Differential effects of acute and prolonged morphine withdrawal on motivational and goal-directed control over reward-seeking behaviour.

Opioid addiction is a relapsing disorder marked by uncontrolled drug use and reduced interest in normally rewarding activities. The current study investigated the impact of spontaneous withdrawal from chronic morphine exposure on emotional, motivational and cognitive processes involved in regulating the pursuit and consumption of food rewards in male rats. In Experiment 1, rats experiencing acute morphine withdrawal lost weight and displayed somatic signs of drug dependence. However, hedonically driven sucrose consumption was significantly elevated, suggesting intact and potentially heightened reward processing. In Experiment 2, rats undergoing acute morphine withdrawal displayed reduced motivation when performing an effortful response for palatable food reward. Subsequent reward devaluation testing revealed that acute withdrawal disrupted their ability to exert flexible goal-directed control over reward seeking. Specifically, morphine-withdrawn rats were impaired in using current reward value to select actions both when relying on prior action-outcome learning and when given direct feedback about the consequences of their actions. In Experiment 3, rats tested after prolonged morphine withdrawal displayed heightened rather than diminished motivation for food rewards and retained their ability to engage in flexible goal-directed action selection. However, brief re-exposure to morphine was sufficient to impair motivation and disrupt goal-directed action selection, though in this case, rats were only impaired in using reward value to select actions in the presence of morphine-paired context cues and in the absence of response-contingent feedback. We suggest that these opioid-withdrawal induced deficits in motivation and goal-directed control may contribute to addiction by interfering with the pursuit of adaptive alternatives to drug use.

Emodin relieves morphine-stimulated BV2 microglial activation and inflammation through the TLR4/NF-κB/NLRP3 pathway.

The objective of this study is to disclose the role of emodin, a natural anthraquinone derivative that has been proposed to suppress microglial activation and inflammation, in morphine tolerance. Here, cell counting kit-8 method assayed the viability of BV2 microglial cells treated by ascending concentrations of emodin. In emodin-pretreated BV2 microglial cells challenged with morphine with or without transfection of toll-like receptor 4 (TLR4) overexpression plasmids, transwell assay measured cell migration. Immunofluorescence staining and western blot detected the expression of microglial markers. Inflammatory levels were subjected to ELISA and western blot. BODIPY 581/591 C11 assay estimated lipid reactive oxygen species activity. Iron assay kit examined total iron content. Western blot tested the expression of ferroptosis- and TLR4/nuclear factor-kappaB (NF-κB)/NOD-like receptor 3 (NLRP3) pathway-associated proteins. Molecular docking predicted the binding affinity of emodin to TLR4. Emodin was noted to obstruct the migration, activation, inflammatory response, and ferroptosis of BV2 microglial cells induced by morphine. In addition, emodin had a high binding affinity with TLR4 and inactivated TLR4/NF-κB/NLRP3 pathway in morphine-challenged BV2 microglial cells. Upregulation of TLR4 partially countervailed the protective role of emodin against morphine-elicited BV2 microglial cell migration, activation, inflammation, and ferroptosis. Accordingly, emodin might target TLR4 and act as an inactivator of TLR4/NF-κB/NLRP3 pathway, thus inhibiting BV2 microglial activation and inflammation to mitigate morphine tolerance.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Inhibition of endoplasmic reticulum stress attenuates morphine protracted abstinence-induced anxiety-like behaviors in the male mice.

The anxiety caused by morphine protracted abstinence is considered to be an important factor contributes to drug-seeking and relapse. Endoplasmic reticulum (ER) stress plays important roles in many kinds of mental disorders including drug addiction and anxiety, but it is unclear whether ER stress is involved in anxiety-like behaviors induced by morphine withdrawal. In this study, by using behavioral test, western blot, immunofluorescence, electron transmission microscope, we found that: (1) Inhibition of endoplasmic reticulum stress by 4-Phenylbutyric acid (4-PBA) could attenuate anxiety-like behaviors induced by morphine withdrawal. (2) The endoplasmic reticulum stress-related proteins in the lateral habenula (LHb) but not in the nucleus accumbens (NAc), ventral pallidum (VP), basolateral amygdala (BLA) and CA1 of hippocampus was upregulated by morphine withdrawal, upregulation of endoplasmic reticulum stress-related proteins in the lateral habenula induced by morphine withdrawal was inhibited by 4-PBA. (3) Endoplasmic reticulum stress-related protein CHOP and eIF2α were expressed in neurons but not in microglia in the LHb. (4) Morphine withdrawal induced neuronal morphological change in the LHb, which was attenuated by 4-PBA.

Fentanyl Analogs Exert Antinociceptive Effects via Sodium Channel Blockade in Mice.

The formalin test is one approach to studying acute pain in rodents. Similar to formalin, injection with glutamate and veratrine can also produce a nociceptive response. This study investigated whether opioid-related compounds could suppress glutamate- and veratrine-induced nociceptive responses in mice at the same dose. The administration of morphine (3 mg/kg), hydromorphone (0.4 mg/kg), or fentanyl (0.03 mg/kg) suppressed glutamate-induced nociceptive response, but not veratrine-induced nociceptive response at the same doses. However, high doses of morphine (10 mg/kg), hydromorphone (2 mg/kg), or fentanyl (0.1 mg/kg) produced a significant reduction in the veratrine-induced nociceptive response. These results indicate that high doses are required when using morphine, hydromorphone, or fentanyl for sodium channel-related neuropathic pain, such as ectopic activity. As a result, concerns have arisen about overdose and abuse if the dose of opioids is steadily increased to relieve pain. In contrast, trimebutine (100 mg/kg) and fentanyl analog isobutyrylfentanyl (iBF; 0.1 mg/kg) suppressed both glutamate- and veratrine-induced nociceptive response. Furthermore, nor-isobutyrylfentanyl (nor-iBF; 1 mg/kg), which is a metabolite of iBF, suppressed veratrine-induced nociceptive response. Besides, the optimal antinociceptive dose of iBF, unlike fentanyl, only slightly increased locomotor activity and did not slow gastrointestinal transit. Cancer pain is a complex condition driven by inflammatory, neuropathic, and cancer-specific mechanisms. Thus, iBF may have the potential to be a superior analgesic than fentanyl.

Intravenous administration of recombinant Phα1ß: Antinociceptive properties and morphine tolerance reversal in a cancer-associated pain model.

Cancer-related pain is considered one of the most prevalent symptoms for those affected by cancer, significantly influencing quality of life and treatment outcomes. Morphine is currently employed for analgesic treatment in this case, however, chronic use of this opioid is limited by the development of analgesic tolerance and adverse effects, such as digestive and neurological disorders. Alternative therapies, such as ion channel blockade, are explored. The toxin Phα1ß has demonstrated efficacy in blocking calcium channels, making it a potential candidate for alleviating cancer-related pain. This study aims to assess the antinociceptive effects resulting from intravenous administration of the recombinant form of Phα1ß (r-Phα1ß) in an experimental model of cancer-related pain in mice, tolerant or not to morphine. The model of cancer-induced pain was used to evaluate these effects, with the injection of B16F10 cells, followed by the administration of the r-Phα1ß, and evaluation of the mechanical threshold by the von Frey test. Also, adverse effects were assessed using a score scale, the rotarod, and open field tests. Results indicate that the administration of r-Phα1ß provoked antinociception in animals with cancer-induced mechanical hyperalgesia, with or without morphine tolerance. Previous administration of r-Phα1ß was able to recover the analgesic activity of morphine in animals tolerant to this opioid. r-Phα1ß was proved safe for these parameters, as no adverse effects related to motor and behavioral activity were observed following intravenous administration. This study suggests that the concomitant use of morphine and r-Phα1ß could be a viable strategy for pain modulation in cancer patients.

Analgesic effect of a cholinergic agonist (carbachol) in a sural nerve ligation-induced hypersensitivity mouse model.

OBJECTIVES: Neuropathic pain is characterized by long-lasting, intractable pain. Sciatic nerve ligation is often used as an animal model of neuropathic pain, and the spared nerve injury (SNI) model, in which the common peroneal nerve (CPN) and tibial nerve (TN) are ligated, is widely used. In the present study, we evaluated the analgesic effect of a cholinergic agonist, carbachol, on a neuropathic pain model prepared by sural nerve (SN) ligation in mice. METHODS: The SN was tightly ligated as a branch of the sciatic nerve. Mechanical and thermal allodynia, and hyperalgesia were assessed using von Frey filaments and heat from a hot plate. The analgesic effects of intracerebroventricularly-administered morphine and carbachol were compared. RESULTS: SN ligation resulted in a significant decrease in pain threshold for mechanical stimulation 1 day after ligation. In response to thermal stimulation, allodynia was observed at 50°C and hyperalgesia at 53 and 56°C 3 days after ligation. Content of thiobarbituric acid reactive substances (TBARS) in the spinal cord increased significantly at 6 and 12 h after ligation. Acetylcholine content of the spinal cord also increased at 5 and 7 days after ligation. Intracerebroventricular administration of carbachol at 7 days after ligation produced a marked analgesic effect against mechanical and thermal stimuli, which was stronger and longer-lasting than morphine at all experimental time points. CONCLUSION: These findings suggest that cholinergic nerves are involved in allodynia and hyperalgesia of the SN ligation neuropathic pain model.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

The effect of morphine administration on GluN3B NMDA receptor subunit mRNA expression in rat brain.

Opioid addiction is critically dependent on the activation of N­methyl­D­aspartate (NMDA) receptors, which are widely found in the mesocorticolimbic system. Meanwhile, opioid addiction may affect the expression level of NMDA receptor subunits. The existence of GluN3 subunits in the NMDA receptor's tetramer structure reduces the excitatory current of the receptor channel. We evaluated the changes in the mRNA expression pattern of the GluN3B subunit of the NMDA receptor in rat brains following acute and chronic exposure to morphine. Chronic, escalating intraperitoneal doses of morphine or saline were administered twice daily to male Wistar rats for six days. Two other groups were injected with a single acute dose of morphine or saline. The mRNA level of the GluN3B subunit of the NMDA receptor in the striatum, hippocampus, and nucleus accumbens (NAc) was measured by real­time PCR. mRNA expression of the GluN3B subunit was considerably augmented (3.15 fold) in the NAc of animals chronically treated with morphine compared to the control group. The difference between rats that were chronically administered morphine and control rats was not statistically significant for other evaluated brain areas. In rats acutely treated with morphine, no significant differences were found for GluN3B subunit expression in the examined brain regions compared to the control group. It was concluded that chronic exposure to morphine notably increased the GluN3B subunit of the NMDA receptor in NAc. The extent of the impact of this finding on opioid addiction and its features requires further evaluation in future studies.

Effects of morphine on conditioned place preference and pain are independent of uptake­2.

Morphine changes neurotransmitter release, including norepinephrine, dopamine, and serotonin. Decynium­22 (D22) inhibits an alternative neurotransmitter removal pathway, namely uptake­2. Uptake­2 includes plasma membrane monoamine transporter (PMAT) and organic cation transporters that have a low affinity, but high capacity for uptake of various monoamines such as norepinephrine, dopamine, and serotonin. This study was done to assess the effect of uptake­2 inhibition on morphine­induced conditioned place preference (CPP) and analgesia. In this study, the effects of morphine and/or D22 on CPP were evaluated following intraperitoneal injection in mice. Afterward, changes in motor activity were evaluated by the open field test. Using the tail­flick model, the effects of D22 and/or morphine were evaluated on the pain threshold. The results showed that 20 mg/kg of morphine induced a place preference response. D22, at the dose of 0.03 mg/kg, caused place avoidance, while at the dose of 0.3 mg/kg, it produced a notable place preference response. Co­administration of D22 and morphine showed that morphine reversed the CPP aversion induced by D22 at the lowest dose. Motor activity did not alter. In the tail­flick test, morphine, at the dose of 3 mg/kg but not 1 mg/kg, increased the pain threshold. D22 induced significant analgesic responses. Co­administration of D22 and morphine caused considerable analgesic effects. The findings revealed that D22 induced both conditioned aversion and preference depending on the dose while morphine induced CPP. Both drugs produced analgesia.

Activation of epidermal growth factor receptors in triple-negative breast cancer cells by morphine; analysis through Raman spectroscopy and machine learning.

Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer, and the analgesic drug morphine has been shown to promote the proliferation of TNBC cells. This article investigates whether morphine causes activation of epidermal growth factor receptors (EGFR), the roles of µ-opioid and EGFR receptors on TNBC cell proliferation and migration. While examining the changes with molecular techniques, we also aimed to investigate the analysis ability of Raman spectroscopy and machine learning-based approach. Effects of morphine on the proliferation and migration of MDA.MB.231 cells were evaluated by MTT and scratch wound-healing tests, respectively. Morphine-induced phosphorylation of the EGFR was analyzed by western blotting in the presence and absence of µ-receptor antagonist naltrexone and the EGFR-tyrosine kinase inhibitor gefitinib. Morphine-induced EGFR phosphorylation and cell migration were significantly inhibited by pretreatments with both naltrexone and gefitinib; however, morphine-increased cell proliferation was inhibited only by naltrexone. While morphine-induced changes were observed in the Raman scatterings of the cells, the inhibitory effect of naltrexone was analyzed with similarity to the control group. Principal component analysis (PCA) of the Raman confirmed the epidermal growth factor (EGF)-like effect of morphine and was inhibited by naltrexone and partly by gefitinib pretreatments. Our in vitro results suggest that combining morphine with an EGFR inhibitor or a peripherally acting opioidergic receptor antagonist may be a good strategy for pain relief without triggering cancer proliferation and migration in TNBC patients. In addition, our results demonstrated the feasibility of the Raman spectroscopy and machine learning-based approach as an effective method to investigate the effects of agents in cancer cells without the need for complex and time-consuming sample preparation. The support vector machine (SVM) with linear kernel automatically classified the effects of drugs on cancer cells with ∼95% accuracy.

Conditioned morphine tolerance promotes neurogenesis, dendritic remodelling and pro-plasticity molecules in the adult rat hippocampus.

Structural neuroplasticity of the hippocampus in the form of neurogenesis and dendritic remodelling underlying morphine tolerance is still less known. Therefore, in this study, we aimed to assess whether unconditioned- and conditioned-morphine tolerance can trigger structural neuroplasticity in the dorsal and ventral parts of the adult male rat hippocampus. Evaluation of the levels of neurogenesis markers (Ki67 and DCX) by immunohistochemistry shows that conditioned morphine tolerance is sufficient to increase the baseline topographic level of hippocampal neurogenesis in adult rats. Dendritic spine visualization by Golgi staining shows that the behavioural testing paradigms themselves are sufficient to trigger the hippocampus subregion-specific changes in the dendritic remodelling along the apical dendrites of hippocampal CA1 pyramidal neurons and dentate granule cells in adult rats. Quantitative reverse transcription polymerase chain reaction of Bdnf, Trkb, Rac-1 and RhoA mRNA levels as pro-plasticity molecules, shows that the conditioned morphine tolerance is effective in changing Bdnf and RhoA mRNA levels in the ventral hippocampus of adult rats. In summary, we demonstrate that the acquisition of morphine tolerance promotes adult neurogenesis, dendritic remodelling and pro-plasticity molecules such as Bdnf/Trkb in the rat hippocampus. Indeed, the structural neuroplasticity of the hippocampus may underlie the newly formed aberrant memory and could provide the initial basis for understanding the neurobiological mechanisms of morphine-tolerance plasticity in the hippocampus.

8-shogaol derived from dietary ginger alleviated acute and inflammatory pain by targeting TRPV1.

Ginger, a well-known spice plant, has been used widely in medicinal preparations for pain relief. However, little is known about its analgesic components and the underlying mechanism. Here, we ascertained, the efficacy of ginger ingredient 8-Shogaol (8S), on inflammatory pain and tolerance induced by morphine, and probed the role of TRPV1 in its analgesic action using genetic and electrophysiology approaches. Results showed that 8S effectively reduced nociceptive behaviors of mice elicited by chemical stimuli, noxious heat as well as inflammation, and antagonized morphine analgesic tolerance independent on opioid receptor function. Genetic deletion of TRPV1 significantly abolished 8S' analgesia action. Further calcium imaging and patch-clamp recording showed that 8S could specifically activate TRPV1 in TRPV1-expressing HEK293T cells and dorsal root ganglion (DRG) neurons. The increase of [Ca2+]i in DRG was primarily mediated through TRPV1. Mutational and computation studies revealed the key binding sites for the interactions between 8S and TRPV1 included Leu515, Leu670, Ile573, Phe587, Tyr511, and Phe591. Further studies showed that TRPV1 activation evoked by 8S resulted in channel desensitization both in vitro and in vivo, as may be attributed to TRPV1 degradation or TRPV1 withdrawal from the cell surface. Collectively, this work provides the first evidence for the attractive analgesia of 8S in inflammatory pain and morphine analgesic tolerance mediated by targeting pain-sensing TRPV1 channel. 8S from dietary ginger has potential as a candidate drug for the treatment of inflammatory pain.

Repeated Morphine Exposure Alters Temporoamonic-CA1 Synaptic Plasticity in Male Rat Hippocampus.

In this study, the electrophysiological and biochemical consequences of repeated exposure to morphine in male rats on glutamatergic synaptic transmission, synaptic plasticity, the expression of GABA receptors and glutamate receptors at the temporoammonic-CA1 synapse along the longitudinal axis of the hippocampus (dorsal, intermediate, ventral, DH, IH, VH, respectively) were investigated. Slice electrophysiological methods, qRT-PCR, and western blotting techniques were used to characterize synaptic plasticity properties. We showed that repeated morphine exposure (RME) reduced excitatory synaptic transmission and ability for long-term potentiation (LTP) in the VH as well as eliminated the dorsoventral difference in paired-pulse responses. A decreased expression of NR2B subunit in the VH and an increased expression GABAA receptor of α1 and α5 subunits in the DH were observed following RME. Furthermore, RME did not affect the expression of NR2A, AMPA receptor subunits, and γ2GABAA and GABAB receptors in either segment of the hippocampus. In sum, the impact of morphine may differ depending on the region of the hippocampus studied. A distinct change in the short- and long-term synaptic plasticity along the hippocampus long axis due to repeated morphine exposure, partially mediated by a change in the expression profile of glutamatergic receptor subunits. These findings can be useful in further understanding the cellular mechanism underlying deficits in information storage and, more generally, cognitive processes resulting from chronic opioid abuse.

Paynantheine more effectively reverses multidrug resistance in malignant EPG85.257RDB and MCF7MX cells than morphine and speciociliatine.

The possible interactions of morphine, paynantheine and speciociliatine alkaloids with ATP-binding cassette (ABC) transporters was investigated. The compounds were docked against ABCG2 and ABCB1 to predict the binding mode of alkaloids in active binding sites. The cytotoxicity of morphine, paynantheine and speciociliatine for EPG85.257RDB and MCF7MX cells was determined and ABCB1 and ABCG2 gene and protein expression were determined. The binding score of paynantheine to ABCB1 was higher in the docking studies. Paynantheine and speciociliatine had similar binding scores to ABCB1, but higher binding scores to ABCG2 than did morphine. Paynantheine and speciociliatine were more effective against MCF7MX and EPG85.257RDB cells and showed greater cyctotoxicity in the MTT assay. The effect of morphine and paynantheine on the ABCB1 gene and protein expression suggests these compounds can reduce resistance in cancer patients, but that speciociliatine may not be a suitable candidate because of its increased ABCB1 expression while speciociliatine decreased the expression of ABCG2 in MCF7MX cells. This indicates that speciociliatine is a better candidate for reducing drug resistance in this cell line. Structural modification, drug-metabolizing enzymes and differences in the binding sites could cause functional differences between these compounds.

