Morphine-induced kinase activation and localization in the periaqueductal gray of male and female mice.

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.

Upregulation of fatty acid amide hydrolase in the dorsal periaqueductal gray is associated with neuropathic pain and reduced heart rate in rats.

Nerve damage can induce a heightened pain response to noxious stimulation, which is termed hyperalgesia. Pain itself acts as a stressor, initiating autonomic and sensory effects through the dorsal periaqueductal gray (dPAG) to induce both sympathoexcitation and analgesia, which prior studies have shown to be affected by endocannabinoid signaling. The present study addressed the hypothesis that neuropathic pain disrupts autonomic and analgesic regulation by endocannabinoid signaling in the dPAG. Endocannabinoid contents, transcript levels of endocannabinoid signaling components, and catabolic enzyme activity were analyzed in the dPAG of rats at 21 days after painful nerve injury. The responses to two nerve injury models were similar, with two-thirds of animals developing hyperalgesia that was maintained throughout the postinjury period, whereas no sustained change in sensory function was observed in the remaining rats. Anandamide content was lower in the dPAG of rats that developed sustained hyperalgesia, and activity of the catabolic enzyme fatty acid amide hydrolase (FAAH) was higher. Intensity of hyperalgesia was correlated to transcript levels of FAAH and negatively correlated to heart rate and sympathovagal balance. These data suggest that maladaptive endocannabinoid signaling in the dPAG after nerve injury could contribute to chronic neuropathic pain and associated autonomic dysregulation. This study demonstrates that reduced anandamide content and upregulation of FAAH in the dPAG are associated with hyperalgesia and reduced heart rate sustained weeks after nerve injury. These data provide support for the evaluation of FAAH inhibitors for the treatment of chronic neuropathic pain.

Ligand-biased activation of extracellular signal-regulated kinase 1/2 leads to differences in opioid induced antinociception and tolerance.

Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates ß-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by administration of PTX or U0126, and was enhanced by administration of dyn-DN. Microinjection of DAMGO, but not fentanyl, into the vlPAG induced phosphorylation of ERK1/2, which was blocked by inhibiting receptor internalization with administration of dyn-DN, but not by inhibition of Gαi/o proteins. ERK1/2 inhibition also prevented the development and expression of tolerance to repeated DAMGO microinjections, but had no effect on fentanyl tolerance. These data reveal that ERK1/2 activation following MOPr internalization contributes to the antinociceptive effect of some (e.g., DAMGO), but not all opioids (e.g., fentanyl) despite the known similarities for these agonists to induce ß-arrestin recruitment and internalization.

Extracellular Signal-Regulated Kinase 5 in the Cerebrospinal Fluid-Contacting Nucleus Contributes to Neuropathic Pain in Rats.

BACKGROUND: The activation of mitogen-activated protein kinases (MAPKs) have been observed in synaptic plasticity processes of learning and memory in neuropathic pain. Cerebrospinal fluid-contacting nucleus (CSF-CN) has been identified with the onset and persistence of neuropathic pain. However, whether extracellular signal-regulated protein kinase 5 (ERK5), a member of MAPKs, in CSF-CN participates in neuropathic pain has not been studied yet. OBJECTIVE: The aim of the present study was to identify the role of ERK5 in CSF-CN on the formation and development of neuropathic pain, and to investigate its possible mechanism. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: After a chronic constriction injury (CCI) model was produced, BIX02188 was dissolved in 1% DMSO and injected into the lateral ventricles LV in a volume of 3 µl with different doses (0.1 µg, 1 µg, 10 µg). Mechanical allodynia and thermal hypersensitivity behavioral test, immunofluorescence, and western blot technique were used in this research. RESULT: Following CCI, mechanical allodynia and thermal hypersensitivity were developed within a day, peaked at 14 days, and persisted for 21 days. ERK5 was remarkably activated by CCI in CSF-CN. Moreover, selective inhibiting of p-ERK5 expression in CSF-CN by BIX02188 could significantly relieve CCI-induced mechanical allodynia and thermal hypersensitivity, accompanying with the decreased phosphorylation of cAMP response-element binding protein (CREB) in CSF-CN. LIMITATIONS: More underlying mechanism(s) of the role of ERK5 in CSF-CN on the formation and development of neuropathic pain will be needed to explore in future research. CONCLUSION: These findings suggest activation of ERK5 in CSF-CN might contribute to the onset and development of neuropathic pain and its role might be partly accomplished by p-CREB.

