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1Departments of Neuroscience and 2Pharmacology, Basic Research Center on Molecular and Cell Biology of Drug Addiction, The University of Minnesota, Minneapolis, Minnesota
Submitted 31 July 2006; accepted in final form 16 November 2006
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ABSTRACT |
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INTRODUCTION |
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; Madison and Nicoll 1988; Williams et al. 2001; Wimpey and Chavkin 1991). AMPA receptors, particularly GluR1 subunits, are reported to be important for morphine tolerance and dependence (Stephens and Mead 2003; Vekovischeva et al. 2001), suggesting that morphine might also modulate the function and morphology of excitatory synapses. Most excitatory glutamatergic synaptic transmission occurs in dendritic spines (Harris and Kater 1994; Hering and Sheng 2001; Hering and Sheng 2001; Kennedy 1997, 2000; Nimchinsky et al. 2002). A recent study showed that self-administration of morphine, which maintained a high plasma concentration of morphine for a prolonged time, decreased the density of dendritic spines by 50% in the hippocampus of adult rats (Robinson et al. 2002). A previous publication by our group also showed that chronic treatment with morphine could alter both the function and morphology of excitatory synapses (Liao et al. 2005). However, chronic postsynaptic effects of opioids other than morphine on mature excitatory synaptic transmission have not yet been published. Here we report the differential effects of three opioids, including morphine, etorphine, and DAMGO, on miniature excitatory postsynaptic currents (mEPSCs) in mature cultured hippocampal neurons (>3 wk in vitro).
Different opioids are known to have differential effects on the internalization of mu opioid receptors (MORs). Agonist-induced internalization of opioid receptors has been suggested to be one of the alterations in opiate addiction and tolerance (Dang and Williams 2004, 2005; von Zastrow et al. 2003). Morphine induces little receptor internalization in most cell types, including cultured hippocampal neurons, whereas other opioids, such as DAMGO and etorphine, cause obvious receptor internalization (Alvarez et al. 2002; Bailey et al. 2003; Kovoor et al. 1998; Minnis et al. 2003; Sternini et al. 1996; von Zastrow 2001; Whistler and von Zastrow 1998; Yu et al. 1997). This property of morphine has been proposed to be responsible for the continued signaling by morphine, which may cause downstream adaptations that mediate addiction and tolerance (Finn and Whistler 2001; He et al. 2002; Whistler et al. 1999). The distinct difference between the abilities of morphine and other opioids in inducing receptor internalization raises an important question: Do "noninternalizing" opioids such as morphine have the same effect on excitatory synaptic transmission as "internalizing" opioids? Primary hippocampal cultures were used in the current study because the hippocampus is one of the regions that contain the highest levels of MOR (Arvidsson et al. 1995) and is a part of the "learning and memory circuits, " which have been implicated in drug addiction in recent models and experiments (Biala et al. 2005; Fan et al. 1999; Kelley 2004; Nestler 2002; Vorel et al. 2001). In this current study, cultured hippocampal neurons were chronically treated with several opioids including morphine, DAMGO, and etorphine for >3 days. Our results revealed that "internalizing" opioids such as etorphine and DAMGO increased the frequency of mEPSCs, an effect that was opposite to morphine's effect. These and other results together indicate that receptor internalization can modulate opioid-induced postsynaptic plasticity of excitatory synaptic transmission.
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METHODS |
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A 25-mm glass coverslip (thickness, 0.08 mm) was glued to the bottom of a 35-mm culture dish with a 22-mm hole using silicone sealant as previously described (Lin et al. 2004). Dissociated neuronal cultures from rat hippocampus and striatum at postnatal days 1–2 were prepared as previously described (Brustovetsky et al. 2001; Liao et al. 1999, 2001). The same method was used to make dissociated neurons from mouse hippocampus. To prepare cortico-striatal cultures, neurons were dissociated from the cortex (frontal and parietal) and the striatum, respectively. Equal number of dissociated cortical and striatal neurons were mixed together and plated at a density of 1 x 106 per dish (each type contained 0.5 x 106). All rat and mouse studies were approved by University of Minnesota IACUC and were performed in accordance with institutional and federal guidelines. Neurons were plated onto prepared culture dishes at a density of 1 x 106 cells/dish. The age of cultured neurons was counted from the day of plating, one day in vitro (DIV). To label dendrites, neurons 5–7 DIV were transfected with plasmids encoding appropriate molecules as described in the text (Lin et al. 2004).
Electrophysiology
mEPSCs were recorded from cultured hippocampal neurons as previously described (Liao et al. 2005). Neurons at 21 DIV were treated with various opioid drugs for 3 days before recording. No opioid drug was present during the recording. Visual fields were randomly moved to approximate the center of the culture dish. Attempts were made to patch the first encountered "spiny" transfected neuron expressing fluorescence proteins or fluorescence protein-tagged proteins. If the establishment of whole cell configuration was not successful, the second encountered transfected neuron was attempted. No more than two neurons were attempted from the same culture dish. mEPSCs were recorded at holding potentials of –55 
–60 mV and filtered at 1 kHz. Input and series resistances were checked before and after the recording of mEPSCs, which lasted 10–30 min. There were no significant difference in the series resistances and input resistance among various groups of experiments. One recording sweep lasting 200 ms was sampled for every 1 s. mEPSCs were recorded in cultured dissociated neurons in standard Earle's balanced salts solution (EBSS) at room temperature with 200 µM APV [ N-methyl-D-aspartate (NMDA) receptor blocker; D,L-form, the concentration is equivalent to active form at 100 µM], 1 µM TTX (sodium current blocker), and 100 µM picrotoxin (GABA receptor antagonist), gassed with 95% O2 -5% CO2. To increase the number of mEPSCs, an extra 2 mM Ca2+ and 1 mM Mg2+ were added to the bath solution. The internal solution in the patch pipette contained (in mM) 100 cesium gluconate, 0.2 EGTA, 0.5 MgCl2, 2 ATP, 0.3 GTP, and 40 HEPES (pH 7.2 with CsOH).