Limited bedding and nesting increases ethanol drinking in female rats.

Prenatal opioid exposure (POE) and postnatal adverse experiences are early life adversities (ELA) that often co-occur and increase problematic alcohol (EtOH) drinking during adolescence. We investigated the relationship between POE, postnatal adversity, and adolescent EtOH drinking in rats. We also sought to determine whether ELAs affect alpha-adrenoceptor density in the brain because the noradrenergic system is involved in problematic alcohol drinking and its treatment. We hypothesized that the combination of POE and postnatal adversity will increase alcohol drinking in rats compared to rats with exposure to either adversity alone or to control. We also predicted that POE and postnatal adversity would increase α1-adrenoceptor density and decrease α2-adrenoceptor density in brain to confer a stress-responsive phenotype. Pregnant rats received morphine (15 mg/kg/day) or saline via subcutaneous minipumps from gestational day 9 until birth. Limited bedding and nesting (LBN) procedures were introduced from postnatal day (PD) 3-11 to mimic early life adversity-scarcity. Offspring rats (PD 31-33) were given opportunities to drink EtOH (20 %, v/v) using intermittent-access, two-bottle choice (with water) procedures. Rats given access to EtOH were assigned into sub-groups that were injected with either yohimbine (1 mg/kg, ip) or vehicle (2 % DMSO, ip) 30 min prior to each EtOH access session to determine the effects of α2-adrenoceptor inhibition on alcohol drinking. We harvested cortices, brainstems, and hypothalami from EtOH-naïve littermates on either PD 30 or PD 70 and conducted radioligand receptor binding assays to quantify α1- and α2-adrenoceptor densities. Contrary to our hypothesis, only LBN alone increased EtOH intake in female adolescent rats compared to female rats with POE. Neither POE nor LBN affected α1- or α2-adrenoceptor densities in the cortex, brainstem, or hypothalamus of early- or late-aged adolescent rats. These results suggest a complex interaction between ELA type and sex on alcohol drinking.

Evaluation of the analgesic effects of diclofenac as a premedication drug in dogs undergoing ovariohysterectomy.

Pre-emptive analgesia consists of administering drugs such as opioids and nonsteroid anti-inflammatory drugs. This study aims to evaluate the intraoperative antinociceptive effects of diclofenac administered alone in premedication or combined with morphine along with its potential influence on recovery of dogs undergoing ovariohysterectomy. A total of 34 dogs (ASA I or II) admitted for ovariohysterectomy were randomly allocated into three groups according to the drugs given in premedication: Diclofenac (D) (n = 11), Morphine (M) (n = 13) and Diclofenac-Morphine (DM) (n = 10) groups. Induction and maintenance of anesthesia were standardized in all dogs. To assess intraoperative nociception, the heart rate (HR) and mean arterial pressure (MAP) were recorded during the surgery and at predefined time points: St (steady-state), Cut (cutaneous incision), P1 (first ovarian manipulation), P2 (second ovarian manipulation) and Cerv (cervical manipulation). The dynamic variation of HR (ΔHR) and MAP (ΔMAP) over 2 min was calculated at each time point. After extubation, early quality of recovery was assessed. Compared to St, a significant increase in HR and MAP at P1, P2 and Cerv was shown in all groups. MAP in the M group was lower at St than in the other groups. The dynamic variation of HR (ΔHR) and MAP (ΔMAP) was significantly less important at P2 and Cerv compared to P1 only in the DM group. Also, a better quality of recovery was shown in the D group compared to the M and DM groups. Diclofenac may be considered a suitable premedication drug and a part of a multimodal anesthetic approach in dogs.

HIV-1 Tat and morphine interactions dynamically shift striatal monoamine levels and exploratory behaviors over time.

Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by co-morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context-dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty-seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.

The impact of multifunctional enkephalin analogs and morphine on the protein changes in crude membrane fractions isolated from the rat brain cortex and hippocampus.

Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.