Injection Of DAMGO Research Articles

Intrathecal RGS4 inhibitor, CCG50014, reduces nociceptive responses and enhances opioid-mediated analgesic effects in the mouse formalin test.

The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation. Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection. Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol). These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.

Interaction Between Endocannabinoid and Opioidergic Systems Regulates Food Intake in Neonatal Chicken

Endocannabinoids and opiates have regulatory role in some physiological functions in mammals but their interaction(s) have not been studied in avian. This survey is designed to investigate interaction of these systems on feeding behavior in neonatal chickens. In experiment 1, chicken intracerebroventricular (ICV) injected with saline, DAMGO (µ-opioid receptors agonist, 125 pmol), SR141716A (CB1 receptors antagonist, 6.25 µg) and SR141716A + DAMGO. In experiment 2, saline, DAMGO, AM630 (CB2 receptors antagonist, 1.25 µg) and DAMGO + AM630. Experiments 3–6 followed the procedure similar to experiments 1 and 2, except DPDPE (δ-opioid receptors agonist, 40 pmol) and U-50488H (κ-opioid receptors agonist, 30 nmol) instead of DAMGO were used. In experiment 7, saline, Naloxone (opioid receptors antagonist, 5 µg), 2-AG (CB1 receptors agonist, 2 µg), Naloxone + 2-AG were used. Experiment 8 was similar to experiment 7, except CB65 (CB2 receptors agonist, 1.25 µg) used instead of 2-AG. Cumulative food intake was recorded until 120 min post injection. Data provided that, ICV injection of DAMGO decreased food intake and its effect amplified by CB1 and CB2 receptors antagonist (P 0.05). Hyperphagia-induced by CB1 and CB2 receptors agonist amplified by naloxone (P < 0.001). Perhaps there is interaction between endocannabinoid and opioidergic systems on appetite regulation in chicken.

Modulation of opioid-induced feeding behavior by endogenous nitric oxide in neonatal layer-type chicks.

The current study was designed to evaluate the effects of central administration of L-arginine (The precursor of nitric oxide), N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, selective opioid receptor agonists and involvement of central nitrergic/opioidergic systems on feeding behavior in neonatal layer-type chicks. The results of this study showed that the intracerebroventricular (ICV) injection of L-arginine (400 and 800 nmol) significantly decreased food intake (P < 0.001) but the injection of 200 nmol L-arginine had no effect on cumulative food intake in FD3 chickens (P > 0.05). The ICV injection of L-NAME (200 and 400 nmol) increased food intake (P < 0.001) but 100 nmol of L-NAME had no significant effect (P > 0.05). On the other hand, the co-injection of 100 nmol L-NAME significantly attenuated the anorexigenic effect of 800 nmol L-arginine (P < 0.001). Moreover, the food intake of chicks was significantly decreased by ICV injection of DAMGO (μ-opioid receptor agonist, 125 pmol) (P < 0.001) while both DPDPE (δ-opioid receptor agonist, 40 pmol) and U-50488H (κ-opioid receptor agonist, 30 nmol) significantly stimulated food intake (P < 0.001). In addition, the hypophagic effect of DAMGO was significantly amplified by administration of L-arginine (P < 0.001) while the administration of L-NAME attenuated the hypophagic effect of DAMGO (P < 0.001). In contrast, co-injection of L-arginine or L-NAME with DPDPE had no effect on the hyperphagia induced by DPDPE as well as the hyperphagic effect of U-50488H on food intake was not affected by concurrent injection of L-arginine or L-NAME (P > 0.05). These results suggest that nitrergic and opioidergic systems have an important role on feeding behavior in the CNS of neonatal layer-type chicks and it seems that interaction between them is mediated by μ-opioid receptor.

Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

P63: Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of CGRP expression in the spine

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ -opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR analysis showed that NaHS significantly reversed the up-regulated spinal calcitonin gene related peptide (CGRP) expression in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pretreatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α , Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. In addition, both repeated DAMGO treatment and naloxone-precipitated withdrawal decreased endogenous H2S level in rat spinal cord, suggesting that the endogenous hydrogen sulfide is important to prevent hyperalgesia. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

Activation of mu‐opioid receptors in commissural nucleus tractus solitarius markedly depresses hypoxic, but not hypercapnic, ventilatory response in conscious rats (712.11)

Activation of cNTS MORs blocks HVR and moderately depresses HCVR (↓30%) in anesthetized rats (Zhang et al, Anesthesiology 2011) in agreement with the cNTS synaptically receiving carotid body afferents and heavily expressing MORs. Because of synergistic effect of anesthesia on opioids’ impacts and projections of bronchopulmonary C‐fibers (PCFs) to the cNTS, we asked whether stimulation of cNTS MORs would alter HVR, HCVR, and PCF‐mediated apnea in conscious state. cNTS catheter guide was instrumented in the anesthetized rats with the right site ensured by lack of HVR following local injection of DAMGO (2.5 mM, 30 nl), a MOR agonist. After recovery from surgery, three groups of conscious animals were individually placed in a whole‐body plethysmograph to: inhale 10% O2 for 2 min or 7% CO2 for 5 min, or receive right atrial bolus injection of capsaicin (CAP, 1 μg/kg) before and 5 min after intra‐cNTS microinjection of DAMGO with and without previous local blockade of MORs by CTAP (10 mM, 30 nl). Microinjection of DAMGO into the cNTS decreased baseline VE (↓16%) mainly via lowering fR and reduced HVR and PCF‐mediated apnea (63% and 20%, P &lt; 0.01) without effect on HCVR. Microinjection of DAMGO following CTAP failed to alter HVR. Our data suggest that cNTS MORs play a critical role in depressing respiratory chemoreflexes in conscious state and this influence is largely strengthened by anesthesia.Grant Funding Source: Supported by HL‐107462

Natriorexigenic effect of DAMGO is decreased by blocking AT1 receptors in the central nucleus of the amygdala

μ-Opioid receptor (μ-OR) activation with agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) in the central nucleus of the amygdala (CeA) induces sodium (0.3M NaCl) intake in rats. The purpose of this study was to examine the effects of pre-injections of losartan (AT1 angiotensin receptor antagonist) into the CeA on 0.3M NaCl and water intake induced by DAMGO injected bilaterally in the same area in rats submitted to water deprivation–partial rehydration (WD–PR) and in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP) injected subcutaneously (FURO/CAP). Male Sprague–Dawley rats with stainless steel cannulas implanted bilaterally into the CeA were used. In WD–PR rats, bilateral injections of DAMGO (2nmol in 0.5μL) into the CeA induced 0.3M NaCl and water intake, and pre-treatment with losartan (108nmol in 0.5μL) injected into the CeA reduced 0.3M NaCl and water intake induced by DAMGO. In FURO/CAP rats, pre-treatment with losartan (108nmol in 0.5μL) injected into the CeA attenuated the increase in 0.3M NaCl and water intake induced by DAMGO (2nmol in 0.5μL) injected into the same site. The results suggest that the natriorexigenic effect of DAMGO injected into the CeA is facilitated by endogenous angiotensin II acting on AT1 receptors in the CeA, which drives rats to ingest large amounts of hypertonic NaCl.

Sensitization by ventral pallidal DAMGO

Repeated injections of morphine into the ventral pallidum of laboratory rats results in the development and expression of motor sensitization. Although morphine and [D-Ala, N-MePhe, Gly(ol)]-enkephalin (DAMGO) both activate μ-opioid receptors, their influence on receptor-mediated signaling differs; therefore, we determined if they differentially influenced ventral pallidal-mediated motor sensitization. Repeated intraventral pallidal injections of DAMGO led to the development of motor sensitization and this behavior persisted for at least 18 days. When DAMGO-sensitized rats were challenged with a morphine treatment (either in the ventral pallidum or systemically), the resulting motor response was similar to that seen in rats with a history of intrapallidal saline, that is, cross-sensitization did not occur. As DAMGO and morphine likely activate different arms of the heterologous signal transduction system associated with μ-opioid receptors, these observations may reflect behavioral consequences of biased agonism at these receptors.

