Acute and Chronic Exposure to Linagliptin, a Selective Inhibitor of Dipeptidyl Peptidase-4 (DPP-4), Has an Effect on Dopamine, Serotonin and Noradrenaline Level in the Striatum and Hippocampus of Rats.

Linagliptin is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor that indirectly elevates the glucagon-like peptide-1 (GLP-1) level. The aim of the present study was to check whether linagliptin has an influence on neurotransmission in rat brain. Rats were acutely and chronically exposed to linagliptin (10 and 20 mg/kg, intraperitoneally (i.p.)). Twenty-four hours later, the striatum and hippocampus were selected for further studies. In neurochemical experiments, using high-performance liquid chromatography with electrochemical detection (HPLC-ED), the concentrations of three major neurotransmitters-dopamine, serotonin and noradrenaline-and their metabolites were measured. The analysis of mRNA expression of dopamine (D1 and D2), serotonin (5-HT-1 and 5-HT-2) and noradrenaline (α1 and α2a) receptors was also investigated using real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in the same brain areas. Linagliptin has the ability to influence the dopaminergic system. In the striatum, the elevation of dopamine and its metabolites was observed after repeated administration of that linagliptin, and in the hippocampus, a reduction in dopamine metabolism was demonstrated. Acute linagliptin exposure increases the serotonin level in both areas, while after chronic linagliptin administration a tendency for the mRNA expression of serotoninergic receptors (5-HT1A and 5-HT2A) to increase was observed. A single instance of exposure to linagliptin significantly modified the noradrenaline level in the striatum and intensified noradrenaline turnover in the hippocampus. The recognition of the interactions in the brain between DPP-4 inhibitors and neurotransmitters and/or receptors is a crucial step for finding novel discoveries in the pharmacology of DPP-4 inhibitors and raises hope for further applications of DPP-4 inhibitors in clinical practices.

The Effect of Prenatal and Neonatal Fluoride Exposure to Morphine-Induced Neuroinflammation.

Physical dependence is associated with the formation of neuroadaptive changes in the central nervous system (CNS), both at the molecular and cellular levels. Various studies have demonstrated the immunomodulatory and proinflammatory properties of morphine. The resulting neuroinflammation in drug dependence exacerbates substance abuse-related behaviors and increases morphine tolerance. Studies prove that fluoride exposure may also contribute to the development of neuroinflammation and neurodegenerative changes. Morphine addiction is a major social problem. Neuroinflammation increases tolerance to morphine, and neurodegenerative effects caused by fluoride in structures related to the development of dependence may impair the functioning of neuronal pathways, change the concentration of neurotransmitters, and cause memory and learning disorders, which implies this element influences the development of dependence. Therefore, our study aimed to evaluate the inflammatory state of selected brain structures in morphine-dependent rats pre-exposed to fluoride, including changes in cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression as well as microglial and astroglial activity via the evaluation of Iba1 and GFAP expression. We provide evidence that both morphine administration and fluoride exposure have an impact on the inflammatory response by altering the expression of COX-1, COX-2, ionized calcium-binding adapter molecule (Iba1), and glial fibrillary acidic protein (GFAP) in brain structures involved in dependence development, such as the prefrontal cortex, striatum, hippocampus, and cerebellum. We observed that the expression of COX-1 and COX-2 in morphine-dependent rats is influenced by prior fluoride exposure, and these changes vary depending on the specific brain region. Additionally, we observed active astrogliosis, as indicated by increased GFAP expression, in all brain structures of morphine-dependent rats, regardless of fluoride exposure. Furthermore, the effect of morphine on Iba1 expression varied across different brain regions, and fluoride pre-exposure may influence microglial activation. However, it remains unclear whether these changes are a result of the direct or indirect actions of morphine and fluoride on the factors analyzed.

The L-arginine-NO-cGMP pathway mediates the locomotor activity alterations during the expression of sensitization to mephedrone in mice.

Mephedrone is a representative of synthetic cathinones that is known from its rewarding and psychostimulant effects. It exerts behavioural sensitization after repeated and then interrupted administration. In our study, we investigated a role of the L-arginine-NO-cGMP-dependent signalling in the expression of sensitization to hