6-cyano-7-nitroquinoxaline-2-3-dione and adenosine-3--5--cyclic-phosphorothioate

We sought to determine if plasticity in the ventral tegmental area (VTA) of the midbrain is involved in learning to associate morphine exposure with a specific environment. For this, we tested whether activation of glutamate receptors and protein kinase A is needed for the acquisition and expression of a morphine-conditioned place preference (CPP). Rats received bilateral microinjections of either the NMDA antagonist AP5 (0.48 nmol/0.3 microl), the AMPA antagonist CNQX (0.21 nmol/0.3 microl), or vehicle into the VTA prior to each of three morphine-conditioning sessions. Both the AMPA and NMDA receptor antagonists blocked the development of morphine CPP when given into the VTA but not when given outside the VTA. In similar studies the protein kinase A (PKA) inhibitor, Rp-cAMPS (13 nmol/0.3 microl), blocked the acquisition of morphine CPP when given into the VTA immediately after morphine conditioning. In separate experiments, glutamate antagonists, or Rp-cAMPS, immediately prior to the preference test blocked the expression of morphine CPP when microinjected into the VTA. These data indicate that the VTA is an important site for synaptic modifications involved in the learning and memory of environmental cues predicting reward, and that glutamate input and PKA activation are crucial to this process.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Behavior, Animal; Conditioning, Operant; Cyclic AMP; Drug Interactions; Environment; Excitatory Amino Acid Antagonists; Glutamic Acid; Male; Morphine; Narcotics; Neuronal Plasticity; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Thionucleotides; Ventral Tegmental Area

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

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Actins; Aniline Compounds; Animals; Calcium; Cells, Cultured; Cyclic AMP; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Genes, Reporter; Green Fluorescent Proteins; Hippocampus; Light; Luminescent Proteins; Microscopy, Video; Neuronal Plasticity; Neurons; Rats; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Signal Transduction; Silicon; Synapses; Thionucleotides; Valine; Xanthenes

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

The intracellular mechanisms underlying the facilitatory action of isoproterenol (Iso) on the NMDA receptor-mediated synaptic potential (EPSPNMDA) was investigated in an in vitro slice preparation of rat amygdala. Intracellular recordings were made from basolateral amygdala neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and picrotoxin (50 microM) which block non-NMDA and GABAA receptors, respectively. Superfusion of Iso (15 microM) produced a sustained increase in EPSPNMDA. Rp-adenosine-3',5'-cyclic monophosphotioate (Rp-cAMPS), a potent inhibitor of protein kinase A (PKA) alone decreased the amplitude of EPSPNMDA below baseline values and prevented the subsequent potentiation by Iso. Forskolin, a direct activator of adenylate cyclase, mimics the effect of Iso, and Rp-cAMPS also reversed forskolin-induced enhancement of EPSNMDA. These results suggest that cAMP-dependent protein kinase mediates the enhancement of EPSPNMDA by Iso in the amygdala.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Evoked Potentials; In Vitro Techniques; Isoproterenol; Neurons; Picrotoxin; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Synapses; Thionucleotides