Activation of μ-opioid receptors in the central nucleus of the amygdala induces hypertonic sodium intake

Opioid mechanisms are involved in the control of water and NaCl intake and opioid receptors (ORs) are present in the central nucleus of the amygdala (CeA), a site of important facilitatory mechanisms related to the control of sodium appetite. Therefore, in the present study we investigated the effects of the activation of μ-ORs in the CeA on 0.3M NaCl and water intake in rats. Male Sprague–Dawley rats with stainless steel cannulas implanted bilaterally in the CeA were used. In rats submitted to water deprivation–partial rehydration, bilateral injections of the selective μ-OR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) in the doses of 1, 2, and 4nmol into the CeA induced a dose-related increase of 0.3M NaCl intake and water intake, and bilateral injections of the selective μ-OR antagonist D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the doses of 0.5, 1, and 2nmol into the CeA produced a dose-related decrease of 0.3M NaCl and water intake induced by DAMGO 2nmol into the same site. In rats treated with the diuretic furosemide (10mg/kgb.w.) combined with the angiotensin-converting enzyme inhibitor captopril (5mg/kgb.w.) injected subcutaneously, bilateral injections of DAMGO 2nmol into the CeA increased 0.3M NaCl intake and water intake and the blockade of μ-ORs with CTAP 1nmol injected into the CeA reduced the increase in 0.3M NaCl intake and water intake induced by DAMGO 2nmol into the same site. Bilateral injections of DAMGO into the CeA did not change urinary volume, sodium urinary excretion and mean arterial pressure, but increased activity. Thus stimulating μ-ORs in the CeA increases hypertonic sodium intake, whereas antagonizing these sites inhibits hypertonic sodium intake. Together, our results implicate μ-ORs in the CeA in a positive regulation of sodium intake.

The effect of DAMGO on the preBötzinger Complex (preBC) in young and adult rabbits

In vitro studies suggest the preBC as the site of opioid‐induced respiratory depression. This study evaluated the effect of injections of the mu‐opioid agonist [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) into the preBC in young (13–24 days) and adult rabbits, which were decerebrate, tracheotomized, ventilated and vagus‐intact. The preBC was identified by injection of D,L‐homocysteic acid (1mM). DAMGO (100μM) was injected into the preBC and 1mm rostral and 1mm caudal on both sides of the brainstem. When bradypnea or apnea ensued, the opioid antagonist naloxone (1mM) was injected into the same locations. In 9 young rabbits, DAMGO caused significant bradypnea or apnea. Local naloxone reversed this depression. In 3/6 adult rabbits DAMGO injection into the preBC caused apnea, which was reversible with local naloxone. In the other three animals, DAMGO had no effect. In conclusion, opioid injection into the preBC causes respiratory depression in young but not in all adult rabbits. It remains to be seen whether this effect is still relevant at clinical opioid concentrations. Supported by FAER grant MRTG‐BS‐02/15/2010‐Stucke.

Effects of muscimol, amphetamine, and DAMGO injected into the nucleus accumbens shell on food-reinforced lever pressing by undeprived rats

Previous studies have shown that large increases in food intake in nondeprived animals can be induced by injections of both the GABAA agonist muscimol and the μ-opioid agonist DAMGO into the nucleus accumbens shell (AcbSh), while injections of the catecholamine agonist amphetamine have little effect. In the current study we examined whether injections of these drugs are able to increase food-reinforced lever pressing in nondeprived rats. Twelve subjects were trained to lever press on a continuous reinforcement schedule while food deprived and were then tested after being placed back on ad libitum feeding. Under these conditions, responding was markedly increased by injections of either muscimol or DAMGO, although the onset of the effects of the latter drug was delayed by 30–40min. In contrast, amphetamine injections failed to increase reinforced lever pressing, although they did enhance responding on a non-reinforced lever, presumably reflecting alterations in behavioral activation. These results demonstrate that stimulation of GABAA and μ-opioid receptors within the AcbSh is able to promote not only food intake, but also food-directed operant behavior. In contrast, stimulation of AcbSh dopamine receptors may enhance behavioral arousal, but does not appear to specifically potentiate behaviors directed toward food procurement.

