RESUMEN

Effective management of chronic pain is demanded by ethical as well as medical considerations. Although opioid analgesics remain among the most effective pharmacotherapies for ameliorating many types of pain, their use is clouded by concerns regarding their addictive properties, underscored by the current epidemic of prescription opioid abuse and attendant deaths. Medicinal harnessing of endogenous opioid antinociception could provide a strategy for continuing to take advantage of the powerful antinociceptive properties of opioids while avoiding their abuse potential. Based on our studies of endogenous mechanism that suppress and facilitate spinal endomorphin 2 antinociception over the rat reproductive cycle, we identified multiple signaling molecules that could serve as targets for activating endogenous opioid analgesia for chronic pain management in women. Our findings emphasize the need for a precision medicine approach that includes stage of menstrual cycle as an important determinant of drug targets for (activating/harnessing) endogenous opioid antinociceptive systems/ capabilities. Utilization of drugs that harness endogenous opioid antinociception in accordance with varying physiological states represents a novel approach for effective pain management.

Asunto(s)

Analgésicos Opioides/farmacología , Oligopéptidos/farmacología , Manejo del Dolor , Analgésicos Opioides/uso terapéutico , Animales , Ciclo Estral , Femenino , Humanos , Oligopéptidos/uso terapéutico , Ratas , Transducción de Señal

RESUMEN

Rapid-signaling membrane estrogen receptors (mERs) and aromatase (Aro) are present throughout the central nervous system (CNS), enabling acute regulation of CNS estrogenic signaling. We previously reported that spinal membrane Aro (mAro) and mERα oligomerize (1). As their organizational relationship would likely influence functions of locally produced estrogens, we quantified the mAro and mERα that are physically associated and nonassociated in two functionally different regions of rat CNS: the spinal cord, which has predominantly neural functionalities, and the hypothalamus, which has both neural and endocrine capabilities. Quantitative immunoprecipitation (IP), coimmunoprecipitation, and Western blot analysis were used to quantify the associated and nonassociated subpopulations of mAro and mERα. Regardless of estrous-cycle stage, virtually all mAro was oligomerized with mERα in the spinal cord, whereas only ∼15% was oligomerized in the hypothalamus. The predominance of nonassociated mAro in the hypothalamus, in combination with findings that many hypothalamic Aro-immunoreactive neurons could be retrogradely labeled with peripherally injected Fluoro-Gold, suggests that a portion of hypothalamic estrogens is secreted, potentially regulating pituitary function. Moreover, circulating estrogens increased hypothalamic Aro activity (quantified by the tritiated water-release assay) in the absence of increased Aro protein, revealing nongenomic regulation of Aro activity in the mammalian CNS. The demonstrated presence of associated and nonassociated mAro and mERα subpopulations in the CNS suggests that their selective targeting could restore impaired estrogen-dependent CNS functionalities while minimizing unwanted effects. The full physiological ramifications of brain-secreted estrogens remain to be explored.

Asunto(s)

Aromatasa/metabolismo , Sistema Nervioso Central/metabolismo , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Animales , Encéfalo/metabolismo , Inmunoprecipitación , Ratas