All mEPSCs were analyzed with the MiniAnalysis program designed by Synaptosoft Inc. Detection criteria for mEPSCs was set as the peak amplitude >3 pA. Each mEPSC event was visually inspected and only events with a distinctly fast-rising phase and a slow-decaying phase were accepted. The frequency and amplitude of all accepted mEPSCs were directly read out by using the analysis function in the MiniAnalysis program. The amplitudes of all events in all neurons in each experimental group were pooled together and plotted as a cumulative frequency curve. The Kolmogorov-Smirnov test was used to test the difference between two cumulative frequency distributions. In further statistical analyses, the averaged amplitude of mEPSCs from each neuron was treated as a single sample. These averaged amplitudes were further averaged to calculate the mean amplitude in each experimental group and plotted as histograms. The MiniAnalysis program often erroneously locates the beginning of the rising phase of a small mEPSC event, which leads to a substantial underestimate of rise time. To correct this potential error, each previously accepted event was visually inspected again. An event would further be accepted to another group for time-course analysis only when the yellow spot was at the beginning of the rising phase and the red spot was at the peak of the response (see the tutorial in MiniAnalysis). The rise and decay times of all events in the new group would be estimated and averaged for neuron. The rise time was defined as the interval between the very beginning of the rising phase and the peak, and the decay time was the interval between the peak and 90% of the decaying phase. The averaged parameters from each neuron were treated as single samples in any further statistical analyses. Student's t-tests were used to test the difference between two experimental groups, and ANOVA was used to examine the difference among multiple groups.
Image analysis
Neurons that had been transfected with GFP alone (Figs. 1 and 5) or MOR-GFP and DsRed (Fig. 3) were photographed immediately after the electrophysiological recording (see Electrophysiology for the selection of neurons). In addition, images of MOR-GFP-expressing neurons cultured from the hippocampus and striatum were also photographed to compare the distribution of MORs between these two types of cultures (Fig. 2). For a further comparison, the distribution of MORs in cortico-striatal neurons was also examined (Fig. 2). All digital images were analyzed with the MetaMorph Imaging System (Universal Imaging). Unless stated otherwise, all images of live neurons were taken as stacks (series of optical sections) and were averaged into one image before further analysis. In addition to simple averaging, stacks of images were also processed by deconvolution analyses using the MetaMorph software with the nearest planes. A stack of deconvoluted images was further averaged into a single composite image. A dendritic protrusion with an expanded head 50% wider than its neck was defined as a spine. The number of spines or nonspine protrusions from one neuron was manually counted and normalized as number per 100 µm of dendritic length. One-way ANOVA was used for comparison among multiple groups of data (n, number of neurons; P < 0.05, significant). If the ANOVA test indicated significant changes, a t-test was used to further test the significance. If a difference passed the ANOVA test and t-test (P < 0.05), we considered this change to be statistically significant. To measure the fluorescence intensity in dendrites and soma, regions of interest were highlighted and selected in the MetaMorph program with the "autothreshold bright objects" function, and the averaged fluorescence intensity in each region was calculated. To highlight dendritic protrusions and spines, the detection threshold was set at 75% of the fluorescent intensity in the center of the dendritic protrusion or spine to be measured. The spine was manually separated from the dendrite using the line tool, and the area and fluorescence intensity of highlighted dendritic spines were measured by the MetaMorph program. In neurons expressing both MOR-GFP and DsRed, all regions were first highlighted and selected in the DsRed image and then transferred to the MOR-GFP image for further analyses. All data are reported as means ± SE, *P < 0.05, **P < 0.01, ***P < 0.001.
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RESULTS |
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It is well known that etorphine and DAMGO can rapidly induce the internalization of MORs, whereas morphine induces little or no internalization in hippocampal neurons (Celver et al. 2004; Cox 2005). This fact led us to hypothesize that various opioid agonists might have differential effects on excitatory synaptic transmission depending on their abilities to internalize the opioid receptors. mEPSCs were recorded in 3-wk-old GFP-labeled neurons with clearly visible dendritic spines (Fig. 1A). Chronic treatment with morphine (10 µM) caused mEPSCs to become smaller and faster than untreated control (Fig. 1B) and significantly decreased the amplitude, frequency, rise time, and decay time of mEPSCs (Fig. 1, C—F, n = 10 in each group; 
and 
). In contrast, DAMGO (1 µM) and etorphine (1 µM) had no significant effect on the amplitude and time kinetics of mEPSCs (Fig. 1, C, D, and F) but significantly increased their frequency (Fig. 1E), indicating that internalizing opioids such as DAMGO and etorphine can induce effects that are distinctly different from those of morphine. These results support our hypothesis that MOR internalization modulates opioid-induced plasticity of dendritic spines.
Distribution of MORs in cultured hippocampal neurons is markedly different from cultured striatal and cortico-striatal neurons
It is still controversial whether morphine's effects are due to pre- or postsynaptic changes (Liao et al. 2005; Williams et al. 2001). One potential reason for this controversy is that the distribution of MORs might be different in various types of neurons. To address this controversy, neurons were cultured from either the hippocampus (Fig. 2A) or the striatum of rats (Fig. 2, B and C) and were transfected with MOR-GFP. In the hippocampal neurons, MOR-GFP was predominantly expressed in dendrites and was clustered and concentrated in dendritic spines after 21 DIV (Fig. 2A). This distribution is very similar to that of endogenous MORs as described in our previous studies (Liao et al. 2005). In these cultured hippocampal neurons, 91% of MOR-GFP-expressing neurons were spiny neurons, which were likely pyramidal neurons (Fig. 2F; n = 10 dishes). This preferential expression of MOR-GFP is not surprising because previous immunohistochemical studies show that MORs are exceptionally abundant in the dendrites of hippocampal pyramidal neurons (Arvidsson et al. 1995). In contrast, MOR-GFP molecules were predominantly expressed in the axon-like processes of nonspiny neurons in primary striatal cultures after 21 DIV (only 24% of MOR-GFP-expressing neurons were spiny neurons; n = 10 dishes; Fig. 2, B–D and F). Strikingly, many dishes of these striatal cultures were often extensively covered by numerous green thin thread-like axons. These results clearly demonstrate that the cellular distribution of MORs might vary in different regions of the brain. Occasionally, MORs also expressed in nonspiny neurons in cultured hippocampal neurons. Interestingly, MORs were exceptionally abundant in axon-like processes in these nonspiny neurons, which might be GABAergic neurons. This observation reveals that the distribution of MORs might vary in different types of neurons even in the same region of the brain. Therefore morphine might induce either pre- or postsynaptic changes in specific types of neurons. The diverse MOR distribution in various types of neurons might provide reconciliation for many previously reported pre- or postsynaptic effects of opioids.
It has been previously reported that cultured striatal neurons contained almost no dendritic spines if they were grown alone probably due to lack of glutamatergic presynaptic inputs (Segal et al. 2003). It is possible that the strong expression of MOR-GFP in axon-like processes in nonspiny neurons (Fig. 2C) might result from the lack of glutamatergic inputs. Therefore we further examined the distribution of MOR-GFP in neurons of cortico-striatal cultures (50% neurons from the frontal and parietal cortex; 50% from the striatum; see Experimental procedures), which should contain abundant glutamatergic inputs (Segal et al. 2003). In cortico-striatal cultures, 45% of MOR-GFP-expressing neurons were spiny neurons and 55% were nonspiny neurons (Fig. 2F, n = 14 dishes). Consistent with results in Fig. 2, A—D, MOR-GFP molecules were clustered in dendritic spines in spiny neurons (see the arrows in Fig. 2E, right) and were strongly expressed in axon-like processes in nonspiny neurons (see 
in Fig. 2E, left). These results indicate that the differential distribution of MOR-GFP in spiny and nonspiny neurons is unlikely to result from the lack of glutamatergic inputs. Nevertheless, further in vivo experiments are needed to test whether endogenous MORs are indeed differentially distributed in spiny and nonspiny neurons in intact animals.
Opioids postsynaptically modulate the morphology of excitatory synapses in cultured hippocampal neurons
A distinct characteristic of molecules that can modulate postsynaptic function of excitatory synapses is that these molecules often cluster in dendritic spines (Kim and Sheng 2004; Malinow et al. 2000). MOR-GFP molecules clustered at dendritic spines in cultured rat hippocampal neurons and most MOR-GFP-expressing hippocampal neurons (91%) were spiny neurons (Fig. 2). The relative homogeneity of these hippocampal neurons gave us a useful tool to test postsynaptic effects of opioids. To address the issue of postsynaptic MOR activities, two plasmids encoding GFP-tagged µ-opioid receptors (MOR-GFP) and DsRed were co-transfected into hippocampal neurons that were cultured from MOR knock-out mice (MOR–/–) (Loh et al. 1998; Fig. 3). mEPSCs were recorded to examine the function of dendritic spines (see the recording pipette in Fig. 3A, left; mEPSC traces in Fig. 4), DsRed was used to label dendritic morphology (Fig. 3A, middle), and MOR-GFP was used to rescue the MOR deficit in cultured neurons (Fig. 3A, right). Similar to cultured rat neurons (Fig. 2), MOR-GFP molecules also clustered in dendritic spines in mouse cultures (Fig. 3, B and E). Chronic treatment with morphine for 3–6 days significantly decreased the density of dendritic protrusions and dendritic spines (n = 10 in each group, Fig. 3, C and F). In contrast, DAMGO, another MOR agonist, significantly increased the density of dendritic protrusions and spines after 3–6 days of treatment (Fig. 3, D and F). Because MORs were absent in all untransfected cells, including nearby neurons and glial cells, these results provide evidence that morphine can directly act on postsynaptic excitatory neurons, causing the collapse of dendritic spines. In contrast, DAMGO, which is a known "internalizing" opioid, causes effects that are opposite to morphine via postsynaptic MORs.