Implication of dopaminergic projection from the ventral tegmental area to the anterior cingulate cortex in μ‐opioid‐induced place preference

Despite the importance of prefrontal cortical dopamine in modulating reward, little is known about the implication of the specific subregion of prefrontal cortex in opioid reward. We investigated the role of neurons projecting from the ventral tegmental area (VTA) to the anterior cingulate cortex (ACG) in opioid reward. Microinjection of the retrograde tracer fluorogold (FG) into the ACG revealed several retrogradely labelled cells in the VTA. The FG-positive reactions were noted in both tyrosine hydroxylase (TH)-positive and -negative VTA neurons. The released levels of dopamine and its major metabolites in the ACG were increased by either the electrical stimulation of VTA neurons or microinjection of a selective μ-opioid receptor (MOR) agonist, (D-Ala²,N-MePhe⁴,Gly-ol⁵) enkephalin (DAMGO), into the VTA. MOR-like immunoreactivity was seen in both TH-positive and -negative VTA neurons projecting to the ACG. The conditioned place preference induced by intra-VTA injection of DAMGO was significantly attenuated by chemical lesion of dopaminergic terminals in the ACG. The depletion of dopamine in the ACG induced early extinction of μ-opioid-induced place preference. The levels of phosphorylated DARPP32 (Thr34) and phosphorylated CREB (Ser133) were increased in the ACG of rats that had maintained the morphine-induced place preference, whereas the increases of these levels induced by morphine were blocked by pre-treatment of a selective dopamine D1 receptor antagonist SCH23390. These findings suggest that VTA-ACG transmission may play a crucial role in the acquisition and maintenance of μ-opioid-induced place preference. The activation of DARPP32 and CREB through dopamine D1 receptors in the ACG could be implicated in the maintenance of μ-opioid-induced place preference.

The Functional Antiopioid Action of the Ventrolateral Periaqueductal Gray Nociceptin/Orphanin FQ and Nociceptin Receptor System Underlies DAMGO Analgesic Tolerance

Nociceptin/Orphanin FQ (N/OFQ) counteracts supraspinal opioid effects and plays a role in antinociceptive morphine tolerance. Therefore, in the present study, the selective µ-opioid agonist [D-Ala²-NMe-Phe⁴-Gly-ol⁵]-enkephalin (DAMGO) was used. Repeated injection of DAMGO (1 µg/ 1 µl) into the ventrolateral periaqueductal gray (vlPAG), a key site for the development of opioid tolerance, induced analgesia that lasted up to 3 days. In DAMGO-tolerant rats, injection of the N/OFQ antagonist (±)-J 113397 (4 µg/1 µl), into the same site, restored the antinociceptive effect of DAMGO. If (±)-J 113397 treatment preceded each DAMGO injection, tolerance did not develop. Inhibition of N/OFQ signaling can reverse and prevent the development of DAMGO tolerance in the vlPAG. The results confirm that N/OFQ acts as a functional opioid antagonist.

Peripheral μ-opioid receptors attenuate the augmented exercise pressor reflex in rats with chronic femoral artery occlusion

Recently, opioid receptors have been shown to be expressed on group III and IV afferents, which comprise the sensory arm of the exercise pressor reflex. Although the stimulation of opioid receptors in the central nervous system has been shown to attenuate the exercise pressor reflex, the effect on the reflex of their stimulation in the periphery is unknown. We therefore tested the hypothesis that the activation of peripheral mu-opioid receptors attenuates the exercise pressor reflex. The pressor responses to static contraction were compared before and after the injection of the mu-opioid receptor agonist [d-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO; 1 microg) into the abdominal aorta of decerebrated rats in which one femoral artery had been occluded 72 h previously (n = 10) and in control rats whose femoral arteries were freely perfused (n = 8). DAMGO attenuated the peak pressor response to contraction in rats whose femoral arteries had been occluded (before: increase of 34 + or - 3 mmHg and after: increase of 22 + or - 2 mmHg, P = 0.008); the inhibitory effect of DAMGO was prevented by the injection of naloxone (100 microg) into the abdominal aorta (before: increase of 29 + or - 5 mmHg and after: increase of 29 + or - 5 mmHg, P = 0.646, n = 7). An intravenous injection of DAMGO (1 microg, n = 6) had no effect on the peak pressor response to contraction in both groups of rats. DAMGO had no effect on the peak pressor response to contraction in rats whose femoral arteries were freely perfused (before: Delta 23 + or - 4 mmHg, after: Delta 23 + or - 3 mmHg, n = 6) but appeared to have a small effect on topography of the response. DAMGO had no effect on the peak pressor response to tendon stretch in both groups of rats (both P > 0.05). We conclude that the stimulation of peripheral mu-opioid receptors attenuates the exercise pressor reflex in rats whose femoral arteries have been ligated for 72 h.