Chewing suppresses the stress-induced increase in the number of pERK-immunoreactive cells in the periaqueductal grey.

We investigated the effects of chewing under immobilization stress on the periaqueductal gray (PAG) matter using phosphorylated extracellular signal-regulated kinase (pERK) as a marker of responding cells. Immobilization stress increased pERK-immunoreactive cells in the PAG. Among four subdivisions of the PAG, the increase of immunoreactive cells was remarkable in the dorsolateral and ventrolateral subdivisions. However, increase of pERK-immunoreactive cells by the immobilization stress was not so evident in the dorsomedial and lateral subdivisions. The chewing under immobilization stress prevented the stress-induced increase of pERK-immunoreactive cells in the dorsolateral and ventrolateral subdivisions with statistical significances (p<0.05). Again, chewing effects on pERK-immunoreactive cells were not visible in the dorsomedial and lateral subdivisions. These results suggest that the chewing alleviates the PAG (dorsolateral and ventrolateral subdivisions) responses to stress.

Regionally selective activation of ERK and JNK in morphine paradoxical hyperalgesia: a step toward improving opioid pain therapy.

In addition to analgesia, opioid agonists may increase pain sensitivity under different conditions varying dose and administration pattern. While opioid hyperalgesia induced by tolerance and withdrawal is largely studied, little is known on the mechanisms underlying ultra-low dose morphine hyperalgesia. This pronociceptive response appears to play an opposing role in morphine analgesia and might have clinical relevance. Ultra-low dose morphine elicited thermal hyperalgesia through activation of µ opioid receptors. To elucidate the intracellular mechanism of morphine nociceptive behaviour, we investigated the mitogen-activated protein kinase (MAPK), crucial pathways in pain hypersensitivity. The catalytic activity of extracellular signal-regulated kinase (ERK), p38, c-Jun-N-terminal kinase (JNK), upstream modulators and transcription factors was investigated in the mouse periaqueductal grey matter (PAG), thalamus and prefrontal cortex by western blotting. Ultra-low dose morphine intensively increased pERK1 contents in the PAG and cortex and, to a lesser extent, increased cortical ERK2 and JNK phosphorylation. No involvement of p38 was detected. Morphine exposure also increased phosphorylation of cortical c-Jun whereas levels of phosphorylated cAMP response element-binding protein (CREB) remained unmodified. Blockade of protein kinase C (PKC) prevented increases in phosphorylation showing a PKC-dependent mechanism of activation. Pharmacological inhibitors of PKC, ERK, and JNK activity prevented morphine hyperalgesia. No modulation of MAPK and transcription factors' activity was detected in the thalamus. These results support the concept that selective activation of ERK and JNK on descending pathways plays an important role in ultra-low dose morphine hyperalgesia. The modulation of these signalling processes might improve pain management with opiate analgesics.

PKC-mediated potentiation of morphine analgesia by St. John's Wort in rodents and humans.

Our purpose was to combine the use of morphine with clinically available inhibitors of protein kinase C (PKC), finally potentiating morphine analgesia in humans. Thermal tests were performed in rodents and humans previously administered with acute or chronic morphine combined or not with increasing doses of the PKC-blocker St. John's Wort (SJW) or its main component hypericin. Phosphorylation of the γ subunit of PKC enzyme was assayed by western blotting in the periaqueductal grey matter (PAG) from rodents co-administered with morphine and hypericin and was prevented in rodent PAG by SJW or hypericin co-administration with morphine, inducing a potentiation of morphine analgesia in thermal pain. The score of pain assessment in healthy volunteers were decreased by 40% when morphine was co-administered with SJW at a dose largely below those used to obtain an antidepressant or analgesic effect in both rodents and humans. The SJW/hypericin potentiating effect lasted in time and preserved morphine analgesia in tolerant mice. Our findings indicate that, in clinical practice, SJW could reduce the dose of morphine obtaining the same analgesic effect. Therefore, SJW and one of its main components, hypericin, appear ideal to potentiate morphine-induced analgesia.

Effects of nitric oxide synthase inhibition in the dorsolateral periaqueductal gray matter on ethanol withdrawal-induced anxiety-like behavior in rats.