Opioids postsynaptically modulate the function of excitatory synapses in cultured hippocampal neurons
To further test whether morphine and DAMGO can postsynaptically modulate the function of dendritic spines, mEPSCs were recorded in neurons co-transfected with DsRed and MOR-GFP (see Figs. 3A, left for configuration, and 4A for sample traces). The transfected neurons (left 3 averaged traces in Fig. 4B) contain exogenous MORs, whereas the untransfected neurons contain neither endogenous nor exogenous MORs (Fig. 4B, right trace). Morphine decreased the frequency, amplitude, rise time, and decay time in neurons that expressed exogenously introduced MORs (Fig. 4, A—F; n = 10 in each group) but did not suppress excitatory synaptic transmission in neurons that did not express MORs (untransfected neurons; Fig. 4, B-F). These results indicate that morphine can directly act on postsynaptic excitatory neurons and postsynaptically suppress excitatory synaptic transmission. In contrast, the frequency of mEPSCs in DAMGO-treated MOR-GFP-expressing neurons was significantly higher than that in untreated MOR-GFP-expressing neurons (Fig. 4E). This effect is clearly opposite to that of morphine. This result further confirms that "internalizing" opioid may cause effects that are opposite to morphine.
As controls, neurons were cultured from the hippocampus of wild-type mice with similar genetic background (for breeding, see Loh et al. 1998). Morphine decreased the density of spines and DAMGO had the opposite effect in these neurons with endogenous MORs (Fig. 5, A and B; n = 10 in each group). Morphine significantly decreased the amplitude, frequency, rise time, and decay time of mEPSCs (Fig. 5, C–F; n = 10 in each group). DAMGO had no effect on the amplitude and time-kinetics of mEPSCs but significantly increased the frequency of mEPSCs, an effect that was opposite to morphine (Fig. 5, C–F). Data in Figs. 3–5 together indicate that exogenously introduced MOR-GFP and endogenous MORs can mediate similar chronic effects on excitatory synapses probably through the same signaling pathway.
Acute effects of morphine on mEPSCs in neurons expressing GFP or MOR-GFP
An important but unanswered question is: can morphine induce acute functional plasticity in excitatory synaptic transmission? To examine the acute effects of morphine, three groups of experiments were performed (methodological details are provided in the legend of Fig. 6A). In the control group, no morphine was applied to the GFP-labeled neurons during the recording of mEPSCs (Fig. 6A, left). In the other two groups, mEPSCs were recorded in neurons expressing GFP or MOR-GFP before and after treatment with 10 µM morphine (Fig. 6A, middle and right). In comparison to the baseline before treatment, morphine significantly decreased the amplitude of mEPSCs in neurons expressing MOR-GFP after 25 min of treatment (Fig. 6, B–D and F). Morphine had no significant effect on mEPSC frequency, rise time, and decay time in neurons overexpressing MOR-GFP (Fig. 6, E–G), indicating that morphine might regulate these parameters through either a different mechanism or the same mechanism but in different temporal phase. Interestingly, morphine had no effect on neurons expressing GFP alone (Fig. 6), indicating that the endogenous level of MORs is relatively low under normal conditions, which normally allow only slow opioid-induced plasticity of dendritic spines.
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DISCUSSION |
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This study demonstrates that different MOR agonists can have differential postsynaptic effects on excitatory synaptic transmission. Previous studies showed that chronic treatment with morphine can cause collapse of dendritic spines and suppression of excitatory synaptic transmission (Liao et al. 2005; Robinson and Kolb 2004; Robinson et al. 2002). In a proposed "RAVE" (receptor activity vs. endocytosis) hypothesis, morphine's inability to induce receptor internalization allows the continuous signaling of MORs, which contributes to addictive liability and tolerance development (He et al. 2002; Whistler et al. 1999). Morphine causes little receptor internalization in most cell types but can strongly lead to drug addiction and tolerance (Alvarez et al. 2002; Bailey et al. 2003; Kovoor et al. 1998; Minnis et al. 2003; Sternini et al. 1996; von Zastrow 2001; Whistler and von Zastrow 1998; Yu et al. 1997). In contrast, DAMGO and etorphine cause robust internalization and desensitization of opioid receptors (Dang and Williams 2004; Kieffer and Evans 2002; von Zastrow et al. 2003). Naloxone increased the density of spines and the frequency of mEPSCs, effects that are opposite to those of morphine (Liao et al. 2005). If DAMGO and etorphine can cause robust internalization of MORs, they should induce effects that are similar to naloxone because the signaling of MORs might be interrupted when MORs are removed from cell surface. This is in fact what we saw. Therefore the observed correlation between the agonist's ability to induce receptor internalization and to alter excitatory synaptic transmission shown in this study suggests that receptor internalization can modulate opioid-induced synaptic plasticity. Activity-dependent alterations in excitatory synaptic transmission is widely believed to be the cellular model of learning and memory (Martin et al. 2000) and addiction is proposed to be a pathological form of learning and memory (Nestler 2002). Therefore opioid-induced plasticity of excitatory synaptic transmission might contribute to opiate addiction. In support of the RAVE hypothesis, this study demonstrates that opioids with different abilities in inducing receptor internalization can cause different changes in excitatory synaptic transmission, which might account for various addictive liabilities among opiates.
Morphological changes and mEPSCs
AMPLITUDE AND FREQUENCY OF MEPSCS.
In this study, chronic treatment with morphine for >3 days decreased both the frequency of mEPSCs and the density of dendritic spines, whereas chronic treatment with DAMGO or etrophine had opposite effects. Clearly, our current results cannot prove the causal relationship between the morphological and functional changes. Nonetheless, a decrease in the frequency of mEPSCs is consistent with a decrease in the density of dendritic spines. In the classical mathematical model (del Castillo and Katz 1954), the strength of evoked excitatory postsynaptic potentials (EPSPs) in neuromuscular junction (NMJ) is determined by quantal content (m) and quantal size (q). Quantal content is the average number of released vesicles per stimulus. m = n*p, where p is the probability of release and n is the number of releasing sites. Similarly, the frequency of mEPSPs in NMJ or mEPSCs in the CNS, Fm = n*p', where p' is the probability of spontaneous release and n is the number of releasing sites. In the CNS, mEPSCs are often recorded by a patch electrode in the soma of a neuron so that the electrode receives signals from all synapses in that neuron. In this case, an increase in the density of dendritic spines would also increase the number of apposing presynaptic termini, leading to an increase in the total number of releasing sites (n). Therefore an increase in mEPSC frequency is consistent with an increase in the density of dendritic spines. The amplitude of mEPSPs or mEPSCs is equivalent to the quantal size (q) in evoked synaptic transmission. Quantal size is the average postsynaptic response per one released vesicle. As the amount of neurotransmitters per vesicle is assumed to be constant, a decrease in the amplitude of mEPSCs would indicate a loss of AMPA receptors in the postsynaptic membrane (Liao et al. 2001).
KINETICS OF MEPSC.
The mechanism underlying the morphine-induced decrease in rise and decay times of mEPSCs is unknown. It is likely that this alteration is largely attributed to by morphine-induced shortening of dendritic spines (see the theoretical model in Fig. 7). Assuming that a dendrite is a passive cable, dendritic membrane is never perfectly voltage-clamped when a voltage clamp is applied using a patch electrode on the soma (Rall and Segev 1985; Spruston et al. 1993). The clamping voltage along a dendrite progressively decreases when the electrotonic distance increases as described by the equation: V (X)/Vo = cosh (L – X)/cosh L (continuous cable model) (Carnevale and Johnston 1982; Rall and Segev 1985). Due to this attenuation of clamping voltage, the rise time of an EPSC from a remote synapse would be slower than that from a proximate synapse (Spruston et al. 1993). The electrical resistance of a spine stem (or neck) is probably the most important parameter that determines the electrical behavior of a dendritic spine (Johnston and Wu 1995; Segev and Rall 1988; Tsay and Yuste 2004). The high electrical resistance of the spine stem is expected to dramatically increase the electrotonic distance from the head of dendritic spines to the dendritic shaft, and consequently the total electrotonic distance from the spine head to the soma (Fig. 7A). When the length of the spine stem is decreased by morphine (Fig. 7 B–D), the electrotonic distance would be decreased, space-clamp errors would be reduced, and the attenuation of the kinetics of mEPSCs would also be decreased. According to our estimate, the shortening of the spine stem alone would decrease the rise time of mEPSCs by 20% and a decrease in Ri' (resistivity of the spine neck) might account for another 10% reduction (Fig. 7D). The most critical assumption in our theoretical model is that the resistivity of spine neck (Ri') is much higher than that in the dendrite (Ri). This difference in resistivity cannot be explained by geometry alone and requires that the neck of spine must be able to obstruct the free movement of ions. The neck of a dendritic spine is now known to pose a barrier to the diffusion of molecules (Bloodgood and Sabatini 2005). In addition, morphine might decrease the membrane capacitance by decreasing the size of the spine head and consequently increase the rise time of mEPSCs by decreasing the charging of the capacitance. Morphine-induced change in glutamate uptake might also contribute to the altered kinetics of mEPSCs (Xu et al. 2003). Perhaps the major contributor to the decrease in mEPSC kinetics is the morphine-induced reduction in the electrical resistance at the spine neck (Fig. 7; due to decreases in bc and Ri'), although other factors such as spine size and glutamate uptake may also play minor roles.
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; Thiagarajan et al. 2005), the kinetics of mEPSCs in this current study is very slow. This is mainly due to the advanced age of our cultured neurons (>24 DIV: 21 DIV +3 days of treatment). As we previously reported, the time course of mEPSCs from 3-wk-old neurons is much slower than that from 2-wk-old neurons (Liao et al. 2005). Dendritic spines start to form at 14 DIV and become elongated and stable after 21 DIV (Liao et al. 1999). According to our theoretical model in Fig. 7, the kinetics of mEPSCs from a dendritic spine should be slower than that from a nonspiny synapse. Therefore it is not surprising that mEPSCs in more mature neurons are slower than those in immature neurons. In addition, mEPSCs were recorded only from spiny neurons in this study, whereas mixed types of neurons were patched in previous studies from other groups. Which comes first—morphology or function?
REMOVAL OF AMPA RECEPTORS.
According to the cable theory, the shortening of a spine stem alone should increase the amplitude of mEPSCs as there is less attenuation of synaptic inputs (Tsay and Yuste 2004). Therefore the morphine-induced decrease in mEPSC amplitude cannot be directly caused by the morphological change. It is instead probably due to the removal of synaptic AMPA receptors (Liao et al. 2005). Interestingly, acute perfusion of morphine altered only the amplitude of mEPSCs in neurons expressing MOR-GFP, not affecting the frequency, rise time and decay time of mEPSCs (Fig. 6). This result suggests that the activation of MORs might be able to remove AMPA receptors from dendritic spines before altering spine size or density. As GluR2 subunits of AMPA receptors are important for the growth and/or stability of dendritic spines (Passafaro et al. 2003), it is possible that the morphine-induced collapse of dendritic spines might be a secondary effect due to the loss of AMPA receptors. It remains to be determined whether the removal of AMPA receptors and the collapse of spines are regulated through the same or a different independent signaling pathway.