Nociceptive behaviour upon modulation of mu-opioid receptors in the ventrobasal complex of the thalamus of rats

The role of mu-opioid receptors (MORs) in the inflammatory pain processing mechanisms within the ventrobasal complex of the thalamus (VB) is not well understood. This study investigated the effect of modulating MOR activity upon nociception, by stereotaxically injecting specific ligands in the VB. Nociceptive behaviour was evaluated in two established animal models of inflammatory pain, by using the formalin (acute and tonic pain) and the ankle-bend (chronic monoarthritic pain) tests. Control (saline intra-VB injection) formalin-injected rats showed acute and tonic pain-related behaviours. In contrast, intrathalamic administration of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate (DAMGO), a MOR-specific agonist, induced a statistically significant decrease of all tonic phase pain-related behaviours assessed until 30–35min after formalin hind paw injection. In the acute phase only the number of paw-jerks was affected. In monoarthritic rats, there was a noticeable antinociceptive effect with approximately 40min of duration, as denoted by the reduced ankle-bend scores observed after DAMGO injection. Intra-VB injection of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a specific MOR antagonist, or of CTOP followed, 10min after, by DAMGO had no effects in either formalin or ankle-bend tests. Data show that DAMGO-induced MOR activation in the VB has an antinociceptive effect in the formalin test as well as in chronic pain observed in MA rats, suggesting an important and specific role for MORs in the VB processing of inflammatory pain.

Activation of Spinal α-2 Adrenoceptors, but Not μ-Opioid Receptors, Reduces the Intrathecal N-Methyl-d-Aspartate-Induced Increase in Spinal NR1 Subunit Phosphorylation and Nociceptive Behaviors in the Rat

A previous study from our laboratories showed that a significant reduction in spinal N-methyl-D-aspartate (NMDA) receptor NR1 subunit phosphorylation (pNR1) is associated with the antiallodynic effect produced by intrathecal (IT) injection of the alpha-2 adrenoceptor agonist, clonidine, in neuropathic rats. In this study, we determined whether the spontaneous pain and increased pNR1 expression induced by NMDA injection are reduced by IT injection of either clonidine or the mu-opioid receptor agonist, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO). We examined the effect of clonidine (20 microg/rat) or DAMGO (1 microg/rat) injection on IT NMDA-induced spontaneous nociceptive behavior and pNR1 expression in the spinal dorsal horn. We also determined whether the effect of clonidine is mediated by alpha-2A or alpha-2C adrenoceptors. Finally, rat spinal cords were immunohistochemically processed for double staining of pNR1 and alpha-2A or alpha-2C adrenoceptors or mu-opioid receptors. The NMDA-induced increase in both pNR1 expression and nociceptive behavior was significantly reduced by IT clonidine but not DAMGO. This analgesic effect of clonidine was blocked by administration of either an alpha-2A (BRL44408, 30 microg/rat) or an alpha-2C (JP-1302, 50 microg/rat) adrenoceptor antagonist. In addition, immunocytochemistry revealed that spinal pNR1 immunoreactive cells co-contain alpha-2A and alpha-2C adrenoceptors. These results demonstrate that the IT NMDA-induced increase in pNR1 expression and nociceptive behavior is significantly reduced by activation of alpha-2 adrenoceptors, but not mu-opioid receptors, in the spinal cord dorsal horn. Furthermore, these findings suggest that the modulation of spinal NR1 phosphorylation is linked to the effect of IT clonidine on postsynaptic neuronal activity.

