Pharmacological activation of dopamine D4 receptor prevents morphine-induced tolerance at the rat dorsal horn level

Morphine is one of the most effective drugs used for pain management. However, prolonged exposition to morphine produces a wide variety of side effects such as tolerance to analgesia, hyperalgesia and addiction. Downregulation of the mu opioid receptor (MOR) and its uncoupling to G-proteins in the dorsal horn are likely to contribute to the development of morphine tolerance. Previous studies demonstrated that dopamine D4 receptor (D4R) activation prevents morphine addiction by modulating dopamine signaling from nigral dopamine cells. This effect seems to be the result of an antagonistic receptor-receptor interaction involving a D4R-MOR heteroreceptor which could exist in the dorsal striatum. As D4R is expressed in dorsal horn neurons, we hypothesize that D4R could interfere in the development of morphine-induced tolerance to its analgesic effects. Here, using a chronic paradigm of combined treatment of morphine with the D4R agonist PD168.077, we investigated the nociceptive response to three noxious stimuli: thermal (tail-flick test), mechanical (von Frey test) and chemical (formalin test). Moreover, using immunohistochemistry, western blot and qRT-PCR, we have evaluated alterations in the primary circuitry of pain and the balance between glutamate and GABA within dorsal horn. Results from the evaluation of analgesic activity of chronic combined treatment of morphine with PD168,077 showed that D4R prevents the development of morphine-induced analgesic tolerance. The present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interaction in the dorsal horn that could help to the development of a new pharmacology strategy in the treatment of pain.CTS161 y UMA20-FEDERJA-122 Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dopamine D4R restores morphine-induced impairment of adult neurogenesis in the subventricular zone

In the adult mammalian brain, neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream into the olfactory bulb, where they differentiate and synaptically integrate to contribute with the maintenance of the olfactory function. It has been established that endogenous as well as exogenous opioid signalling affects proliferation in adult brains. In fact, chronic administration of morphine reduces adult neurogenesis in SVZ although its implication in addiction has not yet been clarified. On other hand, dopamine has been also identified as a regulatory factor of adult neurogenesis as dopaminergic cells from the substantia nigra compacta project toward the dorsal SVZ whereas the ventral tegmental area innervates the ventral SVZ. Previous results demonstrated that morphine increases striatal dopamine signaling, which is restored by the specific stimulation of dopamine D4 receptor (D4R). The mechanisms by which D4R counteracts morphine effects is not completely understood, but the existence of a D4R-MOR heterodimer in the striosomes of the caudate putamen has been proposed . However, it is unknown how this interaction could affect both the adult neurogenesis and olfaction. In the present work, we have studied the effects of a chronic treatment with morphine alone or in combination with a D4R agonist (PD168,077) on adult neurogenesis occurring in the SVZ. Furthermore, the impairment or improvement of odorants discrimination has also been analyzed. Using immunohistochemical techniques, we found that chronic treatment with morphine increases dopamine signalling in the SVZ and promotes a depletion of cell proliferation, affecting both neural and glial precursors. These effects were counteracted by the coadministration of morphine with the D4R agonist. The present results support for a critical role of the D4R to prevent morphine effects in the SVZ.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of dopamine D4 receptor in the develpment of morphine-induced analgesic tolerance

Morphine is one of the most effective drugs used for pain management. However, prolonged exposition to morphine produces a wide variety of side effects such as tolerance to analgesia, hyperalgesia and addiction. Downregulation of the mu opioid receptor (MOR) and its uncoupling to G-proteins in the dorsal horn are likely to contribute to the development of morphine tolerance. Previous studies demonstrated that dopamine D4 receptor (D4R) activation prevents morphine addiction by modulating dopamine signaling from nigral dopamine cells. This effect seems to be the result of an antagonistic receptor-receptor interaction involving a D4R-MOR heteroreceptor which could exist in the dorsal striatum. As D4R is expressed in dorsal horn neurons, we hypothesize that D4R could interfere in the development of morphine-induced tolerance to its analgesic effects. Here, using a chronic paradigms of combined treatment of morphine with the D4R agonist PD168.077, we investigated the nociceptive response to three noxious stimuli: thermal (tail-flick test), mechanical (von Frey test) and chemical (formalin test). Moreover, using immunohistochemical techniques, we have evaluated alterations in the primary circuitry of pain (peptidergic and non-peptidergic C fibers and spinal projections neurons NK1-R) and the balance between glutamate and GABA within dorsal horn. Results from the evaluation of analgesic activity of chronic combined treatment of morphine with PD168,077 showed that D4R prevents the development of morphine-induced analgesic tolerance. The present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interaction in the dorsal horn that could help to the development of a new pharmacology strategy in the treatment of pain. CTS161 and UMA20-FEDERJA-122 (Junta de Andalucía, Spain)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of dopamine D4 receptor in the development of morphine-induced analgesic tolerance

Morphine is one of the most effective analgesic used in the clinical management of pain. However, long-term use of morphine can cause many side effects including respiratory depression, constipation, analgesic tolerance, hyperalgesia and addiction. The mechanisms underlying morphine tolerance are complex and nowadays it is not yet completely understood. As a primary mediator of morphine analgesia, the mu opioid receptor (MOR) contributes to morphine tolerance through downregulating the expression of MOR and its uncoupling from G-proteins in the dorsal horn of the spinal cord. It has been reported that the colocalization of the dopamine D4 receptor with MOR in the dorsal striatum counteracts the addictive effects induced by morphine through a putative D4R-MOR heteroreceptor that modulates dopamine signaling from nigral dopamine nerve cells. As D4R is also expressed in both the dorsal root ganglia (DRG) and dorsal horn neurons, we hypothesize that D4R could interfere the development of morphine-induced tolerance to its analgesic effects at dorsal horn level. Using a chronic treatment paradigm of morphine with the D4R agonist PD168,077, we have first investigated the nociceptive response to noxious thermal stimulation (tail flick), mechanical stimulation (von Frey) and to persistent noxious chemical stimulation (formalin). Furthermore, using immunohistochemical techniques, we have studied primary afferent fibers (peptidergic and non-peptidergic C fibers), spinal interneurons and NK1 spinal projection neurons, and the balance between glutamate and GABA in the dorsal horn. Results from the evaluation of analgesic activity showed that D4R activation prevents the development of morphine-induced analgesic tolerance. In addition, D4R preserves the appropriate balance between glutamate and GABA for a proper analgesic effect by modulating the spinal circuit.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Pharmacological activation of the dopamine D4 receptor prevents morphineinduced impairment of adult neurogenesis in the subventricular zone: functional implications in odor discrimination learning.

Morphine reduces adult neurogenesis in the subventricular zone (SVZ) and therefore the integration of newly generated neurons in the local olfactory bulb circuit. However, the effects of opioids drugs on the olfactory perception have been scarce studied, although some data suggests that they interfere with olfactory function, and the underlying mechanisms are unknown. We have previously shown that the activation of the dopamine D4 receptor (D4R) prevents both morphine addiction -by modulating dopamine signaling from nigral dopamine cells- and the development of morphineinduced analgesic tolerance -by regulating the primary circuit of pain and GABA/glutamate balance within the dorsal horn. Here, we hypothesize that the D4R could also counteract the impairment of olfaction associated with morphine. We investigated the effect of chronic paradigm of combined treatment of morphine with the D4R agonist PD168,077 on SVZ neurogenesis in adult rats using immunohistochemistry and its functional implications by an olfactory discrimination test. Results showed that, in the SVZ, D4R activation counteracted morphine-induced depletion of newly generated glial cells (astrocytes and oligodendrocytes) and neuroblasts as well as the increase of tyrosine hydroxylase expression. Besides, three weeks of chronic administration of morphine impaired olfactory discrimination between a pair of odorants, which was completely prevented by the co-administration with PD168,077. The present results deepen in the dopaminergic regulation of olfaction and give support for the existence of antagonistic functional D4R-MOR interaction in olfactory bulb and SVZ that could help to the development of new pharmacology strategies for the treatment of pain reducing side-effects of morphine.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions.

To assess the possible interactions between the dorsolateral Periaqueductal Gray matter (dlPAG) and the laryngeal motor neurons of the nucleus Ambiguus (nA), we have examined the pattern of double staining c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/Fox-P2-ir) and Tyrosine Hydroxylase (TH) throughout the rostro-caudal extent of nA in spontaneously breathing anaesthetised male Sprague–Dawley rats during dlPAG electrical stimulation. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in the somatas of the loose (p<0.05) and compact formation (p<0.01) within the nA and confirm the expression of FoxP2 bilaterally in all the domains within the nA. A second group of experiments was made to examine the importance of the dlPAG in modulating the laryngeal response evoked after electrical or chemical (glutamate) dlPAG stimulations. Both electrical and chemical stmulations evoked a significant decrease of laryngeal resistance (subglottal pressure) (p<0.001) accompanied with an increase in respiratory rate together with a pressor and tachycardic response. The results of our study contribute with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.Funding for open access charge: Universidad de Málaga / CBU

Pharmacological activation of dopamine D4 receptor prevents morphine-induced impairment of the subventricular adult neurogenesis: functional implications in odor discrimination learning.

Morphine reduces adult neurogenesis in the subventricular zone (SVZ) and therefore the integration of newly generated neurons in the local olfactory bulb (OB) circuit. However, the effects of opioids drugs on the olfactory perception have been scarce studied, although some data suggests that they interfere with olfactory function. We have previously shown that the activation of the dopamine D4 receptor (D4R) prevents both morphine addiction -by modulating dopamine signaling from nigral dopamine cells- and the development of morphine-induced analgesic tolerance -by regulating the primary circuit of pain and GABA/glutamate balance within the dorsal horn. Here, we hypothesize that the D4R could also counteract the impairment of olfaction associated with morphine. We investigated the effect of chronic paradigm of combined treatment of morphine with the D4R agonist PD168,077 on SVZ neurogenesis in adult rats using immunohistochemistry and its functional implications by an olfactory discrimination test. Results showed that, in the SVZ, D4R activation counteracted morphine-induced depletion of newly generated glial cells (astrocytes and oligodendrocytes) and neuroblasts as well as the increase of tyrosine hydroxylase expression. Besides, three weeks of chronic administration of morphine impaired olfactory discrimination between a pair of odorants, which was completely prevented by the co-administration with PD168,077. The present results give support for the existence of antagonistic functional D4R-MOR interaction in olfactory bulb and SVZ that could help to the development of new pharmacology strategies for the treatment of pain reducing side-effects of morphine.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. CTS161, Junta de Andalucía UMA20-FEDERJA-122, Junta de Andalucí

Laryngeal effects of stimulation of the dorsolateral Periaqueductal Grey Matter in spontaneously breathing anaesthetized rats

The stimulation of the Periaqueductal Gray matter (PAG) and nucleus retroambiguus (nRA) produces vocalization. A high expression of FOXP2 protein at mesencephalic (PAG) and pontine regions involved in cardiorespiratory control has been described. The aim of this study was to characterize the possible role of the dlPAG in modulating laryngeal activity and their effects on vocalization. Experimental studies were carried out with non-inbred male rats (n=27), Sprague-Dawley (250-300 g). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2mg/ kg, i.v., as necessary). Neuromorphological study (n=6) The pattern of staining for c-Fos and FOXP2 protein immunoreactivity (c-Fos-ir) were examinated throughout the rostrocaudal extent of the nRa/nA region during electrical stimulation of the dlPAG. Neuropharmacological study (n=21) A double tracheal cannulation was used to obtain an “isolated glottis in situ” and to record respiratory airflow. Bilateral parietostomy allowed access to the dlPAG. Electrical stimulations (n=7) of this region using concentric bipolar electrodes (1ms pulses, 20-40µA, 100Hz for 5s) were performed. Microinjections of PBS-Evans Blue (250nl, pH 7.4±0.1, 5-s duration) (n=7) or glutamate (0,25M, 250nl) (n=7) were performed. Respiratory flow, pleural pressure, blood pressure and heart rate were also recorded. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in nRA/nA somatas (p<0.01) and confirm the expression of FOXP2 bilaterally in both nuclei. dlPAG PBS-Evans Blue microinjections did not produce any significant changes in any of the cardiorespiratory variables recorded. dlPAG stimulations evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,001) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001), together with a pressor (p<0,001) and tachycardic response (p<0,001).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dorsolateral Periaqueductal Grey Matter in the control of laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats.

The stimulation of the dlPAG produces vocalization. The nRA turn passive into active expiration modifying the activity of laryngeal motoneurons located in the nA. We have shown that cPB and A5 Region are involved in changes of laryngeal caliber. A high expression of FOXP2 protein at mesencephalic and pontine regions involved in cardiorespiratory control has been described. The aim is to characterize the role of the dlPAG in modulating laryngeal activity and their effects on vocalization. Experimental studies were carried out with male Sprague-Dawley rats (n=25) (250-300g). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v.). The pattern of staining for c-Fos and FOXP2 protein immunoreactivity were examinated throughout the rostrocaudal extent of the nRa/nA region during electrical stimulation of the dlPAG. Electrical stimulations (n=7) (1ms pulses, 20-40μA, 100Hz for 5s), microinjections of PBS-Evans Blue (250nl, pH 7.4±0.1, 5-s duration) (n=7) or glutamate (0,25M, 250nl) (n=7) were performed. Respiratory flow, pleural pressure, subglottic pressure, blood pressure and heart rate were recorded. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in nRA/nA somatas (p<0.01) and confirm the expression of FOXP2 bilaterally in both nuclei. dlPAG PBS-Evans Blue microinjections did not produce any significant changes in any of the cardiorespiratory variables recorded. dlPAG electrical and chemical (glutamate) stimulations evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,001) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001), together with a pressor (p<0,001) and tachycardic response (p<0,001). Our study contributes with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Exploring laryngeal effects of dorsolateral periaqueductal grey stimulation in anesthetized rats: implications for c-Fos and FOXP2 expression in the nucleus ambiguus subdivisions.

Stimulation of the Periaqueductal Gray matter (PAG) and nucleus retroambiguus (nRA) evokes vocalization (1). The nRA serves as a key target for converting passive into active expiration by modulating the activity of laryngeal motoneurons located in the nucleus ambiguus (nA) (2). Previously, we have demonstrated the involvement of rostral and ventral pontine structures in altering laryngeal caliber (3). Furthermore, a heightened expression of the FOXP2 protein (a transcription factor closely linked to vocalization) has been observed in both mesencephalic (PAG) and pontine regions implicated in cardiorespiratory control (4). To investigate the potential interactions between the dlPAG and laryngeal motor neurons of the nA assessing the double staining patterns of c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/Fox-P2-ir) and Tyrosine Hydroxylase (TH) across the rostro-caudal extent of the nA. Experimental studies were carried out with non-inbred male rats (n=10), SPF, Sprague-Dawley (250-300 g). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2mg/ kg, i.v.). The pattern of immunohistochemical and immunofluorescence staining for c-Fos and FOXP2 protein immunoreactivity (c-Fos-ir/FOXP2-ir) were examinated throughout the rostrocaudal extent of the nA region during electrical stimulation of the dlPAG. Guanethidine was administered to suppress sympathetically mediated cardiovascular responses. Stimulation of the dlPAG induced a specific increase in c-Fos-ir, with ipsilateral predominance in the somatas of both the loose (p<0.05) and compact formation (p<0.01) within the nA. Furthermore, we could confirm the bilateral expression of FoxP2 across all domains within the nA. Our study contributes with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech