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The role of enkephalin and the opioid receptors in modulating GABA release within the rat globus pallidus (GP) was investigated using whole-cell patch recordings made from visually identified neurons. Two major GP neuronal subtypes were classified on the basis of intrinsic membrane properties, action potential characteristics, the presence of the anomalous inward rectifier (Ih), and anode break depolarizations. The mu opioid receptor agonist [D-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) (1 microM) reduced GABAA receptor-mediated IPSCs evoked by stimulation within the striatum. DAMGO also increased paired-pulse facilitation, indicative of presynaptic mu opioid receptor modulation of striatopallidal input. In contrast, the delta opioid agonist D-Pen-[D-Pen2, 5]-enkephalin (DPDPE) (1 microM) was without effect. IPSCs evoked by stimulation within the GP were depressed by application of [methionine 5']-enkephalin (met-enkephalin) (30 microM). Met-enkephalin also reduced the frequency, but not the amplitude, of miniature IPSCs (mIPSCs) and increased paired-pulse facilitation of evoked IPSCs, indicative of a presynaptic action. Both DAMGO and DPDPE reduced evoked IPSCs and the frequency, but not amplitude, of mIPSCs. However, spontaneous action potential-driven IPSCs were reduced in frequency by met-enkephalin and DAMGO, whereas DPDPE was without effect. Overall, these results indicate that presynaptic mu opioid receptors are located on striatopallidal terminals and pallidopallidal terminals of spontaneously firing GP neurons, whereas presynaptic delta opioid receptors are preferentially located on terminals of quiescent GP cells. Enkephalin, acting at both of these receptor subtypes, serves to reduce GABA release in the GP and may therefore act as an adaptive mechanism, maintaining the inhibitory function of the GP in basal ganglia circuitry.

The inhibition of hippocampal pyramidal cells occurs via inhibitory interneurons making GABAergic synapses on distinct segments of the postsynaptic membrane. In area CA1 of the hippocampus, the activation of mu- and delta-opioid receptors inhibits these interneurons, thereby increasing the excitability of the pyramidal cells. Through the use of selective opioid agonists and biocytin-filled whole-cell electrodes, interneurons possessing somata located within stratum oriens of hippocampal slices were classified according to the location of their primary axon termination and the expression of mu- or delta-opioid receptors. Activation of these opioid receptor subtypes resulted in outward currents in the majority of interneurons, which is consistent with their inhibition. Post hoc morphological analysis revealed that those interneurons heavily innervating the pyramidal cell body layer were much more likely to express mu-opioid receptors, whereas cells with axons ramifying in the pyramidal neuron dendritic layers were more likely to express delta-opioid receptors, as defined by the generation of outward currents. This morphological segregation of interneuron projections suggests that mu receptor activation would diminish GABA release onto pyramidal neuron somata, thereby increasing their excitability and output. Conversely, inhibition of interneurons via delta receptor activation would amplify afferent signaling to pyramidal neuron dendrites by reducing GABAergic inhibition of these structures.

There is some doubt as to the effectiveness of opioids in the management of neuropathic pain. We therefore examined the actions of morphine and the opioid-like peptide nociceptin (both 1 mu) on dorsal root ganglion (DRG) neurons that were isolated from control or from nerve-injured rats. Both substances reduced omega-conotoxin (CTX) GVIA-sensitive, N-type Ca2+ channel current and small persistent nifedipine/ CTX-insensitive (non-N, non-L type) current. Nifedipine-sensitive L-type current was unaffected. The effect of nociceptin was antagonized by naloxone benzoylhydrazone (nalbzoh) but not by naloxone. Sciatic nerve section (axotomy) profoundly reduced the effects of morphine and the mu-receptor agonist D-ala2, N-Me-Phe4,Gly-ol5 enkephalin (DAMGO). The effect of the kappa-agonist [(+)-(5alpha,7alpha, 8beta)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)-benzeneacetamide] (U69593) was unchanged, whereas the effect of nociceptin was increased. All agonists produced their strongest effects on the small, putative nociceptive cells and their weakest effects on the largest cells. The delta-receptor agonist, enkephalin D-pen2,5 (DPDPE), was without effect on control or on axotomized cells. These and other data suggest that the functional downregulation of mu-opioid receptors on sensory nerves contributes to the poor efficacy of opioids in neuropathic pain. Also, the increased effectiveness of nociceptin after axotomy supports the hypothesis that its actions are mediated via a "non-opioid" receptor. Pronounced suppression of Ca2+ channel current in axotomized DRG neurons by nociceptin led to a reduction in Ca2+-dependent K+ conductance and a marked increase in excitability. Despite this, the spinal administration of nociceptin or agonists that activate ORL1 (opioid-like orphan receptor) may prove to be of clinical interest in the management of neuropathic pain.

Motor activity elicited pharmacologically from the nucleus accumbens by the mu-opioid receptor agonist D-Ala-Tyr-Gly-NMePhe-Gly-OH (DAMGO) is augmented in rats sustaining dopamine depletions. GABAergic projections from the nucleus accumbens to ventral pallidum and ventral tegmental area (VTA) are involved because stimulation of GABAB receptors in the VTA (by baclofen) or GABAA receptors in the ventral pallidum (by muscimol) inhibit the motor response induced by the microinjection of DAMGO into the nucleus accumbens. The present study was done to determine which of these projections is mediating the augmented DAMGO-induced motor activity that follows 6-hydroxydopamine lesions of the nucleus accumbens. The inhibition of DAMGO-induced activation by pallidal injections of muscimol was markedly attenuated in lesioned animals, whereas the inhibition by VTA injections with baclofen was greatly enhanced. A similar switch in emphasis from pallidal to mesencephalic efferents was not observed for dopamine-induced motor activity, because muscimol microinjections inhibited the response elicited by dopamine microinjection into the nucleus accumbens in all subjects. The stimulation of mu-opioid receptors in the ventral pallidum also elicits motor activation, and this is blocked by baclofen microinjection into the VTA. However, after dopamine depletion in the nucleus accumbens, baclofen in the VTA was ineffective in blocking the motor response by DAMGO in the ventral pallidum. These data reveal that dopamine depletion in the nucleus accumbens produces a lesion-induced plasticity that alters the effect of mu-opioid receptor stimulation on efferent projections from the nucleus accumbens and ventral pallidum.

The actions of mu- and delta-opioid agonists (DAMGO and DPDPE, respectively) on GABAergic interneurons in stratum oriens of area CA1 of the hippocampus were examined by using whole-cell voltage-clamp recordings in brain slices. Both agonists consistently generated outward currents of similar magnitude (15-20 pA) in the majority of cells. However, under control conditions, current-voltage (I/V) relationships revealed that only a small number of these cells (3 of 77) demonstrated clear increases in membrane conductance, associated with the activation of the potassium current known as Girk. These interneurons also exhibited a slowly activating, inwardly rectifying current known as Ih on hyperpolarizing step commands. Ih was blocked by the extracellular application of cesium (3-9 mM) or ZD 7288 (10-100 microM) but was insensitive to barium (1-2 mM). In an effort to determine whether the holding current changes were attributable to the modulation of Girk and/or Ih, we used known blockers of these ion channels (barium or cesium and ZD 7288, respectively). Extracellular application of cesium (3-9 mM) or ZD 7288 (25-100 microM) blocked Ih and significantly reduced the opioid-induced outward currents by 58%. Under these conditions the opioid agonists activated a potassium current with characteristics similar to Girk. Similarly, during barium (1-2 mM) application the opioid-induced outward currents were reduced by 46%, and a clear reduction in Ih and the whole-cell conductance was revealed. These data suggest that the opioids can modulate both Ih and Girk in the same population of stratum oriens interneurons and that the modulation of these ion channels can contribute to the inhibition of interneuron activity in the hippocampus.

Previous pharmacological studies have indicated the possible existence of functional interactions between mu-, delta- and kappa-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of delta- and kappa-opioid receptors in mice lacking the mu-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPgammaS binding and adenylyl cyclase inhibition showed that functional coupling of delta- and kappa-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[D-penicillamine2, D-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR -/- mice. delta-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR -/- mice. Analgesic and respiratory responses produced by the selective kappa-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of delta- and kappa-receptor signaling properties in mice lacking mu-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the kappa-agonist further suggest that the kappa-receptor mainly acts independently from the mu-receptor in vivo. Reduced delta-analgesia and the absence of delta-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between mu-receptors and central delta-receptors in specific neuronal pathways.

The mu-opioid receptor (mu-OR), like most G-protein-coupled receptors, is rapidly internalized after agonist binding. Although opioid peptides induce internalization in vivo, there are no studies that demonstrate mu-OR internalization in response to natural stimuli. In this study, we used laser-scanning microscopy to demonstrate that estrogen treatment induces the translocation of mu-OR immunoreactivity (mu-ORi) from the membrane to an internal location in steroid-sensitive cell groups of the limbic system and hypothalamus. Estrogen-induced internalization was prevented by the opioid antagonist naltrexone, suggesting that translocation was largely dependent on release of endogenous agonists. Estrogen treatment also altered the pattern of mu-ORi at the bright-field light microscopic level. In the absence of stimulation, the majority of immunoreactivity is diffuse, with few definable mu-OR+ cell bodies or processes. After stimulation, the density of distinct processes filled with mu-ORi was significantly increased. We interpreted the increase in the number of mu-OR+ processes as indicating increased levels of internalization. Using this increase in the density of mu-OR+ fibers, we showed that treatment of ovariectomized rats with estradiol benzoate induced a rapid and reversible increase in the number of fibers. Significant internalization was noted within 30 min and lasted for >24 hr after estrogen treatment in the medial preoptic nucleus, the principal part of the bed nucleus, and the posterodorsal medial amygdala. Naltrexone prevented the increase of mu-OR+ processes. These data imply that estrogen treatment stimulates the release of endogenous opioids that activate mu-OR in the limbic system and hypothalamus providing a "neurochemical signature" of steroid activation of these circuits.

Opioidergic inhibition of neurons in the centrolateral nucleus of the thalamus was investigated using an in vitro thalamic slice preparation from young rats. The mu-opioid receptor agonist D-Ala2,N-Me-Phe4,glycinol5-enkephalin (DAMGO) evoked a hyperpolarization and decrease in input resistance that was reversible, concentration-dependent, and persisted in the presence of tetrodotoxin. Application of the specific mu-receptor antagonist Cys2,Tyr3,Orn5,Pen7-amide blocked this response. The respective delta- and kappa-opioid receptor agonists, (D-Pen2, D-Pen5)-enkephalin and (+/-)-trans-U-50488 methanesulfonate had no effect. Voltage-clamp experiments showed that DAMGO activated an inwardly rectifying potassium conductance (GKIR) characterized by rectification at hyperpolarized potentials that increased in elevated extracellular potassium concentrations, a complete block by Ba2+ (1 mM), and a voltage-dependent block by Cs+. The extent of mu-opioid inhibition in other thalamic nuclei was then investigated. Widespread inhibition similar to that seen in the centrolateral nucleus was observed in a number of sensory, motor, intralaminar, and midline nuclei. Our results suggest that the net action of opioids would depend on their source: exogenous (systemically administered) opiates inhibiting the entire thalamus and favoring the shift of cell firing from tonic to bursting mode; and endogenously released opioids acting on specific thalamic nuclei, their release depending on the origin of the presynaptic input.

To characterize how systemic morphine induces Fos protein in dorsomedial striatum and nucleus accumbens (NAc), we examined the role of receptors in striatum, substantia nigra (SN), and ventral tegmental area (VTA). Morphine injected into medial SN or into VTA of awake rats induced Fos in neurons in ipsilateral dorsomedial striatum and NAc. Morphine injected into lateral SN induced Fos in dorsolateral striatum and globus pallidus. The morphine infusions produced contralateral turning that was most prominent after lateral SN injections. Intranigral injections of [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a mu opioid receptor agonist, and of bicuculline, a GABAA receptor antagonist, induced Fos in ipsilateral striatum. Fos induction in dorsomedial striatum produced by systemic administration of morphine was blocked by (1) SN and VTA injections of the mu1 opioid antagonist naloxonazine and (2) striatal injections of either MK 801, an NMDA glutamate receptor antagonist, or SCH 23390, a D1 dopamine receptor antagonist. Fos induction in dorsomedial striatum and NAc after systemic administration of morphine seems to be mediated by dopamine neurons in medial SN and VTA that project to medial striatum and NAc, respectively. Systemic morphine is proposed to act on mu opioid receptors located on GABAergic interneurons in medial SN and VTA. Inhibition of these GABA interneurons disinhibits medial SN and VTA dopamine neurons, producing dopamine release in medial striatum and NAc. This activates D1 dopamine receptors and coupled with the coactivation of NMDA receptors possibly from cortical glutamate input induces Fos in striatal and NAc neurons. The modulation of target gene expression by Fos could influence addictive behavioral responses to opiates.

The mu-opioid [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) exerts a peripheral antinociceptive effect against prostaglandin E2 (PGE2)-induced mechanical hyperalgesia in the hindpaw of the rat. Tolerance and dependence develop to this effect. We have shown previously that tolerance and dependence can be dissociated and are mediated by different second messenger systems. In the present study, we evaluated whether the same or different second messenger systems mediate the development of this peripheral opioid tolerance or dependence compared with the expression of the loss of antinociceptive effect or rebound opioid antagonist hyperalgesia (i. e., expression of tolerance and dependence). DAMGO-induced tolerance was prevented by pretreatment with the nitric oxide synthase inhibitor NG-methyl-L-arginine (NMLA) but not by the protein kinase C (PKC) inhibitor chelerythrine, the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (ddA), or the calcium chelators 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8) and 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (Quin-2). Once established, however, expression of DAMGO tolerance was acutely reversed by TMB-8 or Quin-2 but not by chelerythrine or NMLA. In contrast, naloxone-precipitated hyperalgesia in DAMGO-tolerant paws, a measure of dependence, was blocked by pretreatment with chelerythrine but not by NMLA, ddA, TMB-8, or Quin-2. Naloxone-precipitated hyperalgesia in DAMGO-tolerant paws was acutely reversed by chelerythrine, ddA, TMB-8, or Quin-2 but not by NMLA. Taken together, these results provide the first evidence that different mechanisms mediate the development and expression of both tolerance and dependence to the peripheral antinociceptive effect of DAMGO. However, although the development of tolerance and dependence are entirely separable, the expression of tolerance and dependence shares common calcium-dependent mechanisms.

Agonists acting at alpha2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the alpha2a adrenergic receptor (alpha2aAR) to investigate the role of the alpha2aAR in alpha2 agonist-evoked analgesia and adrenergic-opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective alpha2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased alpha2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the delta-opioid agonist deltorphin II and the micro-opioid agonist [D-ALA2,N-Me-Phe4, Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the alpha2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the alpha2aAR subtype is the primary mediator of alpha2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the alpha2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

Repeated peripheral administration of the micro-opioid agonist [D-Ala2,N-Me-Phe4,gly5-ol] enkephalin (DAMGO) produces acute tolerance and dependence on its peripheral antinociceptive effect against prostaglandin E2 (PGE2)-induced mechanical hyperalgesia. In this study we evaluated the roles of protein kinase C (PKC) and nitric oxide (NO) in the development of this tolerance and dependence. Repeated administration of PKC inhibitors chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride with DAMGO did not alter the tolerance to DAMGO; however, dependence (defined as naloxone-induced withdrawal hyperalgesia) was blocked. Repeated administration of N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide, a PKC activator, which alone did not produce tolerance, mimicked the dependence produced by DAMGO. Repeated administration of the NO synthase inhibitor NG-methyl-L-arginine with DAMGO blocked the development of tolerance to DAMGO but had no effect on the development of dependence. Repeated administration of L-arginine, a NO precursor, mimicked tolerance produced by repeated administration of DAMGO (i.e. , the antinociceptive effect of DAMGO was lost); however, L-arginine did not mimic dependence. These findings suggest that the development of acute tolerance and dependence on the peripheral antinociceptive effects of DAMGO have different, dissociable mechanisms. Specifically, PKC is involved in development of mu-opioid dependence, whereas the NO signaling system is involved in the development of mu-opioid tolerance.

We examined the interactions among three classes of peripherally-acting antinociceptive agents (mu-opioid, alpha 2-adrenergic, and A1-adenosine) in the development of tolerance and dependence to their antinociceptive effects. Antinociception was determined by assessing the degree of inhibition of prostaglandin E2 (PGE2)-induced mechanical hyperalgesia, using the Randall-Selitto paw-withdrawal test. Tolerance developed within 4 hr to the antinociceptive effect of the alpha 2-adrenergic agonist clonidine; dependence also occurred at that time, demonstrated as a withdrawal hyperalgesia that was precipitated by the alpha 2-receptor antagonist yohimbine. These findings are similar to those reported previously for tolerance and dependence to mu and A1 peripheral antinociception (Aley et al., 1995). Furthermore, cross-tolerance and cross-withdrawal between mu, A1, and alpha 2 agonists occurred. The observations of cross-tolerance and cross-withdrawal suggest that all three receptors are located on the same primary afferent nociceptors. In addition, the observations suggest that the mechanisms of tolerance and dependence to the antinociceptive effects of mu, A1, and alpha 2 are mediated by a common mechanism. Although any of the agonists administered alone produce antinociception, we found that mu, A1, and alpha 2 receptors may not act independently to produce antinociception, but rather may require the physical presence of the other receptors to produce antinociception by any one agonist. This was suggested by the finding that clonidine (alpha 2-agonist) antinociception was blocked not only by yohimbine (alpha 2-antagonist) but also by PACPX (A1-antagonist) and by naloxone (mu-antagonist), and that DAMGO (mu-agonist) antinociception and CPA (A1-agonist) antinociception were blocked not only by naloxone (mu-antagonist) and PACPX (A1-antagonist), respectively, but also by yohimbine (alpha 2-antagonist). This cross-antagonism of antinociception occurred at the ID50 dose for each antagonist at its homologous receptor. To test the hypothesis that the physical presence of mu-opioid receptor is required not only for mu antinociception but also for alpha 2 antinociception, antisense oligodeoxynucleotides (ODNs) for the mu-opioid and alpha 2C-adrenergic receptors were administered intrathecally to reduce the expression of these receptors on primary afferent neurons. These studies demonstrated that mu-opioid ODN administration decreased not only mu-opioid but also alpha 2-adrenergic antinociception; A1 antinociception was unaffected. In contrast, alpha 2C-adrenergic ODN decreased antinociception induced by all three classes of antinociceptive agents. In conclusion, these data suggest that peripheral antinociception induced by mu, alpha 2, and A1 agonists requires the physical presence of multiple receptors. We propose that there is a mu, A1, alpha 2 receptor complex mediating antinociception in the periphery. In addition, there is cross-tolerance and cross-dependence between mu, A1, and alpha 2 antinociception, suggesting that their underlying mechanisms are related.

The nucleus accumbens (NAcc) may play a major role in opiate dependence, and central NMDA receptors are reported to influence opiate tolerance and dependence. Therefore, we investigated the effects of the selective mu-opioid receptor agonist [D-Ala2-N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) on membrane properties of rat NAcc neurons and on events mediated by NMDA and non-NMDA glutamate receptors, using intracellular recording in a brain slice preparation. Most NAcc neurons showed a marked inward rectification (correlated with Cs+- and Ba2+-sensitive inward relaxations) when hyperpolarized, as well as a slowly depolarizing ramp with positive current pulses. Superfusion of DAMGO did not alter membrane potential, input resistance, or the inward relaxations. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) used to block non-NMDA glutamate receptors and bicuculline to block GABAA receptors, EPSPs evoked by local stimulation displayed characteristics of an NMDA component: (1) long duration, (2) voltage sensitivity, and (3) blockade by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (D-APV). DAMGO (0.1-1 muM) significantly decreased both NMDA- and non-NMDA-EPSP amplitudes with reversal of this effect by naloxone and the mu-selective antagonist [Cys2-Tyr3-Orn5-Pen7]-somatostatinamide (CTOP). To assess a postsynaptic action of DAMGO, we superfused slices with tetrodotoxin and evoked inward currents by local application of glutamate agonists. Surprisingly, 0.1-1 microM DAMGO markedly enhanced the NMDA currents (with reversal by CTOP) but reduced the non-NMDA currents. At higher concentrations (5 microM), DAMGO reduced NMDA currents, but this effect was enhanced, not blocked, by CTOP. These results indicate a complex DAMGO modulation of the NMDA component of glutamatergic synaptic transmission in NAcc: mu receptor activation decreases NMDA-EPSP amplitudes presynaptically yet increases NMDA currents postsynaptically. These new data may provide a cellular mechanism for the previously reported role of NMDA receptors in opiate tolerance and dependence.

Application of 4-aminopyridine (4AP, 50 microM) to combined slices of adult rat hippocampus-entorhinal cortex-induced ictal and interictal epileptiform discharges, as well as slow field potentials that were abolished by the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAGO, 10 microM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 10 microM); hence, they represented synchronous GABA-mediated potentials. Ictal discharges originated in the entorhinal cortex and propagated to the hippocampus, whereas interictal activity of CA3 origin was usually recorded in the hippocampus. The GABA-mediated potentials had no fixed site of origin or modality of propagation; they closely preceded (0.2-5 sec) and thus appeared to initiate ictal discharges. Only ictal discharges were blocked by the antagonist of the NMDA receptor 3,3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP, 10 microM), whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished all epileptiform activities. The GABA-mediated potentials continued to occur synchronously in all regions even after concomitant application of CNQX and CPP. [K+]o elevations were recorded in the entorhinal cortex during the ictal discharge (peak values = 13.9 +/- 0.9 mM) and the synchronous GABA-mediated potentials (peak values = 4.2 +/- 0.1 mM); the latter increases were presumably attributable to postsynaptic GABAa-receptor activation because they were abolished by DAGO or BMI. Their role in initiating ictal activity was demonstrated by using DAGO, which abolished both GABA-mediated synchronous potentials and ictal discharges. These data indicate that NMDA-mediated ictal discharges induced by 4AP originate in the entorhinal cortex; such a conclusion is in line with clinical evidence obtained in temporal lobe epilepsy patients. 4AP also induces GABA-mediated potentials that spread within the limbic system when excitatory transmission is blocked and may play a role in initiating ictal discharge by increasing [K+]o.

The selective mu-opioid agonist, D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAMGO), or the selective A1-adenosine agonist N6-cyclopentyladenosine (CPA), when coinjected intradermally with prostaglandin E2 (PGE2), dose-dependently inhibited PGE2-induced mechanical hyperalgesia in the rat hindpaw, as determined by the Randall-Selitto paw-withdrawal test. Repeated (hourly x 3) intradermal injections of DAMGO or CPA produced tolerance to the antinociceptive effect of a fourth injection 1 hr later. Furthermore, repeated (hourly x 3) intradermal injections of DAMGO produced cross-tolerance to the antinociceptive effect of CPA, and repeated (hourly x 3) intradermal injection of CPA produced cross-tolerance to the antinociceptive effect of DAMGO. The demonstration of the bidirectional cross-tolerance between the peripheral antinociceptive effects of DAMGO and CPA supports the hypothesis that both these agents produced antinociception by acting on the same cell, presumably the primary afferent nociceptor, and that the development of tolerance involves changes downstream to activation of mu-opioid and A1-adenosine receptors. The opioid antagonist naloxone, which had no effect on paw-withdrawal threshold in normal paws, produced withdrawal threshold in normal paws, produced withdrawal hyperalgesia in DAMGO-tolerant paws. Furthermore, naloxone elicited a cross-withdrawal hyperalgesia response in CPA-tolerant paws. Similarly, the A1-adenosine antagonist 1,3-dipropyl-8-(2-amino-4- chlorophenyl)-xanthine (PACPX), which had no effect on paw-withdrawal threshold in normal paws, elicited a withdrawal hyperalgesia response in CPA-tolerant paws and cross-withdrawal hyperalgesia responses in DAMGO-tolerant paws. These cross-dependence and cross-withdrawal responses suggest that the development of dependence to mu-opioid and A1-adenosine agonists involves changes in the same second messenger system downstream to both mu-opioid and A1-adenosine receptor activation.

We used patch-clamp methods to describe signal transduction between the mu opioid receptor, the binding site for morphine, and high-threshold Ca2+ channels in dorsal root ganglion (DRG) sensory neurons from adult rats. Opioid signaling persists in excised membrane patches, and an activated opioid receptor can only inhibit nearby Ca2+ channels; thus, no readily diffusible second-messenger molecule mediates between the mu receptor and Ca2+ channels. Inhibition of Ca2+ channels begins several hundred msec after application of opioid and it is maximal by 5 sec; this is faster than typical phosphorylation cascades. Blockade of the known serine-threonine kinases and phosphatases does not affect this opioid signaling and, as shown previously by Seward et al. (1991) and Moises et al. (1994a), pertussis toxin eliminates virtually all of the effect. Inhibited channels can open, but their half-activation voltage is unphysiologically positive. The link between the mu receptor and Ca2+ channels is clearly unlike the protein kinase C-dependent paths that couple mu receptors to NMDA channels in dorsal horn neurons (Chen and Huang, 1991) and alpha-adrenergic receptors to Ca2+ channels in DRG neurons (Diversé-Pierluissi and Dunlap, 1993). The rapid kinetics and tight localization of the signaling path are properties expected if receptor and channel are linked directly by a G-protein, but these properties do not constitute proof of such a pathway.

Studies in mice and rats have shown that antinociception produced by intrathecal (i.t.) administration of opioids can be partially inhibited by intracerebroventricular (i.c.v.) administration of naloxone. In this study we tested the hypothesis that this inhibition by i.c.v. naloxone results from antagonism of supraspinal endogenous opioid-mediated antinociception produced by the action of i.t. opioids on an ascending antinociceptive pathway. In rats lightly anesthetized with urethane/alpha-chloralose, i.t. DAMGO, i.t. lidocaine, or spinal transection at T5-T6 all attenuated the trigeminal jaw opening reflex (JOR) (i.e., were antinociceptive), an effect that was antagonized in each case by i.c.v. naloxone. These findings support the suggestion that there exists a pathway that ascends from the spinal cord to a supraspinal site that tonically inhibits antinociception mediated by supraspinal opioids. When activity in this ascending pathway is suppressed (e.g., by i.t. opioids or local anesthetics or by spinal cord transection), antinociception mediated by supraspinal opioids is disinhibited. To determine the supraspinal site(s) at which endogenous opioid-dependent antinociception is evoked by i.t. opioids, we microinjected naloxone methiodide into several supraspinal sites. Microinjection of naloxone methiodide into nucleus accumbens but not into the rostral ventral medulla (RVM) or the periaqueductal gray matter (PAG) antagonized the suppression of the JOR produced by i.t. DAMGO or lidocaine. The possibility that this ascending pathway may represent a source of spinal input to mesolimbic circuitry involved in setting the state of sensorimotor reactivity to noxious stimuli is discussed.

Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

We have investigated the coupling between opioid receptors and different types of Ca2+ channels in neurons acutely isolated from the nucleus tractus solitarius (NTS) of the rat. Using fura-2-based imaging we found that Ca2+ transients evoked by depolarization with 50 mM KCl were suppressed by the mu-opioid receptor agonist D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO) and less effectively by the kappa-receptor agonist U-69,593. The delta-receptor agonist D-Pen2,D-Pen5-enkephalin (DPDPE) was ineffective. In whole-cell voltage-clamp recordings from these neurons, depolarizing voltage steps elicited high-threshold Ca2+ currents that could be distinguished pharmacologically into different components. Part of the current could be blocked by dihydropyridines, part by omega-conotoxin-GVIA and part by omega-agatoxin-IVA. This suggests that the neurons contained L-, N-, and P/Q-type Ca2+ channels. DAMGO and U-69,593 both blocked part of the Ca2+ current but DPDPE was ineffective. Perfusion of GTP-gamma-S into the cells produced a rapid rundown of the Ca2+ current and occluded further effects of the opioid agonists, suggesting the involvement of a G-protein in the coupling mechanism. Inhibition of L-channels did not alter the effect of DAMGO. On the other hand inhibition of N-channels occluded about 80% of the effect of DAMGO. Inhibition of the P/Q-current occluded the remainder of the DAMGO effect. Thus, it appears that activation of opioid receptors can inhibit N- and P/Q-type Ca2+ channels but not L-channels in these cells. It is likely that such effects are important in opioid-mediated inhibition of transmitter release in the brain.

The presence of substance P in primary afferents that terminate in the outer laminae of the spinal cord has led to considerable interest in the function of this neuropeptide in nociception. We have examined the actions of tachykinin agonists on the membrane potential of neurons in lamina II of a neonatal spinal cord slice preparation in vitro. Only 10.5% (n = 75) of these neurons showed any response to the application of a selective NK1 receptor agonist while 48.3% (n = 60) of neurons in deeper dorsal horn laminae responded to this agonist. Lamina II neurons were equally insensitive to selective NK2 and NK3 agonists. Synaptic potentials evoked in lamina II neurons by peripheral nerve stimulation were similarly not altered by the NK1 agonist. Immunocytochemical studies using an antibody raised against the C-terminal of the NK1 receptor revealed that very few lamina II neurons express NK1 receptors, and this offers an explanation for our findings.

The mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) hyperpolarizes the majority of arcuate hypothalamic (ARC) neurons by opening an inwardly rectifying potassium conductance. The EC50 for the DAMGO-induced hyperpolarization was 60 +/- 3 nM in ARC neurons from ovariectomized guinea pigs. Superfusion of 17 beta-estradiol (E2; 100 nM) for 20 min in vitro resulted in a significant decrease in DAMGO potency (EC50 = 212 +/- 16 nM) in 40% of the neurons that were tested. This rapid effect of E2 on the mu-opioid response was not mimicked by the biologically inactive isomer 17 alpha-estradiol. Multiple concentrations of E2 were used to generate an E2 concentration-response curve, with an EC50 of 9 nM and a maximal increase in the DAMGO EX50 of 411% of controls. The membrane properties and firing rate of E2-sensitive and E2-insensitive neurons were not different. Streptavidin-FITC labeling did not reveal any significant morphological differences between the groups, but a higher number of E2-sensitive cells was found in the lateral ARC and cell-poor zone. Moreover, immunocytochemical staining of the recorded cells revealed that beta-endorphin neurons were among those sensitive to E2. Therefore, E2 could increase beta-endorphin release by decreasing the potency of beta-endorphinergic autoinhibition, thus increasing the tonic opioid inhibition of E2-insensitive cells. Furthermore, the diffuse projections of hypothalamic beta-endorphin neurons would allow E2 to alter processes throughout the brain, as well as having local effects in the hypothalamus.

AtT-20 cells, which make and release beta-endorphin, or AtT-20/hENK cells, an AtT-20 cell line transfected with the human proenkephalin gene and secreting enkephalin as well as presumably beta-endorphin, were implanted in mouse spinal subarachnoid space. Cell implants did not affect the basal response to thermal nociceptive stimuli. Administration of isoproterenol, believed to stimulate secretion from these cells, produced antinociception in groups receiving AtT-20 or AtT-20/hENK cell implants but not in control groups receiving no cells. The antinociceptive effect of isoproterenol was dose related and could be blocked by the opioid antagonist naloxone. Implantation of these cells offers a novel approach for the study of tolerance. Mice receiving AtT-20 cell implants developed tolerance to beta-endorphin and the mu-opioid agonist DAMGO, whereas mice receiving genetically modified AtT-20/hENK cell implants developed tolerance to the delta-opioid agonist DPDPE. Genetically modified AtT-20/hENK cell implants, but not AtT-20 cell implants, reduced the development of acute morphine tolerance in the host mice. This finding is consistent with the suggestion that enkephalin alters development of opioid tolerance. These results suggest that opioid-releasing cells implanted around mouse spinal cord can produce antinociception and may provide an alternative therapy for chronic intractable pain.

The contribution of high-frequency synaptic activity to the induction of long-term potentiation (LTP) in the opioid peptide-containing mossy fiber projection was investigated in vivo in anesthetized rats. Because high-frequency mossy fiber activity is essential for both the release of opioid peptides and the induction of mossy fiber LTP, we investigated whether the activation of opioid receptors underlies the requirement of sustained high-frequency mossy fiber activity for LTP induction. Mossy fiber responses were found to have a distinct threshold for the number of 100 Hz pulses necessary to induce LTP, with bursts of 25-30 pulses being the minimum for LTP induction. Application of 1 nmol of the mu-opioid receptor agonist DAMGO to the CA3 region potentiated mossy fiber responses, but, unlike for mossy fiber LTP, this potentiation could be reversed by mu-opioid receptor antagonist CTOP. Stimulation of the mossy fibers with either a single burst of 15 pulses at 100 Hz or application of 100 pmol of DAMGO was ineffective in potentiating mossy fiber responses. However, delivery of a 15 pulse burst 10 min following DAMGO application was effective in potentiating mossy fiber responses. This potentiation was not reversed by CTOP and it occluded stimulation-induced LTP, suggesting that brief bursts delivered in the presence of DAMGO had induced mossy fiber LTP. The release of opioid peptides and the resulting activation of mu-opioid receptors is suggested as one factor that underlies the requirement of sustained high-frequency stimulation for the induction of mossy fiber LTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracranial self-administration of mu- and delta-opioid agonists was demonstrated in male Long-Evans rats. Independent groups were allowed to lever-press for ventral tegmental area (VTA) microinfusions of morphine, the selective mu agonist [D-Ala2,N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO), the selective delta-agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE), or ineffective drug vehicle. Morphine, DAMGO, and DPDPE were each effective in establishing and maintaining lever-pressing habits. Lever-pressing responses were extinguished during a session when vehicle was substituted for drug, and reinstated when drug reinforcement was reestablished. Thus, it appears that VTA mu- and delta-opioid receptors are each involved in reinforcement of opiate self-administration. The effective dose of DAMGO--both for establishing and for maintaining the lever-press habit--was 100 times lower than the effective doses for DPDPE and morphine, suggesting that the major contribution of VTA mechanisms to intravenous heroin self-administration involves an action on mu-opioid receptors.

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

The actions of opioid agonists in the substantia gelatinosa are important for their antinociceptive effects. In order to identify possible mechanisms underlying opioid actions in the substantia gelatinosa, the pre- and postsynaptic effects of opioid agonists on neurons of the substantia gelatinosa were examined using a brain slice preparation. Intracellular recordings were made from neurons of the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis in guinea pig and rat. To correlate morphology and electrophysiology, neurons were filled with biocytin and visualized using HRP. The majority of neurons (86%) were hyperpolarized by [Met]5enkephalin (ME), and this was mimicked by the mu-opioid agonist (D-Ala2,N-Me-Phe4,Gly5-ol)enkephalin (DAMGO) but not the delta-opioid agonist (D-Pen2,5)enkephalin (DPDPE). Naloxone (300 nM) shifted the DAMGO dose-response 213-fold to the right, giving an estimated KD of 1.4 nM. Under voltage clamp, the ME current reversed polarity at the potassium equilibrium potential, indicating the hyperpolarization was due to an increase in potassium conductance. EPSPs mediated by glutamate were evoked by stimulating the spinal trigeminal tract, which contains the primary afferent fibers that synapse in the spinal trigeminal nucleus. The excitation produced by stimulating the spinal trigeminal tract was greatly enhanced in the presence of glycine and GABAA receptor antagonists, indicating that local inhibitory circuitry is activated by exciting the primary afferents. The EPSPs were reduced by mu- but not delta-opioid receptor activation. The degree of inhibition varied from 0 to 100%. These results indicate that opioid agonists cause inhibition in the substantia gelatinosa by both pre- and postsynaptic actions.

Intracellular recordings were made from layer V pyramidal neurons in slices of rat anterior cingulate cortex, using electrodes that contained potassium methylsulfate and biocytin. [Met5]enkephalin (300 nM to 30 microM) reversibly reduced the amplitude of EPSPs evoked by stimulation of the subcortical white matter; the half-maximal concentration was about 800 nM. These EPSPs were blocked by (+/-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. [Met5]enkephalin also reduced the amplitude of bicuculline-sensitive IPSPs evoked by stimulation within layer V; the half-maximal concentration was about 60 nM. Both these actions of [Met5]enkephalin were mimicked by the delta-selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) but not by the mu-selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol); they were blocked by the delta-selective antagonist naltrindole (apparent dissociation constant of about 0.3 nM) but not by the mu-selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). [Met5]enkephalin did not change the amplitudes of depolarizations evoked by direct application of glutamate or hyperpolarizations evoked by direct application of muscimol (at -55 mV). Fifty percent (22 of 45) of pyramidal cells were hyperpolarized by [Met5]enkephalin; this resulted from an increase in potassium conductance, and it was mimicked by DPDPE and blocked by naltrindole. Five of seven nonpyramidal cells were hyperpolarized by [Met5]enkephalin; this was mimicked by DAMGO and blocked by CTOP.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of met-enkephalin on GABAergic spontaneous miniature IPSPs (smIPSPs) was investigated in CA1 neurons from hippocampal slice cultures. In the presence of excitatory amino acid blockers (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, DL-2-amino-5-phosphonovaleric acid) and TTX, a continuous high-frequency bombardment of smIPSPs was recorded. The smIPSPs were blocked by the GABAA antagonist bicuculline. The occurrence of the smIPSPs was random and their amplitude distribution was skewed toward larger smIPSPs. Met-enkephalin (10-20 microM) reversibly reduced the frequency and changed the amplitude distribution of the smIPSPs. The proportion of "large" smIPSPs was reduced, but a loss of "small" smIPSPs also contributed to the reduction in smIPSP frequency. The selective mu-receptor agonist DAGO mimicked the effect of met-enkephalin and naloxone blocked the effect of DAGO. Hyperpolarization of the neuronal membranes, produced by reducing the extracellular K+ concentration, did not reduce the frequency of the smIPSPs, nor did it block the effect of DAGO. Reduction of the extracellular concentration of Ca2+ combined with an increase in extracellular Mg2+ or the addition of Cd2+ did not reduce the smIPSP frequency, nor did it block the effect of DAGO. These results suggest that CA1 pyramidal cells of hippocampal organotypic cultures are tonically inhibited by spontaneous release of GABA, through a release mechanism that is independent of propagated sodium action potentials. Met-enkephalin and DAGO reduce the tonic inhibition by reducing the frequency of the smIPSPs, through a direct action on the presynaptic GABAergic terminals. The effect was probably not mediated by hyperpolarization of the presynaptic membrane or by modulation of presynaptic Ca2+ currents.

