6-cyano-7-nitroquinoxaline-2-3-dione and Substance-Withdrawal-Syndrome

Protracted opiate withdrawal is accompanied by altered responsiveness of midbrain dopaminergic (DA) neurons, including a loss of DA cell response to morphine, and by behavioral alterations, including affective disorders. GABAergic neurons in the tail of the ventral tegmental area (tVTA), also called the rostromedial tegmental nucleus, are important for behavioral responses to opiates. We investigated the tVTA-VTA circuit in rats after chronic morphine exposure to determine whether tVTA neurons participate in the loss of opiate-induced disinhibition of VTA DA neurons observed during protracted withdrawal. In vivo recording revealed that VTA DA neurons, but not tVTA GABAergic neurons, are tolerant to morphine after 2 weeks of withdrawal. Optogenetic stimulation of tVTA neurons inhibited VTA DA neurons similarly in opiate-naive and long-term withdrawn rats. However, tVTA inactivation increased VTA DA activity in opiate-naive rats, but not in withdrawn rats, resembling the opiate tolerance effect in DA cells. Thus, although inhibitory control of DA neurons by tVTA is maintained during protracted withdrawal, the capacity for disinhibitory control is impaired. In addition, morphine withdrawal reduced both tVTA neural activity and tonic glutamatergic input to VTA DA neurons. We propose that these changes in glutamate and GABA inputs underlie the apparent tolerance of VTA DA neurons to opiates after chronic exposure. These alterations in the tVTA-VTA DA circuit could be an important factor in opiate tolerance and addiction. Moreover, the capacity of the tVTA to inhibit, but not disinhibit, DA cells after chronic opiate exposure may contribute to long-term negative affective states during withdrawal.. Dopaminergic (DA) cells of the ventral tegmental area (VTA) are the origin of a brain reward system and are critically involved in drug abuse. Morphine has long been known to affect VTA DA cells via GABAergic interneurons. Recently, GABAergic neurons caudal to the VTA were discovered and named the tail of VTA (tVTA). Here, we show that tVTA GABA neurons lose their capacity to disinhibit, but not to inhibit, VTA DA cells after chronic opiate exposure. The failure of disinhibition was associated with a loss of glutamatergic input to DA neurons after chronic morphine. These findings reveal mechanisms by which the tVTA may play a key role in long-term negative affective states during opiate withdrawal.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Afferent Pathways; Animals; Channelrhodopsins; Disease Models, Animal; Dopaminergic Neurons; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; GABAergic Neurons; Glutamate Decarboxylase; Male; Morphine; Narcotics; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Time Factors; Valine; Ventral Tegmental Area

Neuroadaptations in the nucleus accumbens (NAc) are associated with the development of drug addiction. Plasticity in synaptic strength and intrinsic excitability of NAc medium spiny neurons (MSNs) play critical roles in addiction induced by different classes of abused drugs. However, it is unknown whether morphine exposure influences synaptic strength, intrinsic excitability or both in NAc. Here we show that chronic withdrawal (10 days after the last injection) from repeated morphine exposure elicited potentiation in both glutamatergic synaptic strength and intrinsic excitability of MSNs in NAc shell (NAcSh). The potentiation of synaptic strength was demonstrated by an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs), a decrease in the paired-pulse ratio (PPR), and an increase in the ratio of α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptors (AMPAR)- to N-methyl-D-aspartate receptors (NMDAR)-mediated currents. The potentiation of intrinsic excitability was mediated by inhibition of the sustained potassium currents via extrasynaptic NMDAR activation. The function of the presynaptic group II metabotropic glutamate receptors (mGluR2/3) was downregulated, enhancing the probability of glutamate release on synaptic terminals during chronic morphine withdrawal. Pretreatment with the mGluR2/3 agonist LY379268 completely blocked potentiation of both synaptic strength and intrinsic excitability. These results suggest that chronic morphine withdrawal downregulates mGluR2/3 to induce potentiation of MSN glutamatergic synapse via increased glutamate release, leading to potentiation of intrinsic excitability. Such potentiation of both synaptic strength and intrinsic excitability might contribute to neuroadaptations induced by morphine application.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids; Analysis of Variance; Animals; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Down-Regulation; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Morphine; Narcotics; Neurons; Neuroprotective Agents; Nucleus Accumbens; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Synapses; Xanthines

