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. 2005 Feb 1;102(5):1725-30.
doi: 10.1073/pnas.0406797102. Epub 2005 Jan 19.

Mu-opioid Receptors Modulate the Stability of Dendritic Spines

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Free PMC article

Mu-opioid Receptors Modulate the Stability of Dendritic Spines

Dezhi Liao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Opioids classically regulate the excitability of neurons by suppressing synaptic GABA release from inhibitory neurons. Here, we report a role for opioids in modulating excitatory synaptic transmission. By activating ubiquitously clustered mu-opioid receptor (MOR) in excitatory synapses, morphine caused collapse of preexisting dendritic spines and decreased synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Meanwhile, the opioid antagonist naloxone increased the density of spines. Chronic treatment with morphine decreased the density of dendritic spines even in the presence of Tetrodotoxin, a sodium channel blocker, indicating that the morphine's effect was not caused by altered activity in neural network through suppression of GABA release. The effect of morphine on dendritic spines was absent in transgenic mice lacking MORs and was blocked by CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2), a mu-receptor antagonist. These data together with others suggest that endogenous opioids and/or constitutive activity of MORs participate in maintaining normal morphology and function of spines, challenging the classical model of opioids. Abnormal alteration of spines may occur in drug addiction when opioid receptors are overactivated by exogenous opiates.

Figures

Fig. 1.
Fig. 1.
MORs were clustered in mature synapses in both cultured cortical and hippocampal neurons. (A) A 3-week-old neuron (Left) and a 1-week-old neuron (Center) were stained with a polyclonal rabbit antibody against MORs. (Right) The appropriate peptide blocked the staining of MORs. (BF) MORs (Left, red) were double-stained with synaptophysin proteins (B and C), NMDA receptors (D), or AMPA receptors (E and F Center, green). (Right) Overlays of the Left and Center images. (C and F) Mouse cortical neurons. (B, D, and E) Rat hippocampal neurons. (G) An experiment that was similar to E Center but with peptide block. (H) Quantification of colocalization. Sμ/S, synaptophysin clusters that were colocalized with MORs (Sμ) versus total number of synaptophysin clusters (S). μS/μ, MOR clusters that were colocalized with synaptophysin (μS) versus total number of MORs (μ). Aμ/A and μA/μ, similar quantifications for MORs and AMPA receptors; Nμ/N andμN/μ, quantifications for MORs and NMDA receptors. The last two bars (m) show data from mouse cortical neurons. Arrows denote clusters of MORs and their colocalization with synaptophysin proteins, NMDA receptors, and AMPA receptors. Note that synaptophysin clusters sometimes surrounded MOR clusters (C Left, indicated by two arrows).
Fig. 5.
Fig. 5.
Chronic treatment with morphine changed the morphology of dendritic spines even in the presence of TTX. (A) Changes in dendrites before and after treatment with morphine in a neuron that has been chronically treated with TTX for 1 week. TTX was continuously present during the treatment of morphine. Triangles indicate spines that gradually shrank to become tiny philopodia. Arrows indicate where protrusions disappeared. (B) A control neuron that has been treated with TTX alone for 1 week is shown. Arrows indicate no changes in spines. (C and D) Density of protrusions and spines before (b) and after the application of morphine (1–3 days). (E) Chronic treatment with morphine significantly decreased the size of dendritic protrusions (+Mor). Morphine's effect was blocked by naloxone (N+M) but not by TTX (T+M).
Fig. 2.
Fig. 2.
Morphine and endogenous opioids modulate excitatory synaptic transmission. (A)(Left) Five consecutive traces of mEPSCs that were recorded from a 3-week-old control neuron. (Right) Similar traces from a morphine-treated neuron. (B)(Left) An averaged trace recorded from a morphine-treated neuron for 15 min (black trace) was compared with a similar averaged trace from a control neuron (gray trace). (Right) The black trace was rescaled to have the same amplitude as the gray trace. Note that morphine changed the time course of mEPSC responses by shortening both the rising and decaying phases. (CF) Various parameters of mEPSCs were compared among five groups of experiments (all were 3-week-old neurons). Ctl, untreated; Mor, morphine-treated; M+Ctp, morphine plus CTOP; M+Nal, morphine plus naloxone; Nal, naloxone alone. (GI) Various parameters of mEPSCs were compared between a 1-week-old untreated neuron (open bars) and a morphine-treated neuron (filled bars).
Fig. 3.
Fig. 3.
Morphine facilitated the retraction of dendritic spines, whereas naloxone had opposite effects. (A) Images of an EGFP-labeled cultured rat neuron were taken before and at various times after morphine application. Arrows denote loss of preexisting protrusions. (B) A rat neuron that was treated with both morphine and naloxone. Arrows denote the emergence of new spines after 3 days. (C) An untreated rat neuron. Arrows denote unchanged spines. (D) A neuron that was treated with naloxone alone. Arrows indicate new spines that emerged after only 3 h of naloxone application. The triangle indicates that the general shape of dendrites also was changed after 3 days. (E and F) The density of dendritic protrusions and spines in four groups of neurons. (G)(Upper) Morphine-treated neurons were double-stained with DAPI (Left) and TUNEL (Right). (Lower) Staurosporin-treated neurons were positive control. (H) The proportion of apoptotic cells versus total number of cells in four groups of neuronal cultures: Ctl, untreated control; Act, Actinomycin D; Sta, staurosporin; Mor, morphine. (I) The density of neurons (number of neurons per mm2 area) that are estimated from differential interference contrast images in morphine-treated (+M) and untreated (–M) cultures.
Fig. 4.
Fig. 4.
MORs are important for the morphine modulation of dendritic spines. (AC) Images were processed with deconvolution. (A) A cortical neuron that was cultured from control mice before and after application of morphine is shown. Top two arrows indicate two dendritic spines that gradually retracted and became thin tiny protrusions. Bottom two arrows indicate two dendritic spines that disappeared in 3 days. (B) A neuron that was cultured from MOR knockout mice before and after morphine treatment is shown. (C) A rat neuron before and after the double treatment of CTOP and morphine is shown. Arrows in B and C denote an increase in the density of dendritic spines. (D) The number of dendritic spines per 100 μm of dendrites in morphine-treated control mouse neurons (open bars) and μ receptor knockout mouse neurons (filled bars). (E) The density of spines in three groups of drug-treated rat neurons. (F) An untreated control and a morphine-treated EGFP-labeled neuron were stained alive with Cy3 (red)-conjugated antibody against the N terminus of GluR1 subunits. Arrows denote protrusions that contain N-GluR1; triangles denote protrusions that contain no detectable GluR1. (G) The proportion of dendritic protrusions (Left) and spines (Right) that contain AMPA receptors in morphine-treated neurons (+M) and untreated neurons (–M).

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