Endogenous opioids and LTP: another view.
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Animals↗
Biomedical subjects
C Chavkin
Publications and source records attributed to C Chavkin.
Kappa-opioids decrease excitatory transmission in the dentate gyrus of the guinea pig hippocampus.
In the guinea pig hippocampus, kappa 1-opioid binding sites were primarily localized in the molecular layer of the dentate gyrus as shown by autoradiography using either the kappa 1-selective radioligand 3H-U69,593 or the nonselective radioligand 3H-diprenorphine in the presence of unlabeled mu- and delta-blocking ligands. In this region, the electrophysiological effects of kappa 1-receptor activation were identified using extracellular and intracellular recordings of dentate granule cell responses. The amplitude of the extracellularly recorded population spike was reduced by U69,593 with an EC50 of 26 nM; this effect was reversible and blocked by the opioid antagonist naloxone. The kappa 1-selective antagonist norbinaltorphimine also blocked the effect of U69,593 with an apparent equilibrium dissociation constant (Ki) of 0.26 nM determined by Schild analysis in the physiologic assay. This value agreed well with the Ki for norbinaltorphimine at kappa 1-binding sites measured by radioligand binding displacement (0.24 nM). These results indicate that the electrophysiologic response observed was likely mediated by kappa 1-receptors. As seen with U69,593, dynorphin B, an endogenous opioid peptide that is present in the dentate gyrus, also inhibited the population spike response. mu- and delta-selective opioid agonists had no effect on the amplitude of the maximally evoked response. Intracellular recordings of dentate granule cells showed no direct effects of U69,593 on the granule cells themselves. However, analysis of synaptic potentials revealed that U69,593 significantly reduced the amplitude of glutaminergic EPSPs evoked by afferent stimulation without affecting IPSP amplitudes. The specific effect of U69,593 application on granule cell EPSPs indicates that presynaptic kappa 1-receptor activation inhibits glutamate release from perforant path terminals in the molecular layer of the dentate gyrus. These results suggest that endogenous dynorphins present in the granule cells may act as feedback inhibitors of the major excitatory input to the dentate gyrus.
Action Potentials↗
Ionic zinc may function as an endogenous ligand for the haloperidol-sensitive sigma 2 receptor in rat brain.
In the search for an endogenous sigma transmitter, whose existence was previously suggested by release studies, we tested the effects of releasable substances known to be present in the hippocampus, and we determined that ionic zinc may function as an endogenous ligand for the haloperidol-sensitive sigma 2 site. Zn2+ displaced 1,3-di(2-[5-3H]tolyl)guanidine ([3H]DTG) from two binding sites in rat brain membranes, with an IC50 for the high affinity site of 110 +/- 3 microM and for the low affinity site of 20 +/- 4 mM. The sigma 1-selective ligand (+)-[3H]pentazocine was only weakly displaced from rat brain membranes by Zn2+ (IC50 = 1.4 +/- 0.05 mM). These results indicate that the Zn(2+)-sensitive sigma binding site corresponds to the sigma 2 site. The interaction between Zn2+ and the sigma 2 site may have physiological significance, because ionic zinc is present in synaptic vesicles in the brain and may function to regulate binding at the sigma 2 site. To test this hypothesis, we measured the effects of metallothionein peptide 1, a specific zinc chelator, on the actions of the putative endogenous sigma ligand(s) released in the hippocampus by focal electrical stimulation. Release of the endogenous sigma ligand(s) was measured by competition with specific radioligand binding in live hippocampal slices. High frequency, focal, electrical stimulation of the zinc-containing mossy fibers in the hilar region of the hippocampus caused a decrease in the specific binding of [3H]DTG, (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, or (+)-[3H]pentazocine to sigma sites. The decrease in [3H]DTG binding was largely blocked by metallothionein peptide 1, whereas the decrease in (+)-[3H]pentazocine binding was unaffected. These results suggest that Zn2+ may act as an endogenous ligand at sigma 2 sites in the rat hippocampus.
Animals↗
Characterization of Vicia villosa agglutinin-labeled GABAergic interneurons in the hippocampal formation and in acutely dissociated hippocampus.
The distribution, morphology, and ionic conductances of Vicia villosa agglutinin (VVA)-labeled cells were examined in the rat hippocampal formation. The heaviest labeling and highest density of labeled neurons were found in the subicular complex. Lighter VVA-labeling and fewer labeled cells were found in hippocampal strata pyramidale, oriens, and alveus. VVA-labeled cells were found to be heterogeneous morphologically, including multipolar, bipolar, and basket-like shapes. The majority of VVA-labeled cells contained GABA and parvalbumin immunoreactivity; thus VVA-labeled cells in the hippocampal formation resemble previously described VVA-labeled neurons in cerebral cortex. Electrophysiological properties of subicular VVA-labeled cells were studied in an acutely dissociated neuron preparation. Dissociated cells were labeled in vitro with VVA coupled either to a fluorescent marker or to small beads. The viability of labeled dissociated cells was confirmed, and identified cells were partially characterized electrophysiologically using whole-cell voltage clamp recording. VVA-labeled cells were electrophysiologically similar to pyramidal cells from the same region, except that the VVA-labeled cells showed only small transient outward currents.
Animals↗
Endogenous opioids released from perforant path modulate norepinephrine actions and inhibitory postsynaptic potentials in guinea pig CA3 pyramidal cells.
The stimulus parameters needed for the release of endogenous opioid peptides were investigated using an in vitro radioligand displacement assay in living guinea pig hippocampal slices. Electrical stimulation of the enkephalin-containing fibers in the perforant path caused the release of endogenous opioid peptides and the subsequent displacement of [3H]-[D-Ala2,N-methyl-Phe4,glyol5]enkephalin binding. High frequency trains of stimuli (10 Hz for 1 sec every 10 sec) were more effective than lower frequency stimulation (1 Hz continuous) at evoking opioid peptide release. Having identified an effective stimulation paradigm able to release endogenous opioids, the electrophysiological effects of endogenous opioids on CA3 pyramidal cells were measured in the guinea pig hippocampal slice preparation. Unlike exogenously applied opioids, stimulated release of endogenous opioid peptides from the perforant path did not significantly reduce inhibitory postsynaptic potential (IPSP) amplitudes recorded in CA3 pyramidal cells. However, perforant path stimulation in the presence of naloxone did cause a dramatic increase in IPSP amplitudes. CA3 pyramidal cells were not directly affected by perforant path stimulation. The naloxone-sensitive increase in IPSPs was delayed 3 min in onset and lasted for several minutes. In addition, the increase in the IPSPs was specifically blocked either by the beta adrenergic antagonist propranolol or by pretreating the animals with reserpine. These findings indicate that endogenous opioids regulate the effects of norepinephrine in the CA3 region of the guinea pig hippocampus. In addition, endogenously released norepinephrine appeared to act on GABAergic interneurons to increase the amplitude of the IPSP recorded in CA3 pyramidal cells.
Animals↗
Focal stimulation of specific pathways in the rat hippocampus causes a reduction in radioligand binding to the haloperidol-sensitive sigma receptor.
Focal electrical stimulation of selected excitatory pathways in the hippocampal slice caused a decrease in the binding of [3H]-1,3-di(2-tolyl)guanidine (DTG) or [3H]-(+)-3-[hydroxyphenyl]-N-(1-propyl)piperidine [( 3H )-(+)3-PPP) to haloperidol-sensitive sigma binding sites in the slice. Activation of the mossy fibers or perforant path by high frequency electrical stimulation caused the reduction in [3H]-DTG binding; whereas activation of fibers in the strata radiatum, lacunosum-moleculare, alveus, or oriens did not affect [3H]-DTG binding. The decrease in binding observed was calcium-dependent and tetrodotoxin sensitive and varied with the frequency, intensity, and duration of stimulation. Although haloperidol-sensitive [3H]-DTG binding sites are distributed throughout the hippocampus, stimulation of the perforant path or mossy fibers resulted in a significant reduction in binding only in the dentate region of the slice. The decrease in binding following perforant path stimulation was blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); whereas the decrease in binding caused by mossy fiber stimulation was not affected by CNQX or DL-APV. The results obtained support the hypothesis that activation of the granule cells in the hippocampal slice caused the release of an endogenous ligand which acts at the haloperidol-sensitive sigma binding site in the dentate gyrus.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation.
Opioid receptors were found to activate two different types of membrane potassium conductance in acutely dissociated neurons from the CA1/subiculum regions of the adult rat hippocampal formation. Opioid-responsive neurons were distinguished based on their morphology and electrophysiological responses. In one population of neurons having a multipolar, nonpyramidal cell shape, mu-selective opioid agonists increased an inward rectifying potassium current. Opioid activation of the inward rectifying conductance resulted in small outward potassium currents at resting membrane potentials and increased inward currents at hyperpolarized potentials. In a second population of nonpyramidal neurons, mu opioid agonists increased a novel voltage-gated potassium current. This current was blocked by internal CsCl2, unaffected by external BaCl2 or CdCl2, irreversibly activated by intracellular GTP-gamma-S, and inactivated by sustained depolarization. In contrast to the inward rectifying conductance, the voltage-gated conductance was not activated at resting membrane potentials or hyperpolarized potentials. The opioid-activated, voltage-gated conductance represents a new class of G protein-regulated potassium current in the brain.
Animals↗
Focal stimulation of the mossy fibers releases endogenous dynorphins that bind kappa 1-opioid receptors in guinea pig hippocampus.
Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Chronic morphine exposure blocks opioid effects on both the early and late inhibitory postsynaptic potentials in hippocampal CA1 pyramidal cells.
The mu-opioid agonist, [N-MePhe3,D-Pro4]morphiceptin (PL017), significantly decreased the conductance changes measured during both the early and late inhibitory postsynaptic potentials (IPSP) in CA1 pyramidal cells. Although the conductance change during the early IPSP was much larger than that during the late IPSP, the relative decrease in conductance caused by 1 microM PL017 was similar for both. Chronic morphine treatment of rats prior to hippocampal slice preparation resulted in a loss of PL017 (1 microM) effects on both the early and late IPSPs. These results suggest that opioids have an equal ability to alter both early and late IPSPs in the CA1, that these effects are equally sensitive to chronic morphine, and that these measurements are a sensitive means of determining opioid tolerance in the hippocampus.
Animals↗
The sigma enigma: biochemical and functional correlates emerge for the haloperidol-sensitive sigma binding site.
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Animals↗
Stimulation of endogenous opioid release displaces mu receptor binding in rat hippocampus.
Physiological release of endogenous opioids in the rat hippocampus was detected by an in vitro radioligand displacement assay using [3H][D-Ala2,N-methyl-Phe4,glyol5]enkephalin ([3H]DAGO), a mu selective opioid agonist. In this assay, radioligand binding to opioid receptors in the in vitro hippocampal slice was reduced by competition with endogenous opioids released following tissue depolarization. Veratridine-induced opioid release caused displacement of [3H]DAGO that could be blocked by either tetrodotoxin addition or calcium removal from the incubation buffer. Maximal displacement of [3H]DAGO also required the presence of peptidase inhibitors in the incubation buffer. None of the buffer composition changes directly affected [3H]DAGO binding to rat brain membranes. Calcium-dependent displacement of [3H]DAGO binding from mu receptor sites elicited by focal electrical stimulation depended on the intensity and frequency of stimulation and positioning of the electrode in the slice. Maximal displacement of [3H]DAGO binding was observed following high intensity (150-300 microA), high frequency (10-50 Hz) stimulation of the perforant path, a major afferent fiber system to the hippocampus previously shown to contain proenkephalin-derived opioids. Low frequency stimulation (0.1-1 Hz) was ineffective. Stimulation of the mossy fibers (containing both dynorphins and enkephalins) also significantly reduced mu receptor binding, but to a lesser extent. Electrical stimulation of the hippocampal slice at sites not containing opioid peptides did not cause mu receptor displacement. These results demonstrate that under physiological conditions, the release of endogenous opioids from the major opioid containing pathways can be detected in a single hippocampal slice following high frequency stimulation.
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Mu opioid receptor activation reduces inhibitory postsynaptic potentials in hippocampal CA3 pyramidal cells of rat and guinea pig.
Using intracellular recording techniques, we characterized synaptic responses of CA3 pyramidal cells to mu and kappa agonists in hippocampal slices from rats and guinea pigs. In rat CA3 pyramidal cells, the mu selective agonist (N-MePhe3,D-Pro4)-morphiceptin (PLO17) inhibited both the early and the late inhibitory postsynaptic potentials (IPSPs) and increased excitatory postsynaptic potential (EPSP) amplitudes. Voltage clamp analysis of synaptic currents indicated that the excitatory postsynaptic current were not increased by PLO17, showing that the apparent increase in EPSPs was a result of a decrease in the underlying IPSP. The kappa agonists trans-(+)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide methanesulfonate and dynorphin A (1-17) had no effect on EPSPs or IPSP conductances measured in rat pyramidal cells. Maximal inhibition of IPSPs by PLO17 resulted in a bursting response to stimulation in rat but not guinea pig CA3 pyramidal cells. In guinea pig CA3 pyramidal cells, PLO17 also inhibited IPSP conductances but did not affect EPSP amplitudes. In contrast to the lack of effect in rat pyramidal cells, trans-(+)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide methanesulfonate (100 nM) inhibited the late IPSP conductance without influencing the EPSP or the early IPSP conductance of guinea pig pyramidal cells. Dynorphin A (1-17) (0.01-10 microM) did not affect resting membrane properties or evoked synaptic conductances in either preparation. Mu receptor activationin the CA3 of rats and guinea pigs results in the inhibition of inhibitory synaptic input to pyramidal cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Endogenously released opioids inhibit inhibitory post synaptic potentials in guinea pig CA3 pyramidal cells and rat dentate granule cells.
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Animals↗
Calcium-dependent displacement of haloperidol-sensitive sigma receptor binding in rat hippocampal slices following tissue depolarization.
To evaluate the possible existence of an endogenous ligand for the haloperidol-sensitive sigma receptor, we developed an in vitro competition assay to measure endogenous ligand release. Depolarization of in vitro hippocampal slices by either veratridine or potassium reduced [3H]ditolylguanidine binding in a calcium-dependent and transient manner. None of the drugs or iron substitutions directly affected [3H]ditolylguanidine binding to rat brain membranes. Veratridine-induced depolarization also reduced the binding of [3H](+)3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, another sigma radioligand, in a calcium-dependent manner. Radioligand displacement was not associated with alteration in sigma receptor dissociation kinetics or receptor degradation in the hippocampal slice. In contrast, KC1 depolarization had no effect on [3H]ditolyguanidine binding to sigma receptors in liver slices. The results suggest that a calcium-dependent, depolarization-induced reduction in sigma receptor binding may have been caused by the release of an endogenous sigma ligand in rat hippocampal tissue.
Animals↗
Release of endogenous opioid peptides displaces [3H]diprenorphine binding in rat hippocampal slices.
Pharmacological depolarization by KCl or veratrine reduced [3H]diprenorphine binding to opioid receptors in the hippocampal slice in a transient, calcium-dependent, and peptide-sensitive manner. These results suggest that endogenous opioid peptides were released from synaptic terminals and competitively displaced [3H]diprenorphine binding to opioid receptors. [3H]diprenorphine binding was significantly reduced by calcium-dependent depolarization throughout the hippocampus as determined by subsequent receptor autoradiography and quantitative densitometry. Displacement of binding was evident at sites in the CA1 and CA3 regions, the dentate gyrus, and the subiculum. The most dramatic reduction was evident in stratum lacunosum moleculare of CA3. Correlating the sites of maximal [3H]diprenorphine displacement with the previously described distribution of the opioid peptides suggests that the perforant path fibers release enkephalins in stratum lacunosum moleculare of CA3 and stratum moleculare of the dentate gyrus, and that mossy fibers may release both dynorphins and enkephalins near stratum pyramidale of CA3 and stratum granulosum. The lack of complete overlap between the distribution of opioid terminals and the sites of displacement indicates that these peptides may diffuse a moderate distance to their sites of action. Radioligand displacement defines the sites of endogenous opioid binding, suggests the likely sources of peptide release, and thus predicts the sites of endogenous opioid action within the hippocampus.
Diprenorphine↗
Comparison of opioid and GABA receptor control of excitability and membrane conductance in hippocampal CA1 pyramidal cells in rat.
Opioids are thought to increase the excitability of hippocampal pyramidal cells by decreasing release of neurotransmitter from inhibitory interneurons. This study compared the actions of the opioid agonist normorphine, and the GABA receptor antagonist bicuculline, on the responses of CA1 pyramidal cells to afferent stimulation. Both normorphine and bicuculline increased the sensitivity of pyramidal cells to presynaptic stimulation, increased the number of population spikes and action potentials elicited, increased the duration of the excitatory postsynaptic potential (EPSP) and reduced the change in input conductance during the early inhibitory postsynaptic potential (IPSP). Unlike bicuculline, normorphine also decreased the change in conductance during the late inhibitory postsynaptic potential. The decreased change in the conductance of pyramidal cells caused by normorphine during both early and late inhibitory postsynaptic potentials supports the hypothesis that opioids decrease the release of GABA from inhibitory interneurons. In addition to reducing GABA-mediated changes in conductance, both normorphine and bicuculline unmasked a D-APV-sensitive conductance, measured during the early inhibitory postsynaptic potential. These results demonstrate that activation of opioid receptors enhances the excitability of CA1 pyramidal cells by decreasing GABA-mediated early and late inhibitory postsynaptic potentials and by unmasking NMDA receptors.
2-Amino-5-phosphonovalerate↗
Effects of chronic morphine administration on the mu and delta opioid responses in the CA1 region of the rat hippocampus.
Extracellular recording of population spike amplitudes in the hippocampal CA1 region were compared in slices from control and chronically morphine-treated rats. Morphine treatment resulted in a decrease in the maximal excitation produced by both mu and delta selective agonists, [N-MePhe3,D-Pro4]-morphiceptin and [D-Pen2,L-Pen5]-enkephalin. The opioid antagonist naloxone did not produce apparent signs of withdrawal in hippocampal slices from morphine-treated rats as shown by a lack of change in the evoked population spike in the presence of 500 nM naloxone. Brain slices from morphine-treated rats that were maintained in the absence of morphine showed signs of tolerance reversal when monitored over an 8-hr period after dissection. If morphine-tolerant slices were maintained in vitro in the presence of 5 microM morphine (the concentration found by high-performance liquid chromatography to be present in the cerebrospinal fluid of morphine-treated rats), there was no significant reversal of tolerance. Furchgott analysis of the [N-MePhe3,D-Pro4]-morphiceptin concentration-response curve shifts induced by the irreversible opioid receptor antagonist beta-chlornaltrexamine revealed an apparent 50% spare receptor reserve for the mu selective agonist in slices from drug-naive rats. beta-Chlornaltrexamine (20 nM) treatment and chronic morphine exposure resulted in a similar reduction in the maximal response to [N-MePhe3,D-Pro4]morphiceptin. This observation indicates that the development of morphine tolerance resulted in an elimination of the spare opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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Comparison of the actions of phencyclidine and sigma ligands on CA1 hippocampal pyramidal neurons in the rat.
To compare the actions of prototypic drugs which are selective for phencyclidine and sigma receptors, the electrophysiological effects of phencyclidine (PCP),3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [+)3-PPP), and 1,3-di(2-tolyl)guanidine (DTG) on CA1 hippocampal pyramidal neurons were examined. A wide range of concentrations of drug was tested to differentiate specific, receptor-mediated effects from nonselective, anesthetic-like actions. At relatively large concentrations (0.1-1 mM), each compound reversibly increased the threshold of action potentials driven by Schaffer collaterals, the duration of action potentials and membrane resistance. The low potencies and rank order of potency suggested that phencyclidine, (+)3-PPP, and DTG were not acting through either high affinity sigma or phencyclidine receptors. These compounds did have receptor-mediated effects at smaller concentrations. Since none of the compounds affected evoked excitatory or inhibitory postsynaptic potentials (EPSP or IPSP) or driven action potentials at subanesthetic concentrations (less than 100 microM), no evidence was found to support the hypothesis that the actions of phencyclidine result from enhanced release of transmitter, caused by the inhibition of a presynaptic potassium conductance. As observed in other neurons, phencyclidine blocked excitations in CA1 pyramidal cells mediated by N-methyl-D-aspartic acid (NMDA) at behaviorally relevant concentrations (1-10 microM). However, (+)3-PPP (1 microM-1 mM) enhanced the pyramidal cell response to NMDA. Alone, DTG did not effect the NMDA-induced response but did inhibit the enhancement induced by (+)3-PPP. The agonist and antagonist actions of the sigma-selective ligands, (+)3-PPP and DTG, suggests that they modify NMDA-induced responses by acting at the sigma receptor.
Action Potentials↗