INTRACELLULAR SIGNALING PATHWAYS.
Morphine treatment requires 1–3 days to cause collapse of dendritic spines in neurons with only endogenous opioid receptors being expressed (Liao et al. 2005). Logically, if morphine requires such a long time to alter dendritic spines, the downstream signaling pathway must also remain available for a prolonged period of time. Even though morphine causes little internalization, most known signaling pathways are rapidly desensitized on the application of morphine (Law et al. 2000). Therefore morphine-induced collapse of dendritic spines is likely to be caused by an unconventional signaling pathway that regulates the actin-cytoskeleton. Both filamin A (Onoprishvili et al. 2003) and Rho GTPases are potential signaling candidates in this pathway (Lippman and Dunaevsky 2005; Luo 2000). A recent previous study by us shows that Rac1, a Rho GTPase, can mediate both the clustering of AMPA receptors and the formation and maintenance of dendritic spines (Wiens et al. 2005). Therefore it is possible that opioid treatment might simultaneously affect the trafficking of AMPA receptors and the morphology of spines by altering Rac1 activity.
PRE- AND POSTSYNAPTIC MECHANISMS.
This current study demonstrates that chronic morphine treatment can postsynaptically modulate the function of dendritic spines. In contrast, numerous previous studies demonstrate that morphine can acutely inhibit presynaptic release of GABA (the "disinhibition" theory) (Madison and Nicoll 1988; Williams et al. 2001; Wimpey and Chavkin 1991). As shown in Fig. 2, the distribution of MORs in hippocampal neurons is distinctly different from striatal and cortico-striatal neurons. Therefore it is not surprising that different research groups might obtain either pre- or postsynaptic effects if different cell types were examined. However, we believe that MOR-mediated effects on hippocampal pyramidal neurons are mainly postsynaptic for the following reasons. 1) The pyramidal layer in the hippocampus is one of the regions that contain the highest levels of MORs and the dendritic trees of pyramidal neurons clearly contain abundant MORs (Arvidsson et al. 1995). 2) When adult animals were chronically exposed to self-administrated morphine (the plasma concentration is high for a long time in this group), the strongest effect on dendritic spines was observed in the CA1 region of the hippocampus (Robinson et al. 2002). 3) Endogenous MORs are clustered in dendritic spines in cultured hippocampal neurons (Liao et al. 2005). 4) MOR-GFP molecules are clustered in dendritic spines and are preferentially expressed in spiny neurons in primary hippocampal cultures (Fig. 2). 5) In neurons cultured from MOR knock-out mice, chronic treatment with morphine only suppressed mEPSCs in neurons expressing exogenous MOR-GFP (Figs. 3 and 4).
Conclusions
This study has contributed two key pieces of knowledge explaining normal opioid neurophysiology and pathology. First it provides evidence that opioids can postsynaptically modulate the structure and function of dendritic spines in cultured hippocampal neurons. Endogenous opioids may participate in maintaining normal morphology and function of spines via this postsynaptic plasticity, whereas abnormal alteration of spines may occur when MORs in dendritic spines are overactivated. The normal level of endogenous MORs allows only slow opioid-induced plasticity of dendritic spines. Second, this study demonstrates a correlation between receptor internalization and opioid-induced plasticity of excitatory synaptic transmission by showing that noninternalizing opioids such as morphine suppresses synaptic transmission, whereas internalizing opioids such as DAMGO and etorphine have the opposite effect. Therefore this study suggests that the receptor internalization might modulate opioid-induced plasticity of excitatory synapses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Liao, Dept. of Neuroscience, The University of Minnesota, 321 Church St. S.E., Minneapolis, MN 55455 (E-mail: liaox020{at}umn.edu)
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REFERENCES |
|---|
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Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 15: 3328–3341, 1995.[Abstract]
Bailey CP, Couch D, Johnson E, Griffiths K, Kelly E, Henderson G. Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine. J Neurosci 23: 10515–10520, 2003.
Biala G, Betancur C, Mansuy IM, Giros B. The reinforcing effects of chronic D-amphetamine and morphine are impaired in a line of memory-deficient mice overexpressing calcineurin. Eur J Neurosci 21: 3089–3096, 2005.[CrossRef][Web of Science][Medline]
Bloodgood BL, Sabatini BL. Neuronal activity regulates diffusion across the neck of dendritic spines. Science 310: 866–869, 2005.
Brustovetsky N, Brustovetsky T, Dubinsky JM. On the mechanisms of neuroprotection by creatine and phosphcreatine. J Neurochem 76: 425–434, 2001[CrossRef][Web of Science][Medline]
Carnevale NT, Johnston D. Electrophysiological characterization of remote chemical synapses. J Neurophysiol 47: 606–621, 1982.
Celver J, Xu M, Jin W, Lowe J, Chavkin C. Distinct domains of the mu-opioid receptor control uncoupling and internalization. Mol. Pharmacol 65: 528–537, 2004.
Cox BM. Agonists at mu-opioid receptors spin the wheels to keep the action going. Mol Pharmacol 67: 12–14, 2005.