RATIONALE: Nitric oxide (NO)-mediated transmission in the dorsolateral periaqueductal gray matter (dlPAG) has been involved in the expression of anxiety-like behaviors. Ethanol withdrawal sensitizes the dlPAG and results in increased anxiety-like responses. OBJECTIVES: The objective of the study was to test the hypothesis that NO in the dlPAG is involved in the expression of ethanol withdrawal-induced anxiety. METHODS: Male Wistar rats were implanted with guide cannulae aimed at the dlPAG. The animals were forced to consume a liquid diet containing ethanol 6-8 % (v/v) for 15 days as their only source of diet. Six days after surgery and 24 h after ethanol discontinuation, the animals received microinjections of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), nonselective nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME), selective neuronal nitric oxide synthase inhibitor 1-(2-[trifluoromethyl]phenyl) imidazole (TRIM), or selective inducible nitric oxide synthase (iNOS) inhibitor N-([3-(aminomethyl)phenyl]methyl) ethanimidamide dihydrochloride (1400W) into the dlPAG. Ten minutes later, the animals were tested in the light/dark box. RESULTS: Carboxy-PTIO (1 nmol), L-NAME (200 nmol), TRIM (20 nmol), and 1400W (0.3 and 1 nmol) decreased the anxiogenic-like effects of ethanol withdrawal in rats in the light/dark box test. The NO precursor L-arginine reversed the effects of L-NAME. CONCLUSIONS: NO production in the dlPAG may play a role in the modulation of ethanol withdrawal-induced anxiety-like behavior in rats. Furthermore, iNOS-mediated NO synthesis in the dlPAG is predominantly involved in the behavioral expression of anxiety-like behavior during ethanol withdrawal.

NAAG peptidase inhibition in the periaqueductal gray and rostral ventromedial medulla reduces flinching in the formalin model of inflammation.

BACKGROUND: Metabotropic glutamate receptors (mGluRs) have been identified as significant analgesic targets. Systemic treatments with inhibitors of the enzymes that inactivate the peptide transmitter N-acetylaspartylglutamate (NAAG), an mGluR3 agonist, have an analgesia-like effect in rat models of inflammatory and neuropathic pain. The goal of this study was to begin defining locations within the central pain pathway at which NAAG activation of its receptor mediates this effect. RESULTS: NAAG immunoreactivity was found in neurons in two brain regions that mediate nociceptive processing, the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Microinjection of the NAAG peptidase inhibitor ZJ43 into the PAG contralateral, but not ipsilateral, to the formalin injected footpad reduced the rapid and slow phases of the nociceptive response in a dose-dependent manner. ZJ43 injected into the RVM also reduced the rapid and slow phase of the response. The group II mGluR antagonist LY341495 blocked these effects of ZJ43 on the PAG and RVM. NAAG peptidase inhibition in the PAG and RVM did not affect the thermal withdrawal response in the hot plate test. Footpad inflammation also induced a significant increase in glutamate release in the PAG. Systemic injection of ZJ43 increased NAAG levels in the PAG and RVM and blocked the inflammation-induced increase in glutamate release in the PAG. CONCLUSION: These data demonstrate a behavioral and neurochemical role for NAAG in the PAG and RVM in regulating the spinal motor response to inflammation and that NAAG peptidase inhibition has potential as an approach to treating inflammatory pain via either the ascending (PAG) and/or the descending pain pathways (PAG and RVM) that warrants further study.

Contribution of acid sphingomyelinase in the periaqueductal gray region to morphine-induced analgesia in mice.

Opioids are the most widely used drugs for long-term pain management, but their use is limited by the development of antinociceptive tolerance. The present study investigated the role of ceramide production through acid sphingomyelinase (ASM) activation in the periaqueductal gray region, a brain region implicated in opioid analgesia and tolerance. Morphine treatment was found, using immunohistochemistry, to increase ASM expression and intracellular ceramide in the periaqueductal gray 30 min after an acute injection (10 mg/kg). The effects of acute morphine treatment on ASM expression and ceramide generation in the periaqueductal gray region were completely blocked by pretreatment with naloxone and by silencing the ASM gene by plasmid-mediated transfection of ASM shRNA. In chronic morphine pellet-implanted mice, ASM expression and ceramide generation in the periaqueductal gray region were also significantly increased. Functionally, selective silencing of the ASM gene by local ASM shRNA transfection reduced the analgesic response to acute morphine, but the data on the effect of ASM shRNA on the development of antinociceptive tolerance were inconclusive. These data provide evidence that ASM activation and ceramide generation in the periaqueductal gray region play a major role in the antinociceptive mechanism of morphine.

Fear-induced suppression of nociceptive behaviour and activation of Akt signalling in the rat periaqueductal grey: role of fatty acid amide hydrolase.

The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

Mammal-like organization of the avian midbrain central gray and a reappraisal of the intercollicular nucleus.

In mammals, rostrocaudal columns of the midbrain periaqueductal gray (PAG) regulate diverse behavioral and physiological functions, including sexual and fight-or-flight behavior, but homologous columns have not been identified in non-mammalian species. In contrast to mammals, in which the PAG lies ventral to the superior colliculus and surrounds the cerebral aqueduct, birds exhibit a hypertrophied tectum that is displaced laterally, and thus the midbrain central gray (CG) extends mediolaterally rather than dorsoventrally as in mammals. We therefore hypothesized that the avian CG is organized much like a folded open PAG. To address this hypothesis, we conducted immunohistochemical comparisons of the midbrains of mice and finches, as well as Fos studies of aggressive dominance, subordinance, non-social defense and sexual behavior in territorial and gregarious finch species. We obtained excellent support for our predictions based on the folded open model of the PAG and further showed that birds possess functional and anatomical zones that form longitudinal columns similar to those in mammals. However, distinguishing characteristics of the dorsal/dorsolateral PAG, such as a dense peptidergic innervation, a longitudinal column of neuronal nitric oxide synthase neurons, and aggression-induced Fos responses, do not lie within the classical avian CG, but in the laterally adjacent intercollicular nucleus (ICo), suggesting that much of the ICo is homologous to the dorsal PAG.

Increased pain perception and attenuated opioid antinociception in paradoxical sleep-deprived rats are associated with reduced tyrosine hydroxylase staining in the periaqueductal gray matter and are reversed by L-dopa.

Paradoxical sleep deprivation (PSD) increases pain sensitivity and reduces morphine antinociception. Because dopaminergic neurons in the periaqueductal gray matter (PAG) participate in pain modulation and opioid-induced antinociception, we evaluated the effects of PSD on thermal pain sensitivity, morphine- and L-DOPA-induced antinociception and dopaminergic functionality in the PAG by assessing tyrosine hydroxylase (TH) immunoreactivity. Rats that were subjected to 96h of PSD received vehicle, morphine (2.5, 5 or 10mg/kg), L-DOPA (50 or 100mg/kg) or L-DOPA (50mg/kg)+morphine (2.5 and 5mg/kg) and were tested with a 46°C hot plate 1h after. The paw withdrawal latency responses to the hot plate were decreased in PSD rats and were modified by the highest dose of morphine, L-DOPA and L-DOPA+morphine. Analgesic effects were observed in control groups for all of the morphine doses as well as 100mg/kg of L-DOPA and L-DOPA (50mg/kg)+morphine (5mg/kg). The number of cell bodies that were immunopositive for TH in the PAG was reduced in PSD rats. In conclusion, increased thermal sensitivity was reversed by L-DOPA and could be caused by a reduction TH levels in the PAG. Our data also suggest a relationship between central dopaminergic networks and opiate-induced analgesia in rats.

Distinct glutaminyl cyclase expression in Edinger-Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Abeta pathology in Alzheimer's disease.

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer's disease (AD) Abeta peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger-Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger-Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Abeta immunoreactivity and morphological signs of degeneration as well as extracellular pE-Abeta deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Abeta formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Abeta formation.

Temporal changes in mouse brain fatty acid amide hydrolase activity.

Fatty acid amide hydrolase (FAAH) activity is known to mediate the tone of endogenous fatty acid amides including the endocannabinoid anandamide. FAAH is a potential therapeutic target because genetic or pharmacological ablation of FAAH promotes analgesia and anxiolytic effects without disrupting motor coordination. Little is known about the endogenous temporal fluctuations of brain FAAH activity. This is the first comprehensive study examining temporal fluctuations in mouse brain FAAH activity. Regional mouse brain homogenates were generated at the midpoint of the light ("noon") and dark ("midnight") cycles. While immunoblots revealed no significant changes (P>0.05) in regional activity between these two time points, in vitro activity assays detected a subtle 10% reduction (P<0.05) in cerebellar FAAH activity at midnight. A novel ex vivo autoradiography technique permitted the study of 11 different brain regions, many of which cannot be studied using traditional in vitro methods. The cerebellum and the periaqueductal gray both exhibited significant (P<0.05) reductions in regional FAAH activity in "midnight" brains. These data confirm the need to account for temporal changes in FAAH activity when therapeutically targeting FAAH.

Morphine-induced changes of adenylate and guanylate cyclase in locus ceruleus, periaqueductal gray, and substantia nigra in rats.

OBJECTIVES: To observe the changes of adenylate cyclase (AC) and guanylate cyclase (GC) in the cerebral regions including the locus ceruleus, periaqueductal gray, and substantia nigra in rats that were physiologically dependent on morphine. We also investigated the relationship of enzymatic changes in these cerebral regions to the mechanism of morphine dependence. METHODS: A morphine-dependent rat model was established and withdrawal symptoms evaluated. Enzyme histochemistry was used to detect the variations of AC and GC in cerebral regions. RESULTS: Compared to controls, AC and GC significantly increased in morphine-dependent groups. Comparisons of four different morphine-dependent groups also showed AC and GC significantly differed at weeks 1, 2, 4, and 8. CONCLUSIONS: Results found that the content of AC and GC increased in these cerebral regions in rats that demonstrated morphine dependence and appeared to be closely linked to increases in AC and GC activity.

NMDAR-nNOS generated zinc recruits PKCgamma to the HINT1-RGS17 complex bound to the C terminus of Mu-opioid receptors.

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCgamma, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCgamma to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCgamma-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCgamma to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCgamma with the MORs, effect that was prevented by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCgamma recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCgamma to bind to HINT1/RGSZ complex at the C terminus of the receptor.

Cat odour-induced anxiety--a study of the involvement of the endocannabinoid system.

RATIONALE: Recent evidence suggests the involvement of the endocannabinoid (EC) system in the regulation of anxiety. OBJECTIVES: The aim of present work was to study the role of the EC system in cat odour-induced anxiety in rats. Materials and methods Male Wistar rats were exposed to cat odour in home and motility cages. Exposure of rats to elevated zero-maze was used to determine changes in anxiety. Effect of rimonabant (0.3-3 mg/kg), antagonist of CB1 receptors, was studied on cat odour-induced alterations in exploratory behaviour. Real-time PCR was used to determine gene expression levels of EC-related genes in the brain. RESULTS: Anxiogenic-like action of cat odour was evident in the elevated zero-maze. Cat odour increased the expression of FAAH, the enzyme responsible for the degradation of anandamide, in the mesolimbic area. By contrast, in the amygdala and periaqueductal grey (PAG) levels of NAPE-PLD, the enzyme related to the synthesis of anandamide, and FAAH were remarkably decreased. Cat odour also decreased the expression of enzymes related to metabolism of 2-archidonoyl-glycerol in the amygdala and PAG. Pre-treatment of rats with rimonabant (0.3-3 mg/kg) reduced the exploratory behaviour of rats, but did not affect cat odour-induced changes. CONCLUSION: Exposure to cat odour induces anxiogenic-like effect on the behaviour in rats. Cat odour also causes moderate increase in expression of EC-related genes in the mesolimbic area, whereas significant down-regulation is established in the amygdala and PAG. Relation of predator odour-induced anxiety to the inhibition of the EC system in the amygdala and PAG is supported by behavioural studies where blockade of CB1 receptors by rimonabant induces anxiogenic-like action.

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons.

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [D-Ala(2), N-MePhe(4),Gly-ol(5)] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for beta-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.

[Changes of adenylate cyclase on cerebral regions related to mophine dependence in rats].

OBJECTIVE: To observe the changes of adenylate cyclase(AC) on cerebral regions related to morphine dependence in rats and investigate the relationship between the enzymological changes and the mechanism of morphine dependence. METHODS: The technique of enzyme-histochemistry was used to detect the variations of AC of special seven cerebral regions including frontalis cortex, lenticula, corpus amygdaloideun, substantia nigra, hippocampus, periaqueductal gray and locus coerleus in morphine dependent rats. The enzymological changes were observed by optical microscope. Changes of gray degree of these cerebral regions were also observed by using the image analysis system. RESULTS: Compared with those in control group, the contents of AC in morphine dependent groups were increased. CONCLUSION: The contents of AC are increase in those regions. The mechanism of morphine dependence close related to the increasing of AC. The correlation of the mechanism of morphine dependence and up-regulation of AC/cAMP-PKA system is discussed.