Feeding association between the nucleus of the solitary tract and the ventral tegmental area

Male Sprague–Dawley rats were fitted with two cannulae in the VTA and one cannula in the NTS for co-administration of the μ-opioid receptor agonist DAMGO in one site and the opioid antagonist naltrexone in the other. Injection of DAMGO into the VTA or the NTS stimulated feeding. The increase in food intake after DAMGO injection into the VTA was decreased following injection of naltrexone into the NTS. Furthermore, the increase in food intake after DAMGO injection into the NTS was decreased following injection of naltrexone into the VTA. These results suggest an opioid-mediated feeding association between the VTA and NTS.

μ-Opioid receptor agonist injections into the presumed pre-Bötzinger complex and the surrounding region of awake goats do not alter eupneic breathing

Opioids are clinically important in the alleviation of pain. An undesirable side effect of opioids is depression of breathing. Data from isolated preparations suggest this effect is due to attenuation of discharge activity of neurons in the pre-Bötzinger complex (preBötzC), a medullary area with respiratory rhythmogenic properties. The purpose of this study was to examine how [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, affected breathing after injection into the presumed preBötzC of the adult awake goat. We hypothesized that DAMGO would cause breathing to decrease and become irregular when injected into the presumed preBötzC and the surrounding region of the conscious animal. We further hypothesized that ventilatory sensitivity to CO(2) and hypoxia would be blunted after the injection of DAMGO. Microtubules were bilaterally implanted into the presumed preBötzC of 10 adult female goats. After recovery from the surgery, DAMGO (0.5-10 mul, 1 nM-10 muM) was injected into the presumed preBötzC during the awake state. DAMGO had no effect on pulmonary ventilation [inspiratory minute ventilation (Vi)], respiratory rhythm and pattern, the activation pattern of inspiratory and expiratory muscles, or arterial blood gases during eupneic breathing conditions (P > 0.10). However, DAMGO attenuated (P < 0.05) the evoked increase in breathing frequency when inspired CO(2) was increased, and DAMGO attenuated the Vi response to reduction of inspired O(2) to 10.8% (P < 0.05). We conclude that our data do not provide support for the concept that in awake mammals opioid depression of breathing is due to a directed action of opioids on preBötzC neurons.

Activation of opioid μ-receptors in medullary raphe depresses sighs

Sighs, a well-known phenomenon in mammals, are substantially augmented by hypoxia and hypercapnia. Because (d-Ala(2),N-Me-Phe(4),Gly-ol)-enkephalin (DAMGO), a mu-receptor agonist, injected intravenously and locally in the caudal medullary raphe region (cMRR) decreased the ventilatory response to hypoxia and hypercapnia, we hypothesized that these treatments could inhibit sigh responses to these chemical stimuli. The number and amplitude of sighs were recorded during three levels of isocapnic hypoxia (15%, 10%, and 5% O(2) for 1.5 min) or hypercapnia (3%, 7%, and 10% CO(2) for 4 min) to test the dependence of sigh responses on the intensity of chemical drive in anesthetized and spontaneously breathing rats. The role of mu-receptors in modulating sigh responses to 10% O(2) or 7% CO(2) was subsequently evaluated by comparing the sighs before and after 1) intravenous administration of DAMGO (100 microg/kg), 2) microinjection of DAMGO (35 ng/100 nl) into the cMRR, and 3) intravenous administration of DAMGO after microinjection of d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, 100 ng/100 nl), a micro-receptor antagonist, into the cMRR. Hypoxia and hypercapnia increased the number, but not amplitude, of sighs in a concentration-dependent manner, and the responses to hypoxia were significantly greater than those to hypercapnia. Systemic and local injection of DAMGO into the cMRR predominantly decreased the number of sighs, while microinjection into the rostral and middle MRR had no or limited effects. Microinjecting CTAP into the cMRR significantly diminished the systemic DAMGO-induced reduction of the number of sighs in response to hypoxia, but not to hypercapnia. Thus we conclude that hypoxia and hypercapnia elevate the number of sighs in a concentration-dependent manner in anesthetized rats, and this response is significantly depressed by activating systemic mu-receptors, especially those within the cMRR.

GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol—a GABA A receptors agonist—and DAMGO—a μ-opioid receptors agonist—on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence model. Besides, mechanisms mediated by μ- and GABA A-receptors in the CeA appear to exert an inhibitory influence on the anxiety-like behavior induced by withdrawal from acute morphine treatment.