Dang VC, Williams JT. Chronic morphine treatment reduces recovery from opioid desensitization. J Neurosci 24: 7699–7706, 2004.
Dang VC, Williams JT. Morphine-induced µ-opioid receptor desensitization. Mol Pharmacol 68: 1127–1132, 2005.
del Castillo J, Katz B. Quantal components of the end-plate potential. J Physiol 124: 560–573, 1954.
Fan GH, Wang LZ, Qiu HC, Ma L, Pei G. Inhibition of calcium/calmodulin-dependent protein kinase II in rat hippocampus attenuates morphine tolerance and dependence. Mol Pharmacol 56: 39–45, 1999.
Finn AK, Whistler JL. Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron 32: 829–839, 2001.[CrossRef][Web of Science][Medline]
Harris KM, Kater SB. Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Annu Rev Neurosci 17: 341–371, 1994.[CrossRef][Web of Science][Medline]
He L, Fong J, von Zastrow M, Whistler JL. Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization. Cell 108: 271–282, 2002.[CrossRef][Web of Science][Medline]
Hering H, Sheng M. Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci 2: 880–888, 2001.[CrossRef][Web of Science][Medline]
Jin W, Chavkin C. Mu opioids enhance mossy fiber synaptic transmission indirectly by reducing GABAB receptor activation. Brain Res 13: 286–293, 1999.
Johnston D, Wu SM-S. Synaptic transmission. III. Dendritic spines and their effects on synaptic inputs. Foundations of Cellular Neurophysiology. A Bradford Book, Cambridge, MA: MIT Press, 1995, p. 400–411.
Kelley AE. Memory and addiction: shared neural circuitry and molecular mechanisms. Neuron 44: 161–179, 2004.[CrossRef][Web of Science][Medline]
Kennedy MB. The postsynaptic density at glutamatergic synapses. Trends Neurosci 20: 264–268, 1997.[CrossRef][Web of Science][Medline]
Kennedy MB. Signal-processing machines at the postsynaptic density. Science 290: 750–754, 2000.
Kieffer BL, Evans CJ. Opioid tolerance-in search of the holy grail. Cell 108: 587–590, 2002.[CrossRef][Web of Science][Medline]
Kim E, Sheng M. PDZ domain proteins of synapses. Nat Rev Neurosci 5: 771–781, 2004.[CrossRef][Web of Science][Medline]
Kovoor A, Celver JP, Wu A, Chavkin C. Agonist induces homologous desensitization of mu-opioid receptors mediated by G-protein-coupled receptor kinases is dependent on agonist efficacy. Mol Pharmacol 54: 704–711, 1998.
Law PY, Wong YH, Loh HH. Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol 40: 389–430, 2000.
Liao D, Lin H, Law PY, Loh HH. Mu-opioid receptors modulate the stability of dendritic spines. Proc Natl Acad Sci USA 102: 1725–1730, 2005.
Liao D, Scannevin RH, Huganir R. Activation of silent synapses by rapid activity-dependent synaptic recruitment of AMPA receptors. J Neurosci 21: 6008–6017, 2001.
Liao D, Zhang X, O'Brien R, Ehlers MD, Huganir RL. Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat Neurosci 2: 37–43, 1999.[CrossRef][Web of Science][Medline]
Lin H, Huganir R, Liao D. Temporal dynamics of NMDA receptor-induced changes in spine morphology and AMPA receptor recruitment to spines. Biochem Biophys Res Commun 316: 501–511, 2004.[CrossRef][Web of Science][Medline]
Lippman J, Dunaevsky A. Dendritic spine morphogenesis and plasticity. J Neurobiol 64: 47–57, 2005.[CrossRef][Web of Science][Medline]
Loh H, Liu HC, Cavalli A, Yang W, Chen YF, Wei LN. mu Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality. Brain Res Mol Brain Res 54: 321–326, 1998.[Medline]
Luo L. Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci 1: 73–180, 2000.[CrossRef][Web of Science][Medline]
Madison DV, Nicoll RA. Enkephalin hyperpolarizes interneurones in the rat hippocampus. J Physiol 398: 123–130, 1988.
Malinow R, Mainen ZF, Hayashi Y. LTP mechanisms: from silence to four-lane traffic. Curr Opin Neurobiol 10: 352–357, 2000.[CrossRef][Web of Science][Medline]
Martin SJ, Grimwood PD, Morris RGM. Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci 23: 649–711, 2000.[CrossRef][Web of Science][Medline]
Minnis JG, Patierno S, Kohlmeier SE, Brecha NC, Tonini M, Sternini C. Ligand-induced mu opioid receptor endocytosis and recycling in enteric neurons. Neuroscience 119: 33–42, 2003.[CrossRef][Web of Science][Medline]
Nestler EJ. Common molecular and cellular substrates of addiction and memory. Neurobiol Learn Mem 78: 637–647, 2002.[CrossRef][Web of Science][Medline]
Nimchinsky EA, Sabatini BL, Svoboda K. Structure and function of dendritic spines. Annu Rev Physiol 64: 313–353, 2002.[CrossRef][Web of Science][Medline]
Onoprishvili I, Andria ML, Kramer HK, Ancevska-Taneva N, Hiller JM, Simon EJ. Interaction between µ opioid receptor and filamin A is involved in receptor regulation and trafficking. Mol Pharmacol 64: 1092–1100, 2003.
Passafaro M, Nakagawa T, Sala C, Sheng M. Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2. Nature 424: 677–681, 2003.[CrossRef][Medline]
Rall W, Segev I. Space-clamp problems when voltage clamping branched neurons with intracellular microelectrodes. In: Voltage and Patch-Clamping With Microelectrodes, edited by Smith TG, Lecar H, and Redman SJ. Bethesda, MD: Am. Physiol. Soc, 1985, p. 191–215.
Robinson TE, Gorny G, Savage VR, Kolb B. Widespread but regionally specific effects of experimenter- versus self-administered morphine on dendritic spines in the nucleus accumbens, hippocampus, and neocortex of adult rats. Synapse 46: 271–279, 2002.[CrossRef][Web of Science][Medline]
Robinson TE, Kolb B. Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 47 Suppl 1: 33–46, 2004.
Segal M, Greenberger V, Korkotian E. Formation of dendritic spines in cultured striatal neurons depends on excitatory afferent activity. Eur J Neurosci 17: 2573–2585, 2003.[CrossRef][Web of Science][Medline]
Segev I, Rall W. Computational study of an excitable dendritic spine. J Neurophysiol 60: 499–523, 1988.
Spruston N, Jaffe DB, Williams SH, Johnston D. Voltage- and space-clamp errors associated with the measurement of electrotonically remote synaptic events. J Neurophysiol 70: 781–802, 1993.
Stellwagen D, Beattie EC, Seo JY, Malenka RC. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci 25: 3219–3228, 2005.
Stephens DN, Mead AN. What role do GluR1 subunits play in drug abuse? Trends Neurosci 26: 181–182, 2003.[CrossRef][Web of Science][Medline]
Sternini C, Spann, M, Anton, B, Keith, DE Jr, Bunnett NW, von Zastrow M, Evans C, Brecha NC. Agonist-selective endocytosis of mu opioid receptor by neurons in vivo. Proc Natl Acad Sci USA 93: 9241–9246, 1996.
Thiagarajan TC, Lindskog M, Tsien RW. Adaptation to synaptic inactivity in hippocampal neurons. Neuron 47: 725–737, 2005.[CrossRef][Web of Science][Medline]
Tsay D, Yuste R. On the electrical function of dendritic spines. Trends Neurosci 27: 77–83, 2004.[CrossRef][Web of Science][Medline]
Vekovischeva OY, Zamanillo D, Echenko O, Seppala T, Uusi-Oukari M, Honkanen A, Seeburg PH, Sprengel R, Korpi ER. Morphine-induced dependence and sensitization are altered in mice deficient in AMPA-type glutamate receptor-A subunits. J Neurosci 21: 4451–4459, 2001.
von Zastrow M. Endocytosis and downregulation of G protein-coupled receptors. Parkinsonism Relat Disord 7: 265–271, 2001.[CrossRef][Web of Science][Medline]
von Zastrow M, Svingos A, Haberstock-Debic H, Evans C. Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs. Curr Opin Neurobiol 13: 348–353, 2003.[CrossRef][Web of Science][Medline]
Vorel SR, Liu X, Hayes RJ, Spector JA, Gardner EL. Relapse to cocaine-seeking after hippocampal theta burst stimulation. Science 292: 1175–1178, 2001.
Whister JL, von Zastrow M. Morphine-activated opioid receptors elude desensitization by beta-arrestin. Proc Natl Acad Sci USA 95: 9914–9919, 1998.
Whistler JL, Chuang HH, Chu P, Jan LY, von Zastrow M. Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23: 737–746, 1999.[CrossRef][Web of Science][Medline]
Wiens K, Lin H, Liao D. Rac1 induces the clustering of AMPA receptors during spinogenesis. J Neurosci 25: 10627–10636, 2005.
Williams JT, Christie MJ, Manzoni O. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev 81: 299–343, 2001.
Wimpey TL, Chavkin C. Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation. Neuron 6: 281–289, 1991.[CrossRef][Web of Science][Medline]
Xu NJ, Bao L, Fan HP, Bao GB, Pu L, Lu YJ, Wu CF, Zhang X, Pei G. Morphine withdrawal increases glutamate uptake and surface expression of glutamate transporter GLT1 at hippocampal synapses. J Neurosci 23: 4775–4884, 2003.
Yu Y, Zhang L, Yin X, Sun H, Uhl GR, Wang JB. Mu opioid receptor phosphorylation, desensitization, and ligand efficacy. J Biol Chem 272: 28869–28874, 1997.
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ABSTRACT
INTRODUCTION
), morphine-treated (
), plotted as histograms. D: cumulative frequency curves of mEPSC amplitudes from untreated (
), morphine-treated (
) and etorphine-treated neurons (gray triangle). Bin size = 0.5 pA. E and F: frequency and time kinetics of mEPSCs in the preceding 4 groups of neurons are plotted as histograms.
). B and C: MOR-GFP is highly concentrated in axon-like processes in nonspiny striatal neurons. 
, axon in C is continued from B at this location. D: enlarged image from the same neuron as in B and C shows that this is a nonspiny neuron. E: cortico-striatal cultures contain both spiny (left) and nonspiny neurons (right). The arrows denote that MOR-GFP is clustered and concentrated in dendritic spines (left). 
, clustering of MORs in dendritic spines. C and D: similar neurons had been treated with morphine and DAMGO for >3 days. E: ratio of fluorescence intensity in the spines vs. adjacent dendrites was estimated in untreated neurons. 
+ bc/