The amygdala plays key roles in several aspects of addiction to drugs of abuse. This brain structure has been implicated in behaviours that reflect drug reward, drug seeking, and the aversive effects of drug withdrawal. Using a model that involves repeated cocaine injections to approximate 'binge' intoxication, we show in rats that during cocaine withdrawal, the impact of rewarding brain stimulation is attenuated, as quantified by alterations in intracranial self-stimulation (ICSS) behaviour. These behavioural signs of withdrawal are accompanied by enhancements of glutamatergic synaptic transmission within the lateral amygdala (LA) that occlude electrically induced long-term potentiation (LTP) in tissue slices. Synaptic enhancements during periods of cocaine withdrawal are mechanistically similar to LTP induced with electrical stimulation in control slices, as both forms of synaptic plasticity involve an increase in glutamate release. These results suggest that mechanisms of LTP within the amygdala are recruited during withdrawal from repeated exposure to cocaine. As such, they raise the possibility that the development and maintenance of addictive behaviours may involve, at least in part, mechanisms of synaptic plasticity within specific amygdala circuits.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Behavior, Animal; Cocaine; Dopamine Uptake Inhibitors; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fear; GABA Agonists; Long-Term Potentiation; Male; Muscimol; Rats; Rats, Sprague-Dawley; Reflex, Startle; Self Administration; Substance Withdrawal Syndrome; Synaptic Transmission; Time Factors

Stress impairs hippocampal long-term potentiation (LTP), but it is unknown whether the stress evoked by opiate withdrawal has the same effect. Here the authors report that opiate withdrawal for 4 days does not influence basal synaptic transmission, but results in a greatly increased LTP in hippocampal CA1 area in anesthetized rats. Elevated-platform stress enabled a large LTP in rats withdrawn for only 18 h, but the glucocorticoid receptor antagonist RU38486 (twice per day for 3 days) prevented the large LTP on 4 days withdrawal. Moreover, 4 days withdrawal enhanced the NMDAR-mediated EPSCs, in which the NR2A-containing NMDAR-mediated EPSC was increased but the NR2B-containing NMDAR-mediated EPSC was decreased. These results suggest that adaptive changes of the NMDAR and glucocorticoid receptor functions during 4 days of opiate withdrawal may enable stress to facilitate hippocampal LTP, potentially contributing to the opiate withdrawal experience-dependent modifications of hippocampal functions.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Dose-Response Relationship, Radiation; Drug Administration Schedule; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Hormone Antagonists; In Vitro Techniques; Long-Term Potentiation; Male; Mifepristone; Morpholines; Patch-Clamp Techniques; Phenols; Piperidines; Rats; Rats, Sprague-Dawley; Stress, Psychological; Substance Withdrawal Syndrome; Time Factors

Chronic use of morphine leads to physical and psychological dependence. The amygdala is known to be involved in the expression of emotion such as anxiety and fear, and several studies have shown that the central nucleus of the amygdala (CeA) is involved in morphine dependence. In the present study, we investigated the role of glutamate receptors within the CeA in the negative affective component of morphine abstinence by evaluating naloxone-precipitated withdrawal-induced conditioned place aversion (CPA) in morphine-dependent rats. We found that microinjection of the AMPA/kainate-glutamate-receptor antagonist CNQX (30 nmol/side) into the bilateral CeA significantly attenuated the naloxone-precipitated withdrawal-induced CPA, as well as several somatic signs, in morphine-dependent rats, without preference or aversive effects by itself in non-dependent rats. Furthermore, microinjection of the non-competitive NMDA-receptor antagonist MK-801 (30 nmol/side) or competitive NMDA-receptor antagonist D-CPPene (0.01 and 0.1 nmol/side) into the CeA significantly attenuated the naloxone-precipitated morphine withdrawal-induced CPA, but not somatic withdrawal signs. These results suggest that the activation of AMPA /kainate and NMDA receptors within the CeA play a crucial role in the negative affective component of morphine abstinence.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Analysis of Variance; Animals; Avoidance Learning; Cerebellar Nuclei; Conditioning, Psychological; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Microinjections; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Substance Withdrawal Syndrome

Noradrenergic neurons of the brain nucleus locus coeruleus (LC) become hyperactive during opiate withdrawal. It has been uncertain to what extent such hyperactivity reflects changes in intrinsic properties of these cells. The effects of withdrawal from chronic morphine on the activity of LC neurons were studied using intracellular recordings in rat brain slices. LC neurons in slices from chronically morphine-treated rats exhibited more than twice the frequency of spontaneous action potentials after naloxone compared with LC neurons from control rats. However, after naloxone treatment, the resting membrane potential (MP) of LC neurons from dependent rats was not significantly different from that in control rats. Neither resting MP nor spontaneous discharge rate (SDR) was altered by naloxone in LC neurons from control rats. Neither kynurenic acid nor a cocktail of glutamate and GABA antagonists (6-cyano-7-nitroquinoxalene-2,3-dione + 2-amino-5-phosphonopentanoic acid + bicuculline) blocked the hyperactivity of LC neurons precipitated by naloxone in slices from morphine-dependent rats. The effects of ouabain on MP and SDR were similar in LC neurons from control and morphine-dependent rats. These results indicate that an adaptive change in glutamatergic or GABAergic synaptic mechanisms or altered Na/K pump activity does not underlie the withdrawal-induced activation of LC neurons in vitro. Specific inhibitors of protein kinase A [Rp-cAMPS or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89)] partially suppressed the withdrawal hyperactivity of LC neurons, and activators of cAMP (forskolin) or protein kinase A (Sp-cAMPS) increased the discharge rate of LC neurons from control rats. These results suggest that upregulation of cAMP-dependent protein kinase A during chronic morphine treatment is involved in the withdrawal-induced hyperactivity of LC neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics, Opioid; Animals; Barium; Bicuculline; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Kynurenic Acid; Locus Coeruleus; Male; Membrane Potentials; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Neurons; Ouabain; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Tetrodotoxin; Thionucleotides

We investigated the effect of topical application of capsaicin cream on withdrawal latency in the hind foot of rat in response to radiant heat in an experimental model of neuropathic pain. A neuropathic state was induced by loose ligation of the sciatic nerve with chromic gut suture. A marked thermal hyperalgesia was observed in response to heat stimulus applied to the operated side from 3 days through 2 weeks, followed by a gradual return to the control level by 35 days after surgery. Capsaicin cream applied to both the bilateral hind instep and sole once a day for a continuous period of 2 weeks or 4 weeks alleviated thermal hyperalgesia in a dose-dependent manner. A remarkable effect was observed 2 weeks after the start of the application and this effect proved to be reversible. On the other hand, in sham-operated animals when capsaicin cream was applied once daily from day 7 after the sham operation, from 1 day through 3 weeks following capsaicin application, withdrawal latency of the sham-operated paws of the capsaicin-treated group was significantly increased as compared to that of the vehicle cream-treated group. The effects of antagonists of glutamate receptor and tachykinin receptors were investigated 7 days post surgery. Pretreatment with MK-801 (0.5 mg kg(-1), i.p.), but not with CNQX (0.5 mg kg(-1), i.p.), reversed the thermal hyperalgesia following nerve injury. Neither of RP67580 (1--10 mg kg(-1), i.p.) nor SR48968 (1--10 mg kg(-1), i.p.) had any effect on the withdrawal latency in the injured and non-injured hind paw. These results suggest that although the manifestation of effectiveness may be delayed by changes in networks of neurotransmitters related to the nociceptive pathways following nerve injury, longer-term repetitive application of capsaicin cream has a significant therapeutic effect on subjects with painful peripheral neuropathy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics; Animals; Benzamides; Capsaicin; Carrageenan; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hyperalgesia; Indoles; Isoindoles; Male; Neuroprotective Agents; Pain; Peripheral Nervous System Diseases; Piperidines; Postoperative Period; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Substance Withdrawal Syndrome; Time Factors

Both the locus coeruleus (LC) and the amygdala have been implicated in aspects of opiate dependence and withdrawal. The LC is known to be one of the most sensitive sites for precipitating withdrawal behaviors after local opiate antagonist infusions in morphine-dependent subjects. The amygdala is also known to mediate antagonist-induced withdrawal behaviors and aversive motivational states. The goal of the present study was to evaluate directly the ability of noradrenergic agonists and glutamatergic antagonists to attenuate naloxone-precipitated withdrawal behaviors when infused into the LC or the central nucleus of the amygdala (CeA). The alpha-2-noradrenergic agonists clonidine or ST-91 were infused into the CeA to compare the effects of noradrenergic activation in the CeA to the attenuation of withdrawal previously observed in rats infused with clonidine into the LC, since the LC and CeA are known to contain co-localized opiate and noradrenergic receptors. The effects of microinfusions of the non-NMDA excitatory amino acid antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) were also infused into the LC and CeA since opiate withdrawal is associated with increased glutamatergic transmission. Intra-CeA clonidine or ST-91 (2.4 microg/0.5 microl or 1.0 microl) produced significant reductions primarily in the occurrence of irritability. Conversely, intra-CeA or intra-LC infusions of CNQX (2.5 microg/0.5 microl) significantly attenuated naloxone-precipitated withdrawal, an effect similar to the attenuation previously observed after intra-LC clonidine infusions. These data demonstrate the specific behavioral effects of altering glutamatergic and noradrenergic neurotransmission in the LC or CeA during naloxone-precipitated opiate withdrawal. Elucidation of the neuroanatomical circuitry involved in opiate withdrawal should increase our understanding of the neuroadaptations associated with drug dependence and subsequent withdrawal behavior.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adrenergic alpha-Agonists; Amygdala; Animals; Clonidine; Excitatory Amino Acid Antagonists; Glutamic Acid; Glutamine; Locus Coeruleus; Male; Morphine Dependence; N-Methylaspartate; Naloxone; Narcotic Antagonists; Norepinephrine; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome

Opioid receptors located on interneurons in the ventral tegmental area (VTA) inhibit GABA(A)-mediated synaptic transmission to dopamine projection neurons. The resulting disinhibition of dopamine cells in the VTA is thought to play a pivotal role in drug abuse; however, little is known about how this GABAA synapse is affected after chronic morphine treatment. The regulation of GABA release during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. Slices containing the VTA were prepared and maintained in morphine-free solutions, and GABAA IPSCs were recorded from dopamine cells. The amplitude of evoked IPSCs and the frequency of spontaneous miniature IPSCs measured in slices from morphine-treated guinea pigs were greater than placebo-treated controls. In addition, activation of adenylyl cyclase, with forskolin, and cAMP-dependent protein kinase, with Sp-cAMPS, caused a larger increase in IPSCs in slices from morphine-treated animals. Conversely, the kinase inhibitors staurosporine and Rp-CPT-cAMPS decreased GABA IPSCs to a greater extent after drug treatment. The results indicate that the probability of GABA release was increased during withdrawal from chronic morphine treatment and that this effect resulted from an upregulation of the cAMP-dependent cascade. Increased transmitter release from opioid-sensitive synapses during acute withdrawal may be one adaptive mechanism that results from prolonged morphine treatment.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dopamine; Dopamine Antagonists; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; Guinea Pigs; Interneurons; Morphine; Nerve Tissue Proteins; Organophosphorus Compounds; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Picrotoxin; Receptors, GABA-A; Salicylamides; Serotonin; Signal Transduction; Staurosporine; Strychnine; Substance Withdrawal Syndrome; Tegmentum Mesencephali; Thionucleotides

We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Behavior, Animal; Brain; Dizocilpine Maleate; DNA-Binding Proteins; GABA Modulators; Histocytochemistry; In Situ Hybridization; Kainic Acid; Male; Phenobarbital; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Receptors, Glutamate; RNA, Messenger; Substance Withdrawal Syndrome; Substance-Related Disorders; Transcription Factor AP-1

Opioid dependence in ventrolateral periaqueductal gray (PAG) neurons was studied by using intracellular recordings from brain slices. In slices from morphine-dependent rats maintained in morphine (5 microM) in vitro, action potential frequencies of opioid-sensitive neurons did not differ from untreated control neurons but were greater than in control neurons maintained in morphine in vitro, indicating development of tolerance. Naloxone (100 nM or 1 microM) depolarized 25 of 51 neurons from morphine-dependent rats maintained in morphine in vitro, 19 of which previously had been classified as opioid-sensitive. Action potential frequencies in the presence of naloxone were greater than in control neurons in the absence of opioids, as well as in control neurons in the presence of both morphine and naloxone, demonstrating opioid withdrawal. In slices from control animals, opioid-induced hyperpolarizations and naloxone-induced depolarizations (in the presence of morphine) reversed polarity near expected EK (-111 +/- 3 mV and -113 +/- 3 mV, respectively). In contrast, the reversal potential of naloxone-induced depolarizations was more negative than expected in neurons from dependent animals (-143 +/- 9 mV), indicating that the depolarization was not attributable simply to antagonism of a K-conductance increase. Naloxone-induced depolarizations were not inhibited by tetrodotoxin (1 microM), bicuculline (30 microM), 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), or prazosin (300 nM), suggesting no involvement of major synaptic neurotransmitters. Clonidine (1 microM) and baclofen (30 microM) overcame naloxone-induced depolarizations. These results demonstrate development of both tolerance and withdrawal in PAG neurons and suggest induction of a novel opioid-sensitive current that could be involved in withdrawal behavior.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adrenergic alpha-Agonists; Analgesics, Opioid; Animals; Baclofen; Bicuculline; Clonidine; Drug Tolerance; Electrophysiology; Excitatory Amino Acid Antagonists; GABA Agonists; GABA Antagonists; Male; Membrane Potentials; Morphine; Naloxone; Narcotic Antagonists; Neurons; Organ Culture Techniques; Periaqueductal Gray; Potassium Channels; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Substance Withdrawal Syndrome; Tetrodotoxin; Time Factors; Valine