RESUMEN

We previously showed that intrathecal application of endomorphin 2 [EM2; the highly specific endogenous µ-opioid receptor (MOR) ligand] induces antinociception that varies with stage of the rat estrous cycle: minimal during diestrus and prominent during proestrus. Earlier studies, however, did not identify proestrus-activated signaling strategies that enable spinal EM2 antinociception. We now report that in female rats, increased spinal dynorphin release and κ-opioid receptor (KOR) signaling, as well as the emergence of glutamate-activated metabotropic glutamate receptor 1 (mGluR1) signaling, are critical to the transition from an EM2 nonresponsive state (during diestrus) to an analgesically responsive state (during proestrus). Differential signaling by mGluR1, depending on its activation by membrane estrogen receptor α (mERα; during diestrus) versus glutamate (during proestrus), concomitant with the ebb and flow of spinal dynorphin/KOR signaling, functions as a switch, preventing or promoting, respectively, spinal EM2 antinociception. Importantly, EM2 and glutamate-containing varicosities appose spinal neurons that express MOR along with mGluRs and mERα, suggesting that signaling mechanisms regulating analgesic effectiveness of intrathecally applied EM2 also pertain to endogenous EM2. Regulation of spinal EM2 antinociception by both the nature of the endogenous mGluR1 activator (i.e., endogenous biased agonism at mGluR1) and changes in spinal dynorphin/KOR signaling represent a novel mechanism for modulating analgesic responsiveness to endogenous EM2 (and perhaps other opioids). This points the way for developing noncanonical pharmacological approaches to pain management by harnessing endogenous opioids for pain relief.SIGNIFICANCE STATEMENT The current prescription opioid abuse epidemic underscores the urgency to develop alternative pharmacotherapies for managing pain. We find that the magnitude of spinal endomorphin 2 (EM2) antinociception not only varies with stage of reproductive cycle, but is also differentially regulated during diestrus and proestrus. This finding highlights the need for sex-specific and cycle-specific approaches to pain management. Additionally, our finding that spinal EM2 antinociception in female rats is regulated by both the ebb and flow of spinal dynorphin/κ-opioid receptor signaling over the estrous cycle, as well as the nature of the endogenous mGluR1 activator, could encourage noncanonical pharmacological approaches to pain management, such as harnessing endogenous opioids for pain relief.

Asunto(s)

Receptor alfa de Estrógeno/metabolismo , Ciclo Estral/metabolismo , Oligopéptidos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Opioides kappa/metabolismo , Médula Espinal/metabolismo , Animales , Receptor alfa de Estrógeno/agonistas , Femenino , Plasticidad Neuronal/fisiología , Nocicepción/fisiología , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores Opioides kappa/agonistas , Reproducción/fisiología , Transducción de Señal/fisiología

RESUMEN

The magnitude of antinociception elicited by intrathecal endomorphin 2 (EM2), an endogenous mu-opioid receptor (MOR) ligand, varies across the rat estrous cycle. We now report that phasic changes in analgesic responsiveness to spinal EM2 result from plastic interactions within a novel membrane-bound oligomer containing estrogen receptors (mERs), aromatase (aka estrogen synthase), metabotropic glutamate receptor 1 (mGluR1), and MOR. During diestrus, spinal mERs, activated by locally synthesized estrogens, act with mGluR1 to suppress spinal EM2/MOR antinociception. The emergence of robust spinal EM2 antinociception during proestrus results from the loss of mER-mGluR1 suppression, a consequence of altered interactions within the oligomer. The chemical pairing of aromatase with mERs within the oligomer containing MOR and mGluR1 allows estrogens to function as intracellular messengers whose synthesis and actions are confined to the same signaling oligomer. This form of estrogenic signaling, which we term "oligocrine," enables discrete, highly compartmentalized estrogen/mER-mGluR1 signaling to regulate MOR-mediated antinociception induced by EM2. Finally, spinal neurons were observed not only to coexpress MOR, mERα, aromatase, and mGluR1 but also be apposed by EM2 varicosities. This suggests that modulation of spinal analgesic responsiveness to exogenous EM2 likely reflects changes in its endogenous analgesic activity. Analogous suppression of spinal EM2 antinociception in women (eg, around menses, comparable with diestrus in rats) as well as the (pathological) inability to transition out of that suppressed state at other menstrual cycle stages could underlie, at least in part, the much greater prevalence and severity of chronic pain in women than men.

Asunto(s)

Analgésicos Opioides/administración & dosificación , Estrógenos/metabolismo , Ciclo Estral/efectos de los fármacos , Oligopéptidos/administración & dosificación , Médula Espinal/metabolismo , Animales , Aromatasa/metabolismo , Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Fadrozol/farmacología , Femenino , Inyecciones Espinales/métodos , Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Opioides mu/metabolismo , Médula Espinal/efectos de los fármacos , Tiazoles/farmacología

RESUMEN

In previous studies we have reported that spinal nerve ligation (SNL), a model of neuropathic pain, results in the loss of over 20% of neurons in the rostral portion of the ventromedial medulla (RVM) in rats, 10 days after SNL. The RVM is involved in pain modulation and we have proposed that loss of pain inhibition from the RVM, including loss of RVM serotonin neurons, contributes to the increased hypersensitivity observed after SNL. In the present study we examined whether RVM neuronal loss occurs in two other models of neuropathic pain, chronic constriction injury (CCI) and spared nerve injury (SNI). We found no evidence for neuronal loss 10 days after either nerve injury, a time when robust tactile hypersensitivity is present in both CCI and SNI. We conclude that loss of RVM neurons appears not to be required for expression of tactile hypersensitivity in these models of neuropathic pain.

Asunto(s)

Bulbo Raquídeo/patología , Neuralgia/patología , Neuroglía/patología , Neuronas/patología , Traumatismos de los Nervios Periféricos/patología , Nervio Ciático/lesiones , Animales , Recuento de Células , Enfermedad Crónica , Constricción Patológica , Modelos Animales de Enfermedad , Miembro Posterior/inervación , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Masculino , Neuralgia/fisiopatología , Traumatismos de los Nervios Periféricos/fisiopatología , Estimulación Física , Ratas Sprague-Dawley , Factores de Tiempo , Tacto

RESUMEN

The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α(2)-noradrenergic receptor (α(2)-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid-induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α(2)-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.

Asunto(s)

Dinorfinas/metabolismo , Dolor Nociceptivo/metabolismo , Dimensión del Dolor , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides kappa/metabolismo , Caracteres Sexuales , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Dinorfinas/biosíntesis , Femenino , Inyecciones Espinales , Masculino , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Dolor Nociceptivo/tratamiento farmacológico , Dolor Nociceptivo/etiología , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/biosíntesis , Receptores Opioides kappa/biosíntesis , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Yohimbina/farmacología

RESUMEN

Cell death has been reported in the CNS in models of neuropathic pain (Sugimoto et al., 1990; Whiteside and Munglani, 2001; Scholz et al., 2005; Fuccio et al., 2009). In our present study, we examined the effects of spinal nerve ligation (SNL) on the number of neurons in the rostral ventromedial medulla (RVM), a brainstem region involved in modulation of nociception. In rats receiving SNL, we found that the number of RVM neurons decreased by 23% in the side ipsilateral to the surgery. The loss of RVM neurons was also associated with a bilateral increase in the number of glia as well as bilateral activation of both astrocytes and microglia. Administration of tauroursodeoxycholic acid (TUDCA), which reportedly inhibits apoptosis, significantly reduced the loss of neurons, the increase in glia, and the mechanical hypersensitivity induced by SNL. Among RVM neurons, we found that serotonergic (5-hydroxytryptamine, 5-HT) neurons decreased by 35% ipsilateral to SNL. Consistent with these findings, the density of 5-HT-immunoreactive varicosities in the superficial dorsal horn of the spinal cord was 15-30% lower, ipsilateral to SNL. To test the function of the remaining 5-HT neurons, we administered the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). Interestingly, after 5,7-DHT, mechanical withdrawal thresholds increased significantly. We conclude that nerve injury induces death of antinociceptive RVM neurons that can be reduced or abolished by TUDCA. We propose that the loss of RVM neurons shifts the balance of descending control from pain inhibition to pain facilitation.

Asunto(s)

Modelos Animales de Enfermedad , Bulbo Raquídeo/patología , Neuralgia/patología , Neuronas/patología , Nervios Espinales/lesiones , Animales , Recuento de Células/métodos , Muerte Celular/fisiología , Masculino , Neuralgia/etiología , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Nervios Espinales/patología

RESUMEN

Enhanced glutamatergic neurotransmission in dopamine (DA) neurons of the ventral tegmental area (VTA), triggered by a single cocaine injection, represents an early adaptation linked to the more enduring effects of abused drugs that characterize addiction. Here, we examined the impact of in vivo cocaine exposure on metabotropic inhibitory signaling involving G-protein-gated inwardly rectifying K(+) (Girk) channels in VTA DA neurons. Somatodendritic Girk currents evoked by the GABA(B) receptor (GABA(B)R) agonist baclofen were diminished in a dose-dependent manner in mice given a single cocaine injection. This adaptation persisted for 3-4 d, was specific for DA neurons of the VTA, and occurred in parallel with an increase in spontaneous glutamatergic neurotransmission. No additional suppression of GABA(B)R-Girk signaling was observed following repeated cocaine administration. While total Girk2 and GABA(B)R1 mRNA and protein levels were unaltered by cocaine exposure in VTA DA neurons, the cocaine-induced decrease in GABA(B)R-Girk signaling correlated with a reduction in Girk2-containing channels at the plasma membrane in VTA DA neurons. Systemic pretreatment with sulpiride, but not SCH23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), prevented the cocaine-induced suppression of GABA(B)R-Girk signaling, implicating D(2/3) DA receptor activation in this adaptation. The acute cocaine-induced weakening of somatodendritic Girk signaling complements the previously demonstrated cocaine-induced strengthening of glutamatergic neurotransmission, likely contributing to enhanced output of VTA DA neurons during the early stages of addiction.

Asunto(s)

Cocaína/toxicidad , Dopamina/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Neuronas/efectos de los fármacos , Receptores de GABA-B/fisiología , Transducción de Señal/fisiología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismo , Animales , Modelos Animales de Enfermedad , Inhibidores de Captación de Dopamina/farmacología , Esquema de Medicación , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Transducción de Señal/efectos de los fármacos , Área Tegmental Ventral/patología

RESUMEN

We previously demonstrated that the spinal cord κ-opioid receptor (KOR) and µ-opioid receptor (MOR) form heterodimers (KOR/MOR). KOR/MOR formation and the associated KOR dependency of spinal morphine antinociception are most robust during proestrus. Using Sprague Dawley rats, we now demonstrate that (1) spinal synthesis of estrogen is critical to these processes, and (2) blockade of either estrogen receptor (ER) α-, ß-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception. Effects of blocking ERs were manifest within 15 min, whereas those of PR blockade were manifest after 18 h, indicating the requirement for rapid signaling by estrogen and transcriptional effects of progesterone. Individual or combined blockade of ERs produced the same magnitude of effect, suggesting that they work in tandem as part of a macromolecular complex to regulate KOR/MOR formation. Consistent with this inference, we found that KOR and MOR were coexpressed with ERα and G-protein-coupled ER1 in the spinal dorsal horn. Reduction of KOR/MOR by ER or PR blockade or spinal aromatase inhibition shifts spinal morphine antinociception from KOR dependent to KOR independent. This indicates a sex steroid-dependent plasticity of spinal KOR functionality, which could explain the greater analgesic potency of KOR agonists in women versus men. We suggest that KOR/MOR is a molecular switch that shifts the function of KOR and thereby endogenous dynorphin from pronociceptive to antinociceptive. KOR/MOR could thus serve as a novel molecular target for pain management in women.

Asunto(s)

Estrógenos/biosíntesis , Dolor/metabolismo , Multimerización de Proteína/fisiología , Receptores de Estrógenos/biosíntesis , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Animales , Aromatasa/metabolismo , Antagonistas de Estrógenos/administración & dosificación , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/fisiología , Femenino , Inyecciones Espinales , Morfina/administración & dosificación , Dolor/prevención & control , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Células del Asta Posterior/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/antagonistas & inhibidores , Receptores Opioides kappa/química , Receptores Opioides mu/química , Factores Sexuales , Transducción de Señal/efectos de los fármacos , Médula Espinal/efectos de los fármacos

RESUMEN

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC(1)R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC(1)R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213-1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

Asunto(s)

Dinorfinas/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Médula Espinal/fisiología , Analgésicos/farmacología , Animales , Dinorfinas/metabolismo , Masculino , Fragmentos de Péptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/análisis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/fisiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/análisis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Médula Espinal/metabolismo