Activation of the endogenous opioid system can suppress pain without affecting other sensations, but the cellular mechanism of this selectivity is unclear. The analgesia might be due to inhibitory synapses arranged only on neurons whose activity leads to pain sensations. Alternatively, opioids might be released broadly, with neurons involved in pain sensation being especially sensitive. Therefore, we asked whether different subsets of rat dorsal root ganglion (DRG) sensory neurons vary in their sensitivity to opioids. Dissociated neurons were subdivided according to the spinal laminae to which they likely had projected, and whether they had innervated muscle. Using the patch-clamp method, we measured the inhibition of Ca2+ current by DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), a peptide that selectively activates the mu (morphine) receptor. We also investigated the presence of different types of Ca2+ channels. In DRG neurons chosen at random, Ca2+ currents were inhibited by DAGO to widely varying degrees, with an average inhibition of 38%. Ca2+ currents in neurons in a subset that projects to laminae I and II had a lower average inhibition, and unlike the randomly selected cells, the responses were predictable and tightly distributed about the mean. This indicates that the variability of opioid sensitivity among DRG neurons reflects the presence of different subsets of cells. Since neurons projecting to laminae I and II, the projection site of nociceptive neurons, did not show high opioid sensitivity, there is no evidence that nociceptive neurons have stronger responses to opioids. But a firm conclusion is impossible because projection site does not strictly define sensory modality.

The effects of estrogen on the response of hypothalamic arcuate neurons to mu-opioid and GABAB agonists were investigated. Intracellular recordings were made from arcuate neurons in slices prepared from ovariectomized guinea pigs that were pretreated with estrogen or vehicle. Estrogen shifted the dose-response curve to the mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) by 3.4-fold; the EC50 for DAMGO was 240 +/- 25 nM in estrogen-treated females versus 70 +/- 12 nM in the controls. The maximal hyperpolarization induced by DAMGO was equivalent in neurons from both groups. The Ke for the naloxone antagonism of the DAMGO response was similar in both groups, which would indicate that the affinity of the mu-receptor was unchanged. To explore where in the receptor/G-protein/K+ channel cascade estrogen may be acting to attenuate the mu-opioid-mediated hyperpolarization, the response to the GABAB agonist baclofen was also tested. Estrogen treatment also shifted the dose-response curve for the baclofen-induced hyperpolarization by 3.3-fold without altering the maximum hyperpolarization; the EC50 shifted from 11.0 +/- 4.0 microM to 36.0 +/- 5.0 microM. All of the neurons were identified after linking the intracellular biocytin with streptavidin-FITC, and a subpopulation of cells in both groups were immunoreactive for beta-endorphin. We conclude that estrogen decreases the functional coupling of the mu-opioid and GABAB receptors to the inwardly rectifying K+ channel possibly through an action on the G-protein.

Increased activity of dopamine-containing neurons in the ventral tegmental area is necessary for the reinforcing effects of opioids and other abused drugs. Intracellular recordings from these cells in slices of rat brain in vitro showed that opioids do not affect the principal (dopamine-containing) neurons but hyperpolarize secondary (GABA-containing) interneurons. Experiments with agonists and antagonists selective for opioid receptor subtypes indicated that the hyperpolarization of secondary cells involved the mu-receptor. Most principal cells showed spontaneous bicuculline-sensitive synaptic potentials when the extracellular potassium concentration was increased from 2.5 to 6.5 or 10.5 mM; these were prevented by TTX and assumed to result from action potentials arising in slightly depolarized local interneurons. The frequency of these synaptic potentials, but not their amplitudes, was reduced by opioids selective for mu-receptors. It is concluded that hyperpolarization of the interneurons by opioids reduces the spontaneous GABA-mediated synaptic input to the dopamine cells. In vivo, this would lead to excitation of the dopamine cells by disinhibition, which would be expected to contribute to the positive reinforcement seen with mu-receptor agonists such as morphine and heroin.

The physiological role of opioid peptides in the rat striatum was sought by intracellular recording in vitro. Excitatory synaptic potentials (mediated by glutamate or aspartate) and inhibitory synaptic potentials (mediated by GABA) were isolated pharmacologically and/or by positioning the stimulation electrode over the external capsule. Opioid agonists and antagonists selective for mu-, delta-, and kappa-receptors were applied by superfusion. Two main actions of opioids were observed. First, mu- and delta-selective opioids presynaptically inhibited the excitatory postsynaptic potential, whereas only delta-selective opioids decreased the inhibitory synaptic potential. Second, a small subpopulation of cells (not medium spiny neurons) were hyperpolarized by delta-selective agonists. The results indicate that the main action of opioids on striatal neurons is presynaptic inhibition of the corticostriate excitatory synaptic input.

Opioid agonists are known to inhibit the activity of locus coeruleus (LC) neurons. In this study, microinjections of the mu-opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 1.6 microM) bilaterally into the LC caused a significant impairment in the development of a heart-rate (HR) conditioned response (CR). The adverse effect of DAMGO on the HR CR could be reversed with naltrexone pretreatment. Microinjections of DAMGO into the periaqueductal gray, parabrachial nucleus, or fourth ventricle structures 1-2 mm away from the LC had no effects on the development of an HR CR. We conclude that central noradrenergic activity as mediated by the LC is critically involved in the learning and retention of conditioned cardiovascular responses.

The electrophysiological properties and opioid responsiveness of the dopamine-containing neurons in the arcuate nucleus of the guinea pig hypothalamus were examined. Dopamine-containing neurons, identified immunocytochemically by the presence of tyrosine hydroxylase, had a mean length-to-width profile of 14.9 +/- 4.4 x 11.5 +/- 3.1 microns (N = 14). The Na+ action potential of these neurons was of short duration, and induction of repetitive firing (20-50 Hz) caused an afterhyperpolarization of 6-9 mV in amplitude, with a decay half-time of approximately 1.5 sec. Dopamine-containing cells exhibited a low threshold spike, which induced 1-4 Na+ action potentials. This potential had a threshold close to -65 mV, could not be induced without prior hyperpolarization and was not sensitive to TTX. Dopamine-containing neurons also exhibited a time- and voltage-dependent inward current at potentials negative to -70 mV, and Cs+ blocked this conductance. The mu-opioid agonist Tyr-D-Ala-Gly-mePhe-Gly-ol hyperpolarized (14 +/- 3 mV) dopamine neurons via induction of an outward current (93 +/- 44 pA near the resting membrane potential) which had a reversal potential similar to that expected for a selective potassium conductance. TTX (1 microM) did not block the opioid effects. These results show that dopamine neurons of the arcuate nucleus differ in their intrinsic conductances and their responsiveness to opioids from other CNS dopaminergic neurons. Furthermore, opioid activation of a potassium conductance resulted in a direct hyperpolarization of dopamine neurons of the arcuate nucleus, and we suggest that this mechanism may underlie the effects of opioids on dopamine-mediated prolactin release.

The injection of neurotensin or the enkephalin analog Tyr-D-Ala-Gly-MePhe-Gly(ol) (DAMGO) into the A10 region of rats produces a motor stimulant effect that is associated with an increase in the postmortem levels of dopamine metabolites in the nucleus accumbens. These behavioral and neurochemical effects are augmented following daily administration. In vivo dialysis in the nucleus accumbens of conscious rats was used to determine if the acute and augmented behavioral responses following neurotensin or DAMGO administration are associated with an increase in extracellular dopamine concentrations. The acute injection of DAMGO produced a dose-dependent (0.03-3.3 nmol) elevation in extracellular dopamine and its metabolites in the nucleus accumbens. Neurotensin also produced a dose-related (0.1-3.3 nmol) increase in dopamine and its metabolites. The elevation in extracellular dopamine produced by DAMGO, but not by neurotensin, was positively correlated with the increase in motor activity. Following daily treatment of either neurotensin (1.0 nmol X 4 d) or DAMGO (0.03 nmol X 4 d), a significant elevation in extracellular dopamine levels occurred in the nucleus accumbens compared to an acute injection. The time course of the change in extracellular dopamine after daily injections was similar to the time course of the behavioral stimulation for both compounds. These data demonstrate that enhanced dopamine release in the nucleus accumbens mediates the acute behavioral effect of DAMGO but does not entirely explain the motor effects of neurotensin. However, enhanced dopamine release may mediate the behavioral sensitization produced by daily injection of both peptides into the A10 region.

While there is an abundance of pharmacological and biochemical evidence to suggest the existence of multiple opioid receptors, their precise localization within the brain is unclear. To help clarify this issue, the present study examined the distributions of the mu, delta, and kappa opioid receptor subtypes in the rat forebrain and midbrain using in vitro autoradiography. Mu and delta receptors were labeled with the selective ligands 3H-DAGO (Tyr- D-Ala-Gly-MePhe-Gly-ol), and 3H-DPDPE (D-Pen2, D-Pen5-enkephalin), respectively, while the kappa receptors were labeled with 3H-(-)bremazocine in the presence of unlabeled DAGO and DPDPE. Based on previous findings in our laboratory, the labeling conditions were such that each ligand selectively occupied approximately 75% of each of the opioid sites. The results demonstrated that all 3 opioid receptor subtypes were differentially distributed in the rat brain. Mu binding was dense in anterior cingulate cortex, neocortex, amygdala, hippocampus, ventral dentate gyrus, presubiculum, nucleus accumbens, caudate putamen, thalamus, habenula, interpeduncular nucleus, pars compacta of the substantia nigra, superior and inferior colliculi, and raphe nuclei. In contrast, delta binding was restricted to only a few brain areas, including anterior cingulate cortex, neocortex, amygdala, olfactory tubercle, nucleus accumbens, and caudate putamen. Kappa binding, while not as widespread as observed with mu binding, was densely distributed in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. While all 3 opioid receptor subtypes could sometimes be localized within the same brain area, their precise distribution within the region often varied widely. For example, in the caudate putamen, mu binding had a patchy distribution, while delta and kappa sites were diffusely distributed, with delta sites being particularly dense ventrolaterally and kappa sites being concentrated ventromedially. These results support the existence of at least 3 distinct opioid receptors with possibly separate functional roles.

We investigated the postnatal ontogeny of opioid receptors in rat brain under assay conditions which, when combined with computerized analysis, effectively reflect the developmental profile of high affinity binding to mu, delta, and kappa subpopulations. Concentrations of mu sites were assessed with the selective ligand 3H-[D-ala2,mePhe4,gly-ol5]enkephalin (DAGO). The other two sites were analyzed in binding assays with less selective radioligands but in the presence of specific unlabeled ligands which suppress cross-reactivity. We utilized 3H-[D-ala2,D-leu5]enkephalin (DADL) in the presence of 10 nM DAGO to label delta sites and 3H-ethylketocyclazocine (EKC) in the presence of 100 nM DADL + 100 nM [D-ala2,mePhe4,Met(0)ol5]enkephalin to detect kappa receptors. After birth, the density (femtomoles per milligram of wet weight) of mu sites declined for several days and then rose sharply over the next 2 weeks, increasing 2-fold by adulthood. Delta (delta) sites appeared in the second week postnatal and increased more than 8-fold in the next 2 weeks. Levels of kappa receptors were relatively low at birth and increased slowly (2-fold, overall). Computerized analyses of binding data revealed that DAGO and DADL were binding to single populations of sites throughout the postnatal period. DAGO and EKC affinities did not fluctuate in this period, whereas DADL affinities were low for the first week and then rose to adult levels. In summary, mu, kappa, and delta receptors exhibit differential postnatal developmental profiles. The former two are present at birth, whereas the latter appears in the second week. The postnatal increase for all three sites appear to be preceded by the previously demonstrated emergence of opioid peptides.

Intracellular recordings were made from locus coeruleus neurons in slices taken from rats 8-26 d of age. Neurons from these animals exhibited spontaneous action potentials, which were superimposed on slow (0.3-3 Hz) rhythmic depolarizations. The frequency of these potentials was closely related to the age of the animals from which the slice was taken, the slowest frequencies being observed in tissues from the youngest animals. In adult animals, such rhythmic activity was only rarely observed under normal recording conditions. The rhythmic depolarizations had a slow rate of rise and fall, were 3-15 mV in amplitude, were not affected by tetrodotoxin, and were abolished in solutions that contained elevated magnesium content. When the membrane potential was hyperpolarized by passing current through the recording electrode, the depolarizing rhythmic activity persisted even at very negative potentials (-120 mV). These depolarizations appear to be generated by the inward movement of calcium ions, probably in dendritic regions of the neuron. Superfusion of phenylephrine caused membrane depolarizations, increased the frequency of action potentials and of the slow, rhythmic depolarizations in about 80% of the cells from young rats, whereas it had no effect or a depressant action on cells from adults. Noradrenaline hyperpolarized the cells through an alpha 2-adrenoceptor and abolished the slow depolarizations. In cells from young rats, the hyperpolarization produced by noradrenaline reached a maximum and then declined, such that there was a "sag" in the membrane potential toward the resting potential following the peak of the hyperpolarization. Following the washout of noradrenaline, the membrane potential repolarized before moving toward the resting level.(ABSTRACT TRUNCATED AT 250 WORDS)