 0.53-fold of the control (N = 4,*p<0.05, **p<0.01). (D) The CPP extinction test measured by time spent in the drug-paired chamber in different groups (N = 6–8, *p<0.05). On day 8, all groups injected with the virus developed CPP to the same extent. However, the group injected with siRNA of NeuroD lost their drug-chamber preference on day 29, while the group injected with the control virus and NeuroD virus lost their preference on day 43 and 78, respectively.</p

Morphine decrease co-localization between BrdU-DCX labeled immature neurons, while increase co-localization between BrdU-GFAP labeled glia.

<p>(A-B) Fluorescent images of BrdU labeled cells colocalized with immature neuron marker DCX and glia marker GFAP (colocalization marked by white arrow heads. Scale bar: 50 μm). (C) Measurement of the colocalization between DCX and BrdU between groups (N = 6/per group, *p<0.05). (D) Measurement of the colocalization between GFAP and BrdU between groups (N = 6/per group, **p<0.01). Data represent mean ± SEM of six to eight animals in separate experiments. Statistical significance was determined by two-way ANOVA with a Bonferroni test for post hoc comparisons.</p

Morphine injection decreases adult-born DGCs labeled with EGFP only after going through a CPP paradigm.

<p>(A) Experiment design. On days 1–3, two groups of animals started habituating to the CPP apparatus and received 200 mg/kg BrdU i.p. injections once daily for three days, while the other two groups only received the BrdU injection. Pre-conditioning preference scores were recorded on day 3. From days 4 to 7, mice received saline injections in the morning and saline or morphine injections in the afternoon; after the injection, group 1/2 mice were put back in their homecage, while group 3/4 mice were trained in the CPP chamber for 30 min. After four days of training, group 3 and group 4 mice were tested for their post-conditioning preferences in a 15 min drug-free post-condition test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153628#pone.0153628.s001" target="_blank">S1 Fig</a>). On day 9, a subset of mice that did not receive the BrdU injections before were injected with BrdU one time 2 h prior to perfusion. All animals were sacrificed on day 9. (B) Comparison of the images of EGFP fluorescent from four groups of mice. (C) Fluorescent images of BrdU labeled newborn cells (Red) from four groups (Antigen-retrieval with acid often disrupted morphology of the granular layer). (D) Neural proliferation measured by average number of EGFP labeled newborn granular cells (count every one in eight brain sections, add together) in total dentate gyrus (N = 10/per group, *p<0.05, **p<0.01). (E) Neural progenitors’ survival was measured by the average number of BrdU (injected three days prior to CPP) labeled granular cells in the mouse dentate gyrus (N = 6/per group, *p<0.05). Data represent mean ± SEM of six to 10 animals in separate experiments. (F) Comparison of the fluorescent images of BrdU-labeled cells when injected on day 1–3 or day 9. (White arrow: BrdU-labeled cells colocalized with EGFP fluorescent; White arrowhead: BrdU-labeled cells did not colocalize with EGFP positive cells). (G) Neural proliferation measured by average number of BrdU (injected 2 h before perfusion) labeled granular cells in the mouse dentate gyrus (N = 4/per group, p>0.05). Statistical significance was determined by two-way ANOVA with a Bonferroni test for post hoc comparisons.</p

The expression levels from each respective initiation codon within the mouse MOR uORF region

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> () A schematic summary of various in-frame constructs. Mouse MOR uORF regions were fused in-frame to the luciferase gene. Initiation codons with vertical dotted lines indicate point mutations of ATG to ACG. () Autoradiogram of the proteins translated by a coupled transcription/translation system in the presence of [S]-methionine from representative translations. Proteins were separated by 10% SDS-PAGE (LUC, Fused Protein). Quantification of mRNA levels from the cell-free transcription/translation system was performed by RT-PCR (+/− RT). Expression levels from each initiation codon were normalized against those of SP6-LUC. Normalized mRNA levels (lower panel). M: 100 bp molecular weight markers. () Relative strength of each initiation context within the mouse MOR UTR as determined by translation. The LUC ATG context is designated 1.00

Inhibition of translation by the uORF is independent of its peptide sequence and )

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> Schematic representations of the DNA constructs used in this study. Shaded triangles indicate point mutations to the Stop (S) codon; vertical dotted lines indicate point mutations of ATG to ACG (M) ( and ). Forty-eight hours after transfection, cells were trypsinized and examined for luciferase and ß-galactosidase activities, as well as by RNA extraction and transcript quantification. Relative LUC activity and mRNA levels were expressed as the ratio LUC/ß-gal and LUC/LacZ, as described in Materials and Methods

Toeprinting analyses of mouse MOR containing uORFs that regulate ribosome leaky scanning

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> Synthetic RNA (100 ng) transcripts were used to program translation mixtures derived from RRL. Mouse MOR-LUC RNA containing the wild-type uORFs [uAUG(+)] or deleted of all three uORFs [uAUG(−)] were incubated at 30°C for 15 min in reaction mixtures without cycloheximide (None) or supplemented with cycloheximide (Cyh) either prior to incubation (T0) or after 5 min of incubation at 30°C (T5). Control samples of RNA in reaction mixtures without extract (-EXT) or reaction mixtures containing extract without mRNA (-RNA) are indicated. The positions of the cDNA extension products corresponding to the mRNA 5′ end, uORF initiation (open triangles, asterisks) and LUC initiation codon (filled triangle) by radiolabeled mToe2 primer are indicated. During leaky scanning, ribosomes scan and load at either the upstream or downstream start codons. Adding cycloheximide at T0 traps ribosomes at the positions where they first load on the mRNA. Adding it during steady-state translation (T5) traps ribosomes where they are loaded, following the primary initiation event and subsequent reinitiation events

Additional file 1: of Opioid doses required for pain management in lung cancer patients with different cholesterol levels: negative correlation between opioid doses and cholesterol levels

A list of current patients’ information. The records of 282 patients were listed after removing primary information. ID was the reference number provided by the authors for easier accession. “Chol level” represented the serum cholesterol level measured and recorded. “Year” represented when the initial diagnosis of cancer was made. “Initial dose”, “stable dose” and “converted dose” represented the initial dose opioid administration, the final dose of opioids used for analysis and the dose when converted to equivalent oxycodone hydrochloride, respectively. The “increased” column, “1” or “0” were used to represented that “stable dose” was higher than “initial dose”. If the patient has one or multiple additional measurement of cholesterol during the first month after opioid administration, the cholesterol level with largest difference from initial cholesterol level was selected, recorded in “Chol level with largest difference” column, and normalized to the initial cholesterol level. (XLSX 43 kb