RESUMEN

We previously reported the existence of MOR1(C) mRNA and MOR1(C)-immunoreactivity (-ir) in rats. However, the sequence that we reported for rat MOR1(C) appears not to be present in the rat genome. We have therefore reexamined whether MOR1(C) mRNA or MOR1(C)-ir exist in rats. We used reverse-transcription polymerase chain reaction (RT-PCR) to attempt to amplify MOR1, MOR1(A), MOR1(B), the rat MOR1(C) sequence we previously reported, and MOR1(C1) and MOR1(C2) (which have recently been reported to exist in rats). In RNA extracted from rats, we were able to demonstrate PCR products representing MOR1, MOR1(A), and MOR1(B) splice variants. All three products were confirmed as related to MOR1 by Southern blot. However, we were unable to detect either the MOR1(C) product reported previously by us or the MOR1(C)-like products reported to exist in rats by others. In RNA extracted from mice we were able to detect MOR1, MOR1(A), MOR1(B), and MOR1(D)-like products. To test the specificity of our MOR1(C) antiserum, we examined MOR1(C)-ir in control and knockout mice. MOR1(C)-ir had a distribution in control mice similar to that previously reported in rats, including coexisting with vGLUT2. However, although MOR1-ir was absent in MOR1 knockout mice, the density and distribution of MOR1(C)-ir were unchanged, suggesting that the antiserum crossreacts with another molecule in tissue. We find no evidence for MOR1(C) mRNA in rats. Furthermore, we conclude that MOR1(C)-ir represents crossreactivity.

Asunto(s)

Expresión Génica/fisiología , Isoformas de Proteínas/genética , Receptores Opioides mu/genética , Animales , Corteza Cerebral/metabolismo , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/deficiencia , Receptores Opioides mu/metabolismo

RESUMEN

Pharmacological and behavioral studies suggest that spinal delta- and kappa-opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal kappa-opioid receptors or delta-opioid receptors (DOR). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213-1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374-1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor alpha (ERalpha) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ERalpha by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ERalpha and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ERalpha-ir cells, cells double-labeled for DYN-ir and ERalpha-ir and the proportion of DYN-ir cells coexpressing ERalpha. Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K(+)-evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain.

Asunto(s)

Dinorfinas/biosíntesis , Receptor alfa de Estrógeno/biosíntesis , Hormonas Esteroides Gonadales/biosíntesis , Ovario/anatomía & histología , Ovario/metabolismo , Receptores Opioides delta/biosíntesis , Médula Espinal/anatomía & histología , Médula Espinal/metabolismo , Analgésicos Opioides/metabolismo , Animales , Dinorfinas/análisis , Dinorfinas/genética , Receptor alfa de Estrógeno/análisis , Receptor alfa de Estrógeno/genética , Femenino , Hormonas Esteroides Gonadales/genética , Neuronas/química , Neuronas/metabolismo , Ovario/química , Dolor/genética , Dolor/metabolismo , Dolor/patología , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/análisis , Receptores Opioides delta/genética , Médula Espinal/química

RESUMEN

It has been reported that mu-opioid agonists depress glutamate release in some neurons but the specific receptor subtype mediating this effect is unclear. The purpose of the present study was to examine whether a particular mu-opioid receptor (MOR) splice-variant, MOR(1C), is expressed in rat central nervous system (CNS) by terminals expressing the vesicular glutamate transporter2 (VGLUT2), a marker of glutamatergic neurons. Several MOR splice variants have been identified in mice and MOR(1C) appears mainly to be localized to fibers and terminals, from which most neurotransmitter release would be expected. In addition, VGLUT2 has been found in the CNS and antibodies to it are reliable markers for glutamatergic terminals. Using fluorescence immunohistochemistry and confocal microscopy to examine spatial relationships between MOR(1C) and VGLUT2, we found that MOR(1C) and VGLUT2 puncta were widely distributed throughout the rat CNS; moreover, many regions contained terminals that expressed both. Thus, it appears that coexpression of MOR(1C) and VGLUT2 is common in the rat CNS. We hypothesize that activation of MOR(1C) by mu-opioid agonists at some glutamatergic terminals may be a mechanism by which glutamate release is inhibited.

Asunto(s)

Sistema Nervioso Central/fisiología , Regulación de la Expresión Génica/fisiología , Subunidades de Proteína/biosíntesis , Receptores Opioides mu/biosíntesis , Proteína 2 de Transporte Vesicular de Glutamato/biosíntesis , Animales , Línea Celular , Sistema Nervioso Central/metabolismo , Femenino , Humanos , Masculino , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Conejos , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/fisiología

RESUMEN

The rostral portion of the ventral medial medulla (RVM) is a crucial site for the supraspinal antinociceptive actions of opioids. Previous studies have reported that serotonergic antagonists block the analgesia induced by microinjection of morphine into the RVM (Hammond and Yaksh [1984] Brain Res 298:329-337) and that spinally projecting serotonergic RVM neurons express mu-opioid receptors (MOR) (Kalyuzhny et al. [1996] J Neurosci 16:6490-6503; Wang and Wessendorf [1999] J Comp Neurol 404:183-196). In addition, axons immunoreactive for the endogenous MOR ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) (EM-2) have been reported to be in the RVM (Martin-Schild et al. [1999] J Comp Neurol 405:450-471; Pierce and Wessendorf [2000] J Chem Neuroanat 18:181-207). In the present study we examined the relationship of EM-2-immunoreactive (EM-2-ir) axons to serotonergic and nonserotonergic RVM neurons in rats, including neurons projecting to the dorsal spinal cord. We also examined the origins of EM-2-ir in the RVM. Using unbiased methods we estimated the total number of cells in the RVM to be 1.50 x 10(4) and of these up to 70% were retrogradely labeled from the dorsal spinal cord. EM-2-ir fibers apposed both serotonergic and nonserotonergic RVM neuronal profiles. However, serotonergic profiles were significantly more likely to be apposed than nonserotonergic profiles. Thus, although serotonergic neurons comprise a minority of RVM neurons (23% of the total RVM neurons), they appear to be selectively apposed by EM-2-ir fibers. We also found that hypothalamic EM-2-ir neurons, but not EM-2-ir neurons, in the nucleus of the solitary tract projected their axons to the RVM.

Asunto(s)

Bulbo Raquídeo/citología , Fibras Nerviosas/fisiología , Neuronas/citología , Neuronas/metabolismo , Oligopéptidos/metabolismo , Serotonina/metabolismo , Animales , Recuento de Células , Inmunohistoquímica/métodos , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo , Estilbamidinas/metabolismo , Triptófano Hidroxilasa/metabolismo

RESUMEN

The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.

Asunto(s)

Neuronas/fisiología , Núcleos del Rafe/citología , Núcleos del Rafe/fisiología , Serotonina/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Inmunohistoquímica , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología , Triptófano Hidroxilasa/metabolismo

RESUMEN

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone a cDNA fragment of a putative G-protein-coupled receptor from rat brain total RNA. Nucleotide sequencing of this cDNA fragment showed it to be homologous to that of the mu-opioid receptor splice variant MOR(1C) from mice. We used the cDNA to make an RNA probe for a ribonuclease protection assay (RPA). The results from the RPA showed a protected fragment of the size expected for MOR(1C) mRNA, as well as other RNase-protected fragments that may indicate the existence of other MOR1 transcripts. We then used the RNA probe for in situ hybridization (ISH) experiments. We detected strong autoradiographic labeling over much of the rat telencephalon, diencephalon, mesencephalon, cerebellum, spinal cord, and dorsal root ganglia. These findings suggest that MOR(1C), and possibly other MOR1 splice variants, are important components of the system by which the actions of opioids are transduced.

Asunto(s)

ARN Mensajero/biosíntesis , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Clonación Molecular/métodos , ADN Complementario/biosíntesis , ADN Complementario/genética , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/aislamiento & purificación

RESUMEN

The rostral ventromedial medulla (RVM) forms part of a descending pathway that modulates nociceptive neurotransmission at the level of the spinal cord dorsal horn. However, the involvement of descending RVM systems in opioid analgesia are a matter of some debate. In the present study, patch-clamp recordings of RVM neurons were made from rats that had received retrograde tracer injections into the spinal cord. More than 90% of identified spinally projecting RVM neurons responded to opioid agonists. Of these neurons, 53% responded only to the mu-opioid agonist D-Ala2, N-Me-Phe4, Gly-ol5 enkephalin, 14% responded only to the kappa-opioid agonist U-69593, and another group responded to both mu and kappa opioids (23%). In unidentified RVM neurons, a larger proportion of neurons responded only to mu opioids (75%), with smaller proportions of kappa- (4%) and mu/kappa-opioid (13%) responders. These RVM slices were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. Forty-percent of spinally projecting neurons and 11% of unidentified neurons were TPH positive. Of the TPH-positive spinally projecting neurons, there were similar proportions of mu- (33%), kappa- (25%), and mu/kappa-opioid (33%) responders. Most of the TPH-negative spinally projecting neurons were mu-opioid responders (67%). These findings indicate that functional opioid receptor subtypes exist on spinally projecting serotonergic and nonserotonergic RVM neurons. The proportions of mu- and kappa-opioid receptors expressed differ between serotonergic and nonserotonergic neurons and between retrogradely labeled and unlabeled RVM neurons. We conclude that important roles exist for both serotonergic and nonserotonergic RVM neurons in the mediation of opioid effects.

Asunto(s)

Tronco Encefálico/citología , Tronco Encefálico/metabolismo , Neuronas/metabolismo , Receptores Opioides/metabolismo , Médula Espinal , Animales , Células Cultivadas , Conductividad Eléctrica , Inmunohistoquímica , Modelos Neurológicos , Narcóticos/farmacología , Neuronas/clasificación , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Fenotipo , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Triptófano Hidroxilasa/análisis , Triptófano Hidroxilasa/inmunología

RESUMEN

Endomorphins are opioid tetrapeptides that have high affinity and selectivity for mu-opioid receptors (muORs). Light microscopic studies have shown that endomorphin-1 (EM-1) -containing fibers are distributed within the brainstem dorsal pontine tegmentum. Here, immunoelectron microscopy was conducted in the rat brainstem to identify potential cellular interactions between EM-1 and tyrosine hydroxylase (TH) -labeled cellular profiles in the locus coeruleus (LC) and peri-LC, an area known to contain extensive noradrenergic dendrites of LC neurons. Furthermore, sections through the rostral dorsal pons, from colchicine-treated rats, were processed for EM-1 and corticotropin releasing factor (CRF), a neuropeptide known to be present in neurons of Barrington's nucleus. EM-1 immunoreactivity was identified in unmyelinated axons, axon terminals, and occasionally in cellular profiles resembling glial processes. Within axon terminals, peroxidase labeling for EM-1 was enriched in large dense core vesicles. In sections processed for EM-1 and TH, approximately 10% of EM-1-containing axon terminals (n=269) targeted dendrites that exhibited immunogold-silver labeling for TH. In contrast, approximately 30% of EM-1-labeled axon terminals analyzed (n = 180) targeted CRF-containing somata and dendrites in Barrington's nucleus. Taken together, these data indicate that the modulation of nociceptive and autonomic function as well as stress and arousal responses attributed to EM-1 in the central nervous system may arise, in part, from direct actions on catecholaminergic neurons in the peri-LC. However, the increased frequency with which EM-1 axon terminals form synapses with CRF-containing profiles in Barrington's nucleus suggests a novel role for EM-1 in the modulation of functions associated with Barrington's nucleus neurons such as micturition control and pelvic visceral function.

Asunto(s)

Locus Coeruleus/metabolismo , Neuropéptidos/metabolismo , Norepinefrina/metabolismo , Oligopéptidos/metabolismo , Puente/metabolismo , Terminales Presinápticos/metabolismo , Ratas Sprague-Dawley/metabolismo , Animales , Comunicación Celular/fisiología , Hormona Liberadora de Corticotropina/metabolismo , Inmunohistoquímica , Locus Coeruleus/ultraestructura , Masculino , Microscopía Electrónica , Neuroglía/metabolismo , Neuroglía/ultraestructura , Puente/ultraestructura , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley/anatomía & histología , Estrés Fisiológico/metabolismo , Transmisión Sináptica/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Micción/fisiología

Asunto(s)

Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Animales , Técnica del Anticuerpo Fluorescente/instrumentación , Técnica del Anticuerpo Fluorescente/métodos , Colorantes Fluorescentes/normas , Humanos , Rayos Láser/normas , Microscopía Confocal/instrumentación , Microtomía , Neuronas/citología