Expression of ORL1 mRNA in some brain nuclei in neuropathic pain rats

The present study was designed to investigate changes of opioid receptor like 1 receptor (ORL(1), NOP) mRNA expression in some pain-related brain nuclei of neuropathic pain rats using in situ hybridization technique. Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of ORL(1), plays an important role in neuropathic pain through its receptor. There are ORL(1) mRNA expression in the nucleus of raphe magnus (NRM), ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) of rat mesencephalon. In the sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain model, a significant increase of ORL(1) mRNA expression was observed in these three regions on the 7th day after operation, and the changes lasted for 2 weeks. The result indicated that ORL(1) synthesis was increased in NRM, vlPAG and DRN of neuropathic pain rats, suggesting that ORL(1) was involved in nociceptive transmission of neuropathic pain.     

The role of endogenous dynorphin in ethanol-induced state-dependent CPP

The aim of this study was to determine the role of the endogenous dynorphin/kappa opioid receptor (DYN/KOP) system in ethanol-induced state-dependent conditioned place preference (CPP). To this end, mice lacking the pro-DYN gene and their wild-type littermates/controls were tested for baseline place preference on day 1, received 15-min morning and afternoon conditionings with saline or ethanol (2 g/kg) each day for three consecutive days and were then tested for CPP under a drug-free state on day 5 and following a saline or ethanol (1 or 2 g/kg) challenge on day 8. Given that compensatory developmental changes may occur in knockout mice, the effect of nor-binaltorphimine (nor-BNI), a KOP antagonist, on state-dependent CPP induced by ethanol was also studied in wild-type mice. On day 1, mice were tested for baseline place preference and, 4 h later, treated with saline or nor-BNI (10 mg/kg). On days 2-4, mice received 15-min morning and afternoon conditionings and were tested for CPP under a drug-free state on day 5 and following an ethanol (1 g/kg) challenge on day 8. A comparable CPP was observed in mice lacking the pro-DYN gene and their wild-type littermates/controls as well as in wild-type mice treated with nor-BNI and their saline-treated controls. However, these mice compared to their respective controls exhibited a greater CPP response following an ethanol (1 g/kg) challenge, suggesting that the endogenous DYN/KOP system may negatively regulate ethanol-induced state-dependent CPP.     

Endogenous nociceptin/orphanin‐fq in the dorsal hippocampus facilitates despair‐related behavior

Nociceptin/orphanin‐FQ (N/OFQ) peptide and its receptor (NOP: N/OFQ opioid peptide receptor) are highly expressed in the hippocampus, but their functional role remains poorly understood. We recently showed that hippocampal N/OFQ inhibits learning and memory abilities in mice. Here, we investigated whether the endogenous peptide also regulated emotional responses at the level of the hippocampus. Bilateral infusions of the selective NOP receptor antagonist, UFP‐101 (1–3 nmol/side), into the dorsal hippocampus produced antidepressant‐like effects in the mouse forced swim and tail suspension tests comparable with those obtained with the prototypical antidepressant, fluoxetine (10–30 mg/kg, intraperitoneal). In the light‐dark test, neither UFP‐101 (1–3 nmol/side) nor N/OFQ peptide (1–3 nmol/side) modified anxiety measures when injected at behaviorally active doses in the dorsal hippocampus. These findings show a clear dissociation in the involvement of hippocampal N/OFQ system in anxiety‐ and despair‐related behaviors. We conclude that the dorsal hippocampus is a brain region in which there is an important N/OFQ modulation of mnemonic processes and adaptive emotional responses associated to despair states. © 2010 Wiley‐Liss, Inc.     

Prolonged inflammatory pain modifies corticotropin-releasing factor-induced opioid peptide release in the hypothalamus

The influence of prolonged pain upon hypothalamic opioid peptide release in vitro was examined in rats subjected to Freund's adjuvant (FA)-induced unilateral inflammation of the hindlimb. Basal release of enkephalin (ENK) but not beta-endorphin (END) or dynorphin (DYN) was increased 10 days following FA treatment. Superfusion of corticotropin-releasing factor (CRF; 10(-8) M) stimulated the release of opioid peptides in control hypothalami. CRF, however, failed to modify beta-END and DYN release in hypothalami of FA-treated rats, whereas ENK release was markedly reduced. In contrast, KCl-stimulated opioid peptide release did not differ between FA and control hypothalami. These data demonstrate that prolonged inflammatory pain alters the responsiveness of hypothalamic opioid systems to CRF. It is suggested that this effect is mediated at the level of the CRF neuron or its receptor.     

The Therapeutic Potential of Nociceptin/Orphanin FQ Receptor Agonists as Analgesics without Abuse Liability

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Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain

Peripheral delta opioid (DOP) receptors are essential for the antiallodynic effect of the tricyclic antidepressant nortriptyline. However, the population of DOP‐expressing cells affected in neuropathic conditions or underlying the antiallodynic activity of antidepressants remains unknown. Using a mouse line in which DOP receptors were selectively ablated in cells expressing Nav1.8 sodium channels (DOP cKO), we established that these DOP peripheral receptors were mandatory for duloxetine to alleviate mechanical allodynia in a neuropathic pain model based on sciatic nerve cuffing. We then examined the impact of nerve cuffing and duloxetine treatment on DOP‐positive populations using a knock‐in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP). Eight weeks postsurgery, we observed a reduced proportion of DOPeGFP‐positive small peptidergic sensory neurons (calcitonin gene‐related peptide (CGRP) positive) in dorsal root ganglia and a lower density of DOPeGFP‐positive free nerve endings in the skin. These changes were not present in nerve‐injured mice chronically treated with oral duloxetine. In addition, increased DOPeGFP translocation to the plasma membrane was observed in neuropathic conditions but not in duloxetine‐treated neuropathic mice, which may represent an additional level of control of the neuronal activity by DOP receptors. Our results therefore established a parallel between changes in the expression profile of peripheral DOP receptors and mechanical allodynia induced by sciatic nerve cuffing.     

Effects of immunoneutralization of dynorphin1-17 and dynorphin1-8 on the activity of central dopaminergic neurons in the male rat.

The effects of administration of antibodies against dynorphin1-17 (DYN1-17-AB) and dynorphin1-8 (DYN1-8-AB) were examined on the activity of dopaminergic (DA) neurons comprising the nigrostriatal, mesolimbic, tuberoinfundibular and periventricular-hypophysial systems in the male rat brain. DA neuronal activity was estimated by measuring the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in brain (striatum, nucleus accumbens, median eminence) and pituitary regions (intermediate lobe) containing terminals of these neurons. The intracerebroventricular administration of either DYN1-17-AB or DYN1-8-AB produced a time-related increase in the activity of tuberoinfundibular and periventricular-hypophysial DA neurons, but failed to alter the activity of nigrostriatal or mesolimbic DA neurons. The ability of both DYN1-17-AB and DYN1-8-AB to enhance the activity of tuberoinfundibular and periventricular-hypophysial DA neurons was reversed by the kappa opioid agonist U-50,488. These results indicate that DYN1-17-AB and DYN1-8-AB, presumably by binding endogenous dynorphins, remove a tonic inhibitory action of these opioid peptides on tuberoinfundibular and periventricular-hypophysial DA neurons.     

Paradoxical effects of prodynorphin gene deletion on basal and cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens

Quantitative and conventional microdialysis were used to investigate the effects of constitutive deletion of the prodynorphin gene on basal dopamine (DA) dynamics in the nucleus accumbens (NAc) and the responsiveness of DA neurons to an acute cocaine challenge. Saline- and cocaine-evoked locomotor activity were also assessed. Quantitative microdialysis revealed that basal extracellular DA levels were decreased, while the DA extraction fraction, an indirect measure of DA uptake, was unchanged in dynorphin (DYN) knockout (KO) mice. The ability of cocaine to increase NAc DA levels was reduced in KO. Similarly, cocaine-evoked locomotor activity was decreased in KO. The selective kappa opioid receptor agonist U-69593 decreased NAc dialysate DA levels in wildtype mice and this effect was enhanced in KO. Administration of the selective kappa opioid receptor (KOPr) antagonist nor-binaltorphimine to KO mice attenuated the decrease in cocaine-induced DA levels. However, it was ineffective in altering the decreased locomotor response to cocaine. These studies demonstrate that constitutive deletion of prodynorphin is associated with a reduction of extracellular NAc DA levels and a decreased responsiveness to acute cocaine. Data regarding the effects of U-69593 and nor-binaltorphimine in KO suggest that the kappa opioid receptor is up-regulated as a consequence of prodynorphin gene deletion and that this adaptation underlies the decrease in basal DA dynamics and cocaine-evoked DA levels observed in DYN KO mice. These findings suggest that the phenotype of DYN KO mice is not solely due to loss of endogenous opioid peptide but also reflects developmental compensations that occur at the level of the opioid receptor.     

Dynorphin and stress-related peptides in rat locus coeruleus: contribution of amygdalar efferents

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress-related peptide corticotropin-releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN- and CRF-containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA-positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA-containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light-level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN- and CRF-labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel.     

Coexpression of prodynorphin and corticotrophin-releasing hormone in the rat central amygdala: evidence of two distinct endogenous opioid systems in the lateral division

The lateral subdivision of the central nucleus of the amygdala (CeA) comprises two groups of gamma-aminobutyric acid (GABA) neurons that express corticotrophin-releasing hormone (CRH) and enkephalin. Regulation of the expression and release of these neuropeptides by glucocorticoids and other factors has been suggested to have a regulatory function on the diverse somatic, autonomic, and neuroendocrine responses that are coordinated by the CeA. Because another opioid peptide, dynorphin, has been reported to be also expressed by neurons in the lateral CeA, this study examined the neuronal expression of this kappa-opioid (KOP) receptor-preferring ligand by using immunohistochemistry for the precursor peptide prodynorphin. Prodynorphin neurons in the extended amygdala were observed mostly in the medial and central regions of the lateral CeA and the oval of the bed nucleus of the stria terminalis (BST). About one-third of the prodynorphin neurons in the CeA coexpressed CRH, whereas no coexpression with CRH was detected in the BST. Prodynorphin was not expressed by calbindin neurons in the medial part of the lateral CeA, and indirect evidence suggested that it was not expressed by enkephalin neurons. Coexpression of prodynorphin in extrahypothalamic CRH neurons in the CeA could provide an anatomical basis for regulation of the stress responses and other CRH-related functions by the brain dynorphin/KOP receptor system.     

Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain regulation. In the present study, the involvement of N/OFQ-NOP receptor system in electroacupuncture (EA)-produced anti-hyperalgesia was investigated in rats with peripheral inflammation. Intrathecal (i.t.) administration of N/OFQ (15 nmol) or EA at acupoints GB30 and GB34 could significantly attenuate hyperalgesia which was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into one hindpaw of rats, manifesting as decreased paw withdrawal latency (PWL) to the noxious thermal stimulus. The anti-nociceptive effect of N/OFQ or EA was significantly blocked by intrathecal injection of [Nphe(1)]nociceptin(1-13)NH(2) (20 nmol), a selective antagonist of the NOP receptor, indicating the NOP-receptor-mediated mechanism. Additionally, the combination of N/OFQ injection with EA treatment could enhance anti-hyperalgesia compared to that produced by each component alone. These findings suggested that the spinal N/OFQ-NOP system might be involved in EA analgesia, which may be one of the mechanisms underlying the anti-nociceptive effect of EA in rat's peripheral inflammatory pain.     

Inflammatory pain is enhanced in delta opioid receptor-knockout mice

To examine the involvement of opioid receptors in inflammatory pain, we compared Complete Freund's Adjuvant-induced hyperalgesia in mice lacking mu, delta or kappa receptors under the same experimental conditions. Mechanical allodynia and thermal hyperalgesia were measured using von Frey filaments and the plantar test, respectively. All three receptor-knockout mice, as well as wild-type animals, developed inflammatory hyperalgesia following Complete Freund's Adjuvant administration. Mu-receptor mutants showed similar hyperalgesia to wild-types in the two tests. Kappa-receptor mutants exhibited enhanced mechanical allodynia compared with wild-type mice but similar thermal hyperalgesia. In contrast, mechanical allodynia and thermal hyperalgesia were both markedly augmented in delta-receptor mutants, indicating a role for an endogenous delta-receptor tone in the control of inflammatory pain. Treatment with the delta-selective agonist SNC80 produced antihyperalgesia, and this effect was abolished in the delta-receptor knockout mice. Altogether, these data demonstrate that delta receptors inhibit inflammatory pain when activated either endogenously or exogenously. We have previously shown enhanced neuropathic pain in delta-receptor knockout mice. The delta receptor definitely represents a promising target for treating chronic pain conditions.     

Effects of electroacupuncture on orphanin FQ immunoreactivity and preproorphanin FQ mRNA in nucleus of raphe magnus in the neuropathic pain rats

Orphanin FQ (OFQ) is an endogenous ligand for opioid receptor-like-1 (ORL1) receptor. Previous studies have shown that both OFQ immunoreactivity and preproorphanin FQ (ppOFQ) mRNA expression could be observed in the brain regions involved in pain modulation, e.g., nucleus of raphe magnus (NRM), dorsal raphe nucleus (DRN), and ventrolateral periaqueductal gray (vlPAG). It was reported that electroacupuncture (EA) has analgesic effect on neuropathic pain, and the analgesic effect was mediated by the endogenous opioid peptides. In the present study, we investigated the effects of EA on the changes of OFQ in the neuropathic pain rats. In the sciatic nerve chronic constriction injury (CCI) model, we investigated the changes of ppOFQ mRNA and OFQ immunoreactivity in NRM after EA by in situ hybridization (ISH) and immunohistochemistry methods, respectively. Then, the ppOFQ mRNA-positive and OFQ immunoreactive cells were counted under a computerized image analysis system. The results showed that expression of ppOFQ mRNA decreased and OFQ immunoreactivity increased after EA treatment in the neuropathic pain rats. These results indicated that EA modulated OFQ synthesis and OFQ peptide level in NRM of the neuropathic pain rats.     

Opioid receptor endocytosis and activation of MAP kinase pathway

Opioid receptors, members of the G-protein coupled receptor (GPCR) super family, bind to endogenous opioid peptides or opiate drugs and induce a wide variety of signal transduction processes by inhibiting adenylyl cyclase, modulating cation channels, and activating the mitogen-activated protein (MAP) kinases. Similar to other GPCRs, agonist binding causes rapid internalization and down-regulation of opioid receptors. The interdependence between receptor endocytosis and activation of MAP kinase pathway are increasingly being examined. We have examined these using ligands that exhibit differential extent of endocytosis as well as mutants of mu and delta opioid receptors that are unable to internalize. We find that ligands, including morphine, that do not induce receptor internalization are able to stimulate MAP kinase phosphorylation not only in heterologous cells but also in neuronal cell lines that express endogenous mu and delta receptors. Moreover, mutant receptors that fail to undergo agonist-mediated internalization are able to efficiently phosphorylate MAP kinases. Taken together, these data are consistent with the notion that the activation of MAP kinase pathway is an internalization independent phenomenon in the case of opioid receptors and that GPCR internalization and activation of MAP kinase are governed by complex regulatory mechanisms.     

Polyarthritis-associated changes in the opioid control of spinal CGRP release in the rat

As a model of chronic inflammatory pain, Freund's adjuvant-induced polyarthritis has been shown to be associated with marked alterations in the activity of opioid- and calcitonin gene-related peptide (CGRP)-containing neurons in the dorsal horn of the spinal cord in rats. Possible changes in the interactions between these two peptidergic systems in chronic inflammatory pain were investigated by comparing the effects of various opioid receptor ligands on the spinal outflow of CGRP-like material (CGRPLM) in polyarthritic and age-paired control rats. Intrathecal perfusion of an artificial cerebrospinal fluid in halothane-anaesthetized animals allowed the collection of CGRPLM released from the spinal cord and the application of opioid receptor ligands. The blockade of κ-opioid receptors similarly increased CGRPLM release in both groups of rats as expected of a κ-mediated tonic inhibitory control of CGRP-containing fibres in control, as well as in polyarthritic rats. In contrast, the higher increase in CGRPLM outflow due to the preferential blockade of μ opioid receptors by naloxone in polyarthritic rats as compared to non-suffering animals supports the idea of a reinforced μ opioid receptor-mediated tonic inhibitory control of CGRP-containing fibres in rats suffering from chronic pain. Even more strikingly, the differences observed in the effects of ∂-opioid receptor ligands on CGRPLM outflow suggest that ∂ receptors are functionally shifted from a participation in a phasic excitatory control in non-suffering rats to a tonic inhibitory control in polyarthritic rats. These data indicate that agonists acting at the three types of opioid receptors all exert a tonic inhibitory influence on CGRP-containing nociceptive primary afferent fibres within the spinal cord of polyarthritic rats. Such a convergence probably explains why morphine and other opioids are especially potent to reduce pain in subjects suffering from chronic inflammatory diseases.     

Kappa opioid receptor activation blocks progressive neurodegeneration after kainic acid injection

We recently demonstrated that endogenous prodynorphin-derived peptides mediate anticonvulsant, antiepileptogenic and neuroprotective effects via kappa opioid receptors (KOP). Here we show acute and delayed neurodegeneration and its pharmacology after local kainic acid injection in prodynorphin knockout and wild-type mice and neuroprotective effect(s) of KOP activation in wild-type mice. Prodynorphin knockout and wild-type mice were injected with kainic acid (3 nmoles in 50 nl saline) into the stratum radiatum of CA1 of the right dorsal hippocampus. Knockout mice displayed significantly more neurodegeneration of pyramidal cells and interneurons than wild-type mice 2 days after treatment. This phenotype could be mimicked in wild-type animals by treatment with the KOP antagonist GNTI and rescued in knockout animals by the KOP agonist U-50488. Minor differences in neurodegeneration remained 3 weeks after treatment, mostly because of higher progressive neurodegeneration in wild-type mice compared with prodynorphin-deficient animals. In wild-type mice progressive neurodegeneration, but not acute neuronal loss, could be mostly blocked by U-50488 treatment. Our data suggest that endogenous prodynorphin-derived peptides sufficiently activate KOP receptors during acute seizures, and importantly in situations of reduced dynorphinergic signaling-like in epilepsy-the exogenous activation of KOP receptors might also have strong neuroprotective effects during excitotoxic events.     

Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine release in the mouse neocortex

A synaptosomal preparation was employed to pharmacologically characterize the role of presynaptic nociceptin/orphanin FQ (N/OFQ) receptors (NOP receptors) in the regulation of 5-hydroxytryptamine release in the Swiss mouse neocortex. In the present study, the NOP receptor ligands N/OFQ, Ac-RYYRWK-NH(2) and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) inhibited the K(+)-induced [(3)H]-5-HT overflow with similar maximal effects ( approximately -35%) but different potencies (pEC(50) of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg(14),Lys(15)]N/OFQ also inhibited [(3)H]-5-HT overflow, but the concentration-response curve was biphasic and the efficacy higher ( approximately -45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg(14),Lys(15)]N/OFQ and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) but maintained full responsiveness to endomorphin-1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe(1)]N/OFQ(1-13)-NH(2) and UFP-101) and nonpeptide (J-113397 and JTC-801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 microm) N/OFQ concentrations. This phenomenon was prevented by the protein kinase C inhibitor, bisindolylmaleimide. Moreover, N/OFQ-induced desensitization did not affect mu opioid receptor responsiveness. Finally, it was observed in a similar preparation of rat cerebrocortical synaptosomes, although it was induced by higher N/OFQ concentrations than that used in the mouse. Together, these findings indicate that presynaptic NOP receptors inhibit 5-hydroxytryptamine release in the mouse neocortex. Based on present and previous studies, we conclude that NOP receptors in the mouse are subtly different from the homologous receptor population in the rat, strengthening the view that there exist species differences in the pharmacology of central NOP receptors.     

UFP‐112 a Potent and Long‐Lasting Agonist Selective for the Nociceptin/Orphanin FQ Receptor

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe⁴Aib⁷Arg¹⁴Lys¹⁵]N/OFQ‐NH₂ (UFP‐112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP‐112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP‐112 and compared with other peptide and nonpeptide NOP receptor ligands.     

Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers

The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity.