Re-exposure to morphine-associated context facilitated long-term potentiation in the vSUB-NAc glutamatergic pathway via GluN2B-containing receptor activation.

The glutamatergic projection from the ventral subiculum of the hippocampus (vSUB) to the nucleus accumbens (NAc) shell has been reported to play a key role in drug-related behavior. The GluN2B subunit of N-methyl-D-aspartate receptors (NMDARs) in the NAc can be selectively elevated after the retrieval of drug-conditioned memory. However, whether the increased GluN2B-containing NMDARs (GluN2B-NMDARs) are able to alter the synaptic plasticity of the vSUB-NAc glutamatergic pathway remains unclear. Here, we found that the long-term potentiation (LTP) in the vSUB-NAc pathway was facilitated and the GluN2B subunit protein level was elevated in synaptoneurosomes of the NAc shell, but not in the core, following morphine-induced conditioned place preference (CPP) expression in rats. The facilitated LTP was prevented by the GluN2B-NMDAR antagonist RO25-6981. Also, a neurochemical disconnection following microinjection of RO25-6981 into the NAc shell, plus microinfusion of GABA agonist baclofen and muscimol into the contralateral vSUB prevented the expression of morphine-induced CPP. These findings suggest that the retrieval of drug-associated memory potentiated synaptic plasticity in the vSUB-NAc pathway, which was dependent on GluN2B-NMDAR activation in the NAc shell. These findings provide a new explanation for the mechanisms that underlie the morphine-associated-context memory. The GluN2B-NMDARs may be regarded as a potential target for erasing morphine-related memory.

NAc Shell Arc/Arg3.1 Protein Mediates Reconsolidation of Morphine CPP by Increased GluR1 Cell Surface Expression: Activation of ERK-Coupled CREB is Required.

BACKGROUND: Relapse into drug abuse evoked by reexposure to the drug-associated context has been a primary problem in the treatment of drug addiction. Disrupting the reconsolidation of drug-related context memory would therefore limit the relapse susceptibility. METHODS: Morphine conditioned place preference (CPP) was used to assess activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and correlative molecule expression in the Nucleus accumbens (NAc) shell during the reconsolidation of morphine CPP. U0126 and Arc/Arg3.1 antisense oligodeoxynucleotide were adapted to evaluate the role and the underlying mechanism of Arc/Arg3.1 during the reconsolidation. RESULTS: The retrieval of morphine CPP in rats specifically increased the Arc/Arg3.1 protein level in the NAc shell, accompanied simultaneously by increases in the phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2), the phosphorylation of Cyclic Adenosine monophosphate (cAMP) response element-binding (pCREB), and the up-regulation of the membrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors GluR1 subunit level. Intra-NAc shell infusion U0126, an inhibitor of the Mitogen-activated protein kinase kinase (MEK), prevented the retrieval-induced up-regulation of pERK1/2, pCREB, Arc/Arg3.1, and membrane GluR1 immediately after retrieval of morphine CPP. The effect of disrupting the reconsolidation of morphine CPP by U0126 could last for at least 14 days, and could not be evoked by a priming injection of morphine. Furthermore, the specific knockdown of Arc/Arg3.1 in the NAc shell decreased the membrane GluR1 level, and impaired both the reconsolidation and the reinstatement of morphine CPP. CONCLUSIONS: Arc/Arg3.1 in the NAc shell mediates the reconsolidation of morphine-associated context memory via up-regulating the level of membrane of GluR1, for which the local activation of the ERK-CREB signal pathway, as an upstream mechanism of Arc/Arg3.1, is required.

Transcutaneous electrical acupoint stimulation for the treatment of withdrawal syndrome in heroin addicts.

OBJECTIVE: To assess the therapeutic effect of transcutaneous electric acupoint stimulation (TEAS) for the treatment of withdrawal syndrome in heroin addicts. METHODS: A total of 63 male heroin addicts with withdrawal score higher than 20 were recruited in the Detoxification Center of Zhongshan city, Guangdong province, China. They were randomly distributed into two groups: TEAS group (n = 31) received TEAS by using a Han's acupoint nerve stimulator (HANS) model 200A with two output channels, 2-3 sessions per day, 30 minutes per session for 10 consecutive days. Electrical stimulation of alternating frequencies of 2- and 100-Hz with 3 second each, and with intensity of 10-15 mA was applied on Hegu (LI-4) and Laogong (PC-8) points on one hand, and Neiguan (PC-6) and Waiguan (SJ-5) points on the other forearm via electroconductive skin pads of 4 cm × 4 cm in size. The control group (n = 32) was treated with similar procedure except that the leads of the output of the stimulator was disconnected. Assessments of the severity of the withdrawal syndrome were conducted one day before and on each day during the whole treatment period of 10 days. Buprenorphin of 1 mg per day sublingually was provided to all subjects in the first two days, and then to those with withdrawal score over 20 in the following days. RESULTS: The TEAS treatment dramatically alleviated the withdrawal syndrome during heroin detoxification. No significant difference was found in withdrawal scores between the two groups at the beginning of the observation. Withdrawal scores showed a more marked drop in TEAS group than the control starting from the second day, and maintained at a lower level for the whole course of treatment. The area under the curve of withdrawal score in TEAS group was only 40% of that in the control (P < 0.001, two way repeated measures analysis of variance), and the requirement of buprenorphine was only 10% of that in the control. No adverse effects were observed in either group. CONCLUSION: TEAS of 2/100 Hz for 10 days in abrupt abstinence of the heroin addicts resulted in a marked reduction of the withdrawal syndrome as well as a reduced requirement for rescue opioids.

Low- and high-frequency transcutaneous electrical acupoint stimulation induces different effects on cerebral µ-opioid receptor availability in rhesus monkeys.

Although systematic studies have demonstrated that acupuncture or electroacupuncture (EA) analgesia is based on their accelerating endogenous opioid release to activate opioid receptors and that EA of different frequencies is mediated by different opioid receptors in specific areas of the central nervous system, there is little direct, real-time evidence to confirm this in vivo. The present study was designed to investigate the effects of transcutaneous electrical acupoint stimulation (TEAS), an analogue of EA, at low and high frequencies on µ-opioid receptor (MOR) availability in the brain of rhesus monkeys. Monkeys underwent 95-min positron emission tomography (PET) with (11) C-carfentanil three times randomly while receiving 0, 2, or 100 Hz TEAS, respectively. Each TEAS was administered in the middle 30 min during the 95-min PET scan, and each session of PET and TEAS was separated by at least 2 weeks. The results revealed that 2 Hz but not 100 Hz TEAS evoked a significant increase in MOR binding potential in the anterior cingulate cortex, the caudate nucleus, the putamen, the temporal lobe, the somatosensory cortex, and the amygdala compared with 0 Hz TEAS. The effect remained after the end of TEAS in the anterior cingulate cortex and the temporal lobe. The selective increase in MOR availability in multiple brain regions related to pain and sensory processes may play a role in mediating low-frequency TEAS efficacy.

NMDA receptors in the midbrain play a critical role in dopamine-mediated hippocampal synaptic potentiation caused by morphine.

A single exposure to drugs of abuse produces an NMDAR (N-methyl-D-aspartate receptor)-dependent synaptic potentiation at excitatory synapses of dopamine (DA) neurons in the ventral tegmental area (VTA) of the midbrain. All addictive drugs can increase DA concentrations in projection areas of the midbrain, including the hippocampus. Hippocampal DA release subsequently modulates hippocampal plasticity and drug-associated memories. Using in vivo electrophysiological recording techniques in anesthetized rats, we show that systemic injection of morphine induced hippocampal synaptic potentiation in a dose-dependent manner. Intra-VTA but not intra-hippocampus injection of morphine evoked this potentiation. Local hippocampal dopamine D1 receptors (D1R) are required in the morphine-induced synaptic potentiation and conditioned place preference (CPP). Moreover, both NMDAR activation in the VTA and VTA/hippocampus dopaminergic connections are essential for the morphine-evoked potentiation and CPP. These findings suggest that NMDAR signalings in the midbrain play a key role in regulating dopamine-mediated hippocampal synaptic plasticity underlying drug-induced associative memory.

Electroacupuncture frequency-related transcriptional response in rat arcuate nucleus revealed region-distinctive changes in response to low- and high-frequency electroacupuncture.

Electroacupuncture (EA) has been clinically applied for treating different medical conditions, such as pain, strain, and immune diseases. Low- and high-frequency EAs have distinct therapeutic effects in clinical practice and experimental studies. However, the molecular mechanism of this difference remains obscure. The arcuate nucleus (Arc) is a critical region of the hypothalamus and is responsible for the effect of EA stimulation to remote acupoints. Gene expression profiling provides a powerful tool with which to explore the basis of physiopathological responses to external stimulus. In this study, using cDNA microarray, we investigated gene expressions in the rat Arc region induced by low-frequency (2-Hz) and high-frequency (100-Hz) EAs to two remote acupoints, zusanli (ST36) and sanyinjiao (SP6). We have found that more genes were differentially regulated by 2-Hz EA than 100-Hz EA (154 vs. 66 regulated genes/ESTs) in Arc, especially those related to neurogenesis, which was confirmed by qRT-PCR. These results demonstrate that the expression level of genes in the Arc region could be effectively regulated by low-frequency EA, compared with high-frequency EA, helping to uncover the mechanisms of the therapeutic effects of the low-frequency EA. Our results also indicate different-frequency EAs are spatially specific.

Role of the NO/sGC/PKG signaling pathway of hippocampal CA1 in morphine-induced reward memory.

Evidence suggests that the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP dependent protein kinase (PKG) signaling pathway plays a key role in memory processing, but the actual participation of this signaling cascade in the hippocampal CA1 during morphine-induced reward memory remains unknown. In this study, we investigated the role of the NO/sGC/PKG signaling pathway in the CA1 on morphine-induced reward memory using a conditioned place preference (CPP) paradigm. We found that rats receiving an intraperitoneal (i.p.) injection of 4mg/kg morphine exhibited CPP, whereas rats treated with only 0.2mg/kg morphine failed to produce CPP. Intra-CA1 injection of the neuronal NO synthase (nNOS) inhibitor 7-NI, the sGC inhibitor ODQ or the PKG inhibitor Rp-8-Br-PET-cGMPS had no effect on the acquisition of CPP by 4mg/kg morphine. Intra-CA1 injection of 7-NI blocked the consolidation of CPP induced by 4mg/kg morphine, and this amnesic effect of 7-NI was mimicked by ODQ and Rp-8-Br-PET-cGMPS. Intra-CA1 injection of the NOS substrate L-arg or the sGC activator YC-1 with an ineffective dose of morphine (0.2mg/kg, i.p.) elicited CPP. This response induced by L-arg or YC-1 was reversed by pre-microinjection of Rp-8-Br-PET-cGMPS in the CA1. These results indicated that the activation of the NO/sGC/PKG signaling pathway in the CA1 is necessary for the consolidation of morphine-related reward memory.

Dynamic changes of tyrosine hydroxylase and dopamine concentrations in the ventral tegmental area-nucleus accumbens projection during the expression of morphine-induced conditioned place preference in rats.

Our previous study demonstrated that morphine dose- and time-dependently elevated dopamine (DA) concentrations in the nucleus accumbens (NAc) during the expression of morphine-induced conditioned place preference (CPP) in rats. However, still unknown are how DA concentrations dynamically change during the morphine-induced CPP test and whether tyrosine hydroxylase (TH) activity in the ventral tegmental area (VTA) plays a vital role in this process. In the present study, we measured dynamic changes in TH and phosphorylated TH serine 40 (pTH Ser(40)) and pTH Ser(31) proteins in the VTA, and DA concentrations in the NAc at 5 min intervals during a 30 min morphine-induced CPP test. Rats that underwent morphine-induced CPP training significantly preferred the morphine-paired chamber during the CPP expression test, an effect that lasted at least 30 min in the drug-free state. DA concentrations in the NAc markedly increased at 15 min when the rats were returned to the CPP boxes to assess the expression of preference for the previously drug-paired chamber. DA concentrations then declined 2 h after the CPP test. TH and pTH Ser(40) levels, but not pTH Ser(31) levels, in the VTA were enhanced during the CPP test. These results indicated that TH and the phosphorylation of TH Ser(40) in the VTA may be responsible for DA synthesis and release in the NAc during the behavioral expression of conditioned reward elicited by a drug-associated context.

Effects of ifenprodil on morphine-induced conditioned place preference and spatial learning and memory in rats.

Drug addiction, as well as learning and memory, share common mechanisms in terms of neural circuits and intracellular signaling pathways. In the present study, the role of N-methyl-D-aspartate (NMDA) receptors, particularly those containing NR2B subunits, in morphine-induced conditioned place preference (CPP) and Morris water maze (MWM) learning and memory task was investigated. CPP was used as a paradigm for assessing the rewarding effect of morphine, and MWM was used to measure spatial learning and memory in male Sprague-Dawley rats. We found that ifenprodil, an antagonist highly selective for NR2B-containing NMDA receptors, dose-dependently blocked the development, maintenance and reinstatement of morphine-induced CPP, without evident impairment of the acquisition and retrieval of spatial memory in the MWM task. However, the consolidation of spatial memory was disrupted by a high dose (10 mg/kg) of ifenprodil. These results clearly demonstrate that NR2B-containing NMDA receptors are actively involved in addiction memory induced by morphine conditioning, but not in the acquisition and retrieval of spatial learning and memory. In conclusion, NR2B-containing NMDA receptors can be considered potential targets for the treatment of opiate addiction.

Damage of splenic T lymphocyte proliferation and differentiation and its normalization by electroacupuncture in morphine-dependent mice mode.

In a previous paper we reported that electroacupuncture (EA) could suppress opioid withdrawal syndrome and increase the appetite, sleep, and body weight in heroin addicts or morphine dependent animals. Considering that opioids were known to inhibit immune function, the present study was designed to observe whether EA could modulate the immune status of morphine dependent and withdrawal mice. We found that chronic morphine-induced decrease of splenic T lymphocyte proliferation and IL-2 production can be significantly raised by 2 Hz EA, and the fluctuation of CD4(+)/CD8(+) ratio was also run to the baseline level by the EA. These findings indicated that chronic morphine exposure-induced immune dysfunction in mice could be normalized by 2 Hz EA.

Electroacupuncture of 2 hz has a rewarding effect: evidence from a conditioned place preference study in rats.

Electroacupuncture (EA) has been used to suppress heroin craving in addicts and the conditioned place preference (CPP) for morphine in the rat. The question remained whether EA by itself will produce some rewarding effect. This was investigated using the CPP procedure in the present study. The results indicated that rats showed a significant preference to the 2 Hz EA-paired compartment. This rewarding effect of EA was prevented by pre-treatment with the opioid receptor antagonist naloxone [2 mg kg(-1), intraperitoneally (i.p.)], CB1 cannabinoid antagonist AM251 (3 µg per rat, intracerebroventricularly) or D1 dopamine receptor antagonist SCH23390 (0.1 mg kg(-1), i.p.), respectively. TempspacetempspaceIt is concluded that 2 Hz EA is capable of inducing CPP in the rat via the activation of the endogenous opioid-, cannabinoid- and dopamine-systems.

Electroacupuncture treatment normalized sleep disturbance in morphine withdrawal rats.

Sleep disturbance is considered as an important symptom of acute and protracted opiate withdrawal. Current results suggest that sleep disturbance may be taken as a predictor of relapse. Appropriate sleep enhancement therapy will be in favor of the retention in treatment for opiate addicts. Our previous studies have shown that electroacupuncture (EA) is effective in suppressing morphine withdrawal syndrome. The aim of the present study is to investigate the effect of 2 and 100 Hz EA on the sleep disturbance during morphine withdrawal. Rats were made dependent on morphine by repeated morphine injections (escalating doses of 5-80 mg kg(-1), subcutaneously, twice a day) for 5 days. EA of 2 or 100 Hz was given twice a day for 3 days, starting at 48 h after the last morphine injection. Electroencephalogram and electromyogram were monitored at the end of the first and the last EA treatments, respectively. Results showed that non-rapid eye movement (NREM) sleep, REM sleep and total sleep time decreased dramatically, while the sleep latency prolonged significantly during acute morphine withdrawal. Both 2 and 100 Hz EA produced a significant increase in NREM sleep, REM sleep and total sleep time. It was suggested that EA could be a potential treatment for sleep disturbance during morphine withdrawal.

Transcriptome profiling analysis reveals region-distinctive changes of gene expression in the CNS in response to different moderate restraint stress.

It is generally believed that temporary moderate stress to a living organism has protective and adaptive effects, but little is known about the responses of CNS to the moderate stresses at molecular level. This study aims to investigate the gene expression changes induced by moderate stress in CNS stress- and nociception-related regions of rats. Moderate restraint was applied to rats for 50 min and cDNA microarrays were used to detect the differential gene expression in different CNS regions. Transcriptome profiling analysis showed that at acute stage stress-related genes were up-regulated in arcuate nucleus; fight-or-flight behavior-related genes were up-regulated in periaqueductal gray, while nitric oxide and GABA signal transmission-related genes were up-regulated in spinal dorsal horn. In addition, immune-related genes were broadly regulated, especially at the late stage. These results suggested that specific genes of certain gene ontology categories were spatiotemporally regulated in specific CNS regions related to relevant functions under moderate external stimuli at acute stage, while immune response was broadly regulated at the late stage. The co-regulated genes among the three different CNS regions may play general roles in CNS when exposed to moderate stress. Furthermore, these results will help to elucidate the physiological processes involved in moderate stress in CNS.

Astrocytes express N-methyl-D-aspartate receptor subunits in development, ischemia and post-ischemia.

The expression of the N-methyl-D-aspartate receptor (NMDA-R) in astrocytes is controversial. The receptor is commonly considered neuron-specific. We showed that astrocytes in primary cultures differentially expressed mRNA of NMDA-R subunits, NR1, NR2A and NR2B, in development, ischemia and post-ischemia. One-week-old cultures expressed detectable NR1 mRNA, which fell significantly at 2 weeks and became barely detectable at 4 weeks. NR2A and NR2B mRNA were both significantly up-regulated from 1 to 2 weeks. In 4 weeks, 2 h of ischemia caused a significant up-regulation of NR1 and NR2B mRNA; while 6 h caused down-regulation of NR2A mRNA. Under 3 h of post-ischemia, only NR1 mRNA was increased. Ischemia induced the expression of major NMDA-R effecter, nitric oxide synthase 1, which was unaffected by AMPA-R antagonist CNQX, but dose-dependently inhibited by NMDA-R specific antagonist MK-801. These findings reflected that astrocyte could express inducible functional NMDA receptors without the presence of neurons.

[Prospective study of Han's acupoint nerve stimulator for preventing relapse of detoxified heroin addicts].

OBJECTIVE: To assess the efficacy of transcutaneous electrical acupoint stimulation (TEAS) in preventing the relapse of detoxified heroin users in a period of 12 months. METHODS: A total of 164 rehabilitating heroin users in Shanghai area were recruited after compulsory detoxification treatment for a period ranging from 3 months to 3 years. The TEAS was executed by the device named Han's acupoint nerve stimulator (HANS). The patients were treated with HANS for at least 3 months. All the subjects were then followed up for one year and relapse was monitored by monthly heroin/morphine urinalysis. RESULTS: Of 164 rehabilitating former heroin addicts, 53 remained drug-free at the end of 12 months observation period as judged by negative urinalysis while 35 become relapsed as documented by positive urine tests. The rest 76 dropped off due to various reasons and were all counted into the category of "relapsed". Thus, our data indicated 32.3% rehabilitating heroin users could stay sober for at least one year, which is significantly higher than the existing reports that less than 5% detoxified abusers would stay drug free for one year with no further intervention. No obvious correlation was found between the susceptibility of relapse and the duration of compulsory detoxification. CONCLUSION: Compared to the existing literatures, our results indicate that HANS could produce a marked decrease of the relapse rate of rehabilitating heroin users after compulsory detoxification.

Electroacupuncture treatment reverses morphine-induced physiological changes in dopaminergic neurons within the ventral tegmental area.

Chronic morphine administration decreases the size of dopamine (DA) neurons in the ventral tegmental area (VTA). These transient morphological changes are accompanied by a reduced sensitivity of morphine-induced conditioned place preference (CPP) after chronic exposure to the drug. In this study we examined alterations in the firing rate of DAergic neurons by means of extracellular recording following chronic morphine exposure and applied 100 Hz electroacupuncture (EA) treatment to reverse the reduced firing rate of these neurons. In the first set of experiments we show that in rats, which received chronic morphine treatment for 14 days, a small dose of morphine was not able to induce a CPP response anymore. However, the sensitivity to morphine was reinstated by consecutive EA treatment for 10 days. The electrophysiological response of VTA DA neurons to morphine was markedly reduced in chronic morphine-treated rats compared to saline-treated controls. A substantial recovery of the reactivity of VTA DA neurons to morphine was observed in rats that received 100 Hz EA for 10 days. Our findings suggest that 100 Hz EA is a potential therapy for the treatment of opiate addiction by normalizing the activity of VTA DA neurons.

The Neuroscience Research Institute at Peking University: a place for the solution of pain and drug abuse.

Neuroscience research in China has undergone rapid expansion since 1980. The Neuroscience Research Institute of Peking University, one of the most active neuroscience research groups in China, was founded in 1987. Currently, the institute is overseeing four research areas, i.e., (1) pain and analgesia, (2) drug abuse and acupuncture treatment for drug addiction, (3) the mechanism of neurological degenerative disorders, and (4) the role of neuroglia in central nervous system injury. The institute is simultaneously investigating both theoretical and clinical studies. Acupuncture remains the core of research, while pain and drug abuse form the two disciplines.

Acupuncture for the treatment of drug addiction.

Over the last three decades there has been an increasing interest in acupuncture treatment of substance abuse around the world. Three important steps can be identified in this field. Dr. Wen of Hong Kong was the first (1972) to report that acupuncture at 4 body points and 2 ear points combined with electrical stimulation can relieve opioid withdrawal signs in the addicts. The second major step was made by Dr. M. Smith in New York, the head of the National Acupuncture Detoxification Association (NADA) of the USA, who finalized a protocol (1985), using only ear points without electrical stimulation for the treatment of drug abuse. The recent advance in this field was made by Dr. Han of the Peking University, Beijing, who characterized a protocol (2005), using electrical stimulation of identified frequencies on body points to ameliorate heroin withdrawal signs and prevent relapse of heroin use. In this review, the efficacy of acupuncture and related techniques for the treatment of drug dependence in experimental settings and clinical practice will be reviewed, and the possible mechanisms underlying this effect be discussed.

Electroacupuncture reduces voluntary alcohol intake in alcohol-preferring rats via an opiate-sensitive mechanism.

Electroacupuncture (EA) has been shown to modify the effects of various drugs of abuse, including alcohol. Inbred P rats were trained to drink alcohol voluntarily and then subjected to two periods of alcohol deprivation lasting 3 days. During the second deprivation, the rats received either EA or sham EA. The rats were pretreated with naltrexone (5 mg/kg) or saline 30 min before each of the EA or sham EA sessions. Approximately 6 h after the last naltrexone or saline treatment, the alcohol tubes were returned and alcohol and water intakes were recorded later at 2, 4, 6, and 24 h. Only EA led to a decrease in alcohol intake, which was most prominent at 6 and 24 h, and this inhibitory effect of EA was blocked by naltrexone, suggesting that activation of the endogenous opiate system may be responsible for EA's effects on alcohol intake in the alcohol-dependent iP rats.

Peripheral electrical stimulation-induced suppression of morphine-induced CCP in rats: a role for dopamine in the nucleus accumbens.

In the previous study we reported that morphine-induced conditioned place preference (CPP) could be suppressed by peripheral electric stimulation (PES), an effect related to the increased gene expression of opioid peptides in the central nervous system. Considering that opioids were known to elevate dopamine (DA) activity in the mesolimbic brain, the present study was designed to further analyze the possible involvement of the mesolimbic dopaminergic system (MLDS) in the suppressive effect of PES on the rewarding effects of morphine in SD male rats. We found that morphine-induced CPP can be successfully suppressed by PES, an effect accompanied by a reversal of the increased tissue contents of DA and its metabolites in the nucleus accumbens (NAc) of morphine-induced CPP rats. Our results suggest that MLDS seems to play important roles in the mechanisms underlying PES's suppression of the rewarding effect of drug-associated environmental cues in the rat.