Kappa Opioid Receptors in Mesolimbic Terminals Mediate Escalation of Cocaine Consumption.

Increases in drug consumption over time, also known as escalation, is a key behavioral component of substance use disorder (SUD) that is related to potential harm to users, such as overdose. Studying escalation also allows researchers to investigate the transition from casual drug use to more SUD-like drug use. Understanding the neurobiological systems that drive this transition will inform therapeutic treatments in the aim to prevent increases in drug use and the development of SUD. The kappa opioid receptor (KOR) system is typically known for its role in negative affect, which is commonly found in SUD as well. Furthermore, the KOR system has also been implicated in drug use and importantly, modulating the negative effects of drug use. However, the specific neuronal subpopulation expressing KOR involved has not been identified. Here, we first demonstrated that pharmacologically inhibiting KOR in the nucleus accumbens core (NAcC), as a whole, blocks cocaine escalation under long-access self-administration conditions. We then demonstrated that KOR expressed on ventral tegmental area (VTA) neurons but not NAcC neurons is sufficient for blocking cocaine escalation by utilizing a novel virally-mediated CRISPR-SaCas9 knock-out of the oprk1 gene. Together, this suggests that activation of KOR on VTA terminals in the NAcC drives the transition to the SUD-like phenotype of escalation of cocaine consumption.

Dynorphin is a specific endogenous ligand of the kappa opioid receptor.

In the guinea pig ileum myenteric plexus--longitudinal muscle preparation, dynorphin-(1--13) and the prototypical kappa agonist ethylketocyclazocine had equally poor sensitivity to naloxone antagonism and showed selective cross protection in receptor inactivation experiments with the alkylating antagonist beta-chlornaltrexamine. In binding assays with membranes from guinea pig brain, ethylketocyclazocine and dynorphin-(1--13) amide were more potent in displacing tritium-labeled ethylketocyclazocine than in displacing typical mu and delta opioid receptor ligands. In the two preparations studied, the dynorphin receptor appears to be the same as the kappa opioid receptor.

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.

L-type calcium channels mediate dynorphin neuropeptide release from dendrites but not axons of hippocampal granule cells.

Granule cells in the guinea pig dentate gyrus release kappa opioid neuropeptides, dynorphins, from dendrites as well as from axon terminals. We have found that both L- and N-type calcium channel antagonists inhibited dendritic dynorphin release. In contrast, N-type but not L-type calcium channel antagonists inhibited axonal dynorphin release. Neither L- nor N-type channel antagonists directly altered the effects of kappa opioid receptor activation. By inhibiting dynorphin release, L-type channel antagonists also facilitated the induction of long-term potentiation of the perforant path-granule cell synapse. These studies establish that a single cell type can release a transmitter from two different cellular domains and provide new distinction between axonal and dendritic transmitter release mechanisms.

Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus.

Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE-mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, we made mice transgenic for a CRE-regulated reporter construct. We focused on long-lasting long-term potentiation (L-LTP), because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus.

Conducting states of a mammalian serotonin transporter.

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Calcium-stimulated adenylyl cyclase activity is critical for hippocampus-dependent long-term memory and late phase LTP.

It is hypothesized that Ca2+ stimulation of calmodulin (CaM)-activated adenylyl cyclases (AC1 or AC8) generates cAMP signals critical for late phase LTP (L-LTP) and long-term memory (LTM). However, mice lacking either AC1 or AC8 exhibit normal L-LTP and LTM. Here, we report that mice lacking both enzymes (DKO) do not exhibit L-LTP or LTM. To determine if these defects are due to a loss of cAMP increases in the hippocampus, DKO mice were unilaterally cannulated to deliver forskolin. Administration of forskolin to area CA1 before training restored normal LTM. We conclude that Ca2+-stimulated adenylyl cyclase activity is essential for L-LTP and LTM and that AC1 or AC8 can produce the necessary cAMP signal.

Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization.

Desensitization of cannabinoid receptor signaling by a G-protein coupled receptor kinase (GRK) was examined using the Xenopus oocyte expression system. Application of a CB1 agonist, WIN 55,212-2, evoked a concentration-dependent increase in K+ conductance (Kir3) in oocytes coexpressing rat CB1 with the G-protein-gated, inwardly rectifying K+ channels Kir3.1 and Kir3.4. Desensitization was slight during continuous agonist application in the absence of GRK and arrestin. However, coexpression of GRK3 and beta-arrestin 2 (beta-arr2) caused profound homologous CB1 receptor desensitization, supporting the hypothesis that GRK3 and beta-arr2 effectively produce CB1 receptor desensitization. To identify the regions of the CB1 receptor responsible for GRK3- and beta-arr2-mediated desensitization, we constructed several CB1 receptor mutants. Truncation of the C-terminal tail of CB1 receptor at residue 418 (Delta418) almost completely abolished desensitization but did not affect agonist activation of Kir3. In contrast, truncation at residues 439 and 460 did not significantly affect GRK3- and beta-arr2-dependent desensitization. A deletion mutant (Delta418-439) did not desensitize, indicating that residues within this region are important for GRK3- and beta-arr2-mediated desensitization. Phosphorylation in this region was likely involved in desensitization, because mutation of either of two putative phosphorylation sites (S426A or S430A) significantly attenuated desensitization. CB1 receptors rapidly internalize after activation by agonist. Phosphorylation of S426 or S430 was not necessary for internalization, because the S426A/S430A CB1 mutant internalized when stably expressed in AtT20 cells. These studies establish that CB1 desensitization can be regulated by a GRK and that different receptor domains are involved in GRK- and beta-arrestin-dependent desensitization and CB1 internalization.

Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation.

Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR-like immunoreactivity (-LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity-purified KT antibody was confirmed by Western blotting, enzyme-linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT-LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT-LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT-labeled processes were also present in stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT-LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT-LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation.

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.

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.

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.

Immunocytochemical localization of delta opioid receptors in mouse brain.

An affinity-purified anti-peptide antibody generated against the carboxy-terminal region of the delta opioid receptor was used to localize delta opioid receptors in mouse brain. delta Opioid receptor immunoreactivity was found in axons and nerve terminals in regions of the olfactory bulb, hippocampal formation, cerebral and cerebellar cortex, midbrain and hindbrain. The immunocytochemical distribution correlated well, though not completely with autoradiographic distribution of delta opioid receptors in mouse brain using either [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) or [3H]naltrindole. Confocal microscopy of double-labeled tissue provided direct evidence that delta opioid receptors are principally expressed on GABAergic terminals in the hippocampus. These anatomical findings complement extensive physiological studies to provide a more detailed description of endogenous opioid circuitry.

Opiate antagonists do not alter neuronal responses to stimulation of opioid-containing pathways in rat hippocampus.

The opioid receptor antagonists, naloxone and beta-chlornaltrexamine, were used to determine whether activation of endogenous opioid peptide containing pathways produced pharmacologically reversible opioid actions. Extracellularly recorded responses of the hippocampal CA3 pyramidal cells were evoked by stimulation of the dynorphin-containing mossy fiber pathway. Neither naloxone nor beta-chlornaltrexamine pretreatment significantly changed the evoked response. However, both antagonists blocked the effect of applied dynorphin-A(1-17) on CA1 pyramidal cell evoked responses. Thus, our data demonstrate that if endogenous opioids are released from this pathway, the peptides cannot be responsible for the evoked response measured in hippocampal CA3 cellular field. With no direct evidence for endogenous opioid peptides acting through opioid receptors, the neurotransmitter role of dynorphins in rat hippocampus remains obscure.

Mu opioids enhance mossy fiber synaptic transmission indirectly by reducing GABAB receptor activation.

The cellular mechanisms underlying mu opioid facilitation of mossy fiber (MF) long-term potentiation (LTP) and synaptic transmission were investigated in the rat hippocampal slice. Naloxone (10 microM) significantly inhibited the induction of mossy fiber LTP, an effect attributed by Derrick and Martinez [B.E. Derrick, J.L.J. Martinez, Opioid receptor activation is one factor underlying the frequency dependence of mossy fiber LTP induction, J. Neurosci. 14 (1994) 4359-4367] to antagonism of endogenous opioid peptide action. We found that the inhibitory effects of naloxone were not blocked by bicuculline, suggesting that endogenous opioids did not enhance mossy fiber LTP by depressing GABAA inhibition. [d-Ala2, NMePhe4, Glyol5] enkephalin, DAMGO (300 nM), a mu opioid agonist, mimicked the action of endogenous opioids, enhancing both mossy fiber LTP induction and paired-pulse facilitation. DAMGO potentiation of the paired-pulse facilitation of mossy fiber response was also insensitive to bicuculline but was blocked by the mu selective antagonist CTOP. Further analysis of the cellular mechanism showed that the depletion of internal Ca2+ stores by thapsigargin (1 microM), or inhibition of protein kinases by application of staurosporine (1 microM) did not block the DAMGO facilitation of mossy fiber-CA3 synaptic transmission. However, application of phaclofen (100 microM GABAB receptor antagonist or SCH 50911, a more potent GABAB antagonist significantly inhibited the DAMGO effect (49+/-15%; 51+/-19% inhibition, P<0.05). The data indicate that the DAMGO effect on the mossy fiber pathway is partially mediated by a reduction in GABA activation of GABAB receptors. These findings further suggest that endogenous opioid peptides activate mu opioid receptors to facilitate mossy fiber LTP and synaptic transmission in rat hippocampus partially by GABAB receptor-mediated disinhibitory mechanism.

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.

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.

Selective inactivation of opioid receptors in rat hippocampus demonstrates that dynorphin-A and -B may act on mu-receptors in the CA1 region.

Dynorphin-A1-17 and dynorphin-B increased the evoked response of hippocampal CA1 pyramidal cells, as did other opioids tested. Treatment of the hippocampal slice with beta-funaltrexamine, a mu-receptor selective antagonist, blocked the effects of normorphine, dynorphin-A and dynorphin-B, but did not change the response to D-Ala2, D-Leu5-enkephalin. The low potency of kappa selective agonists and the antagonism by beta-funaltrexamine of the dynorphins' effect indicate that kappa-opioid receptors may not be involved in these observed responses. Our data suggest that both mu- and delta-receptors are functionally represented and provide evidence that the dynorphins or their derivatives may also be agonists at the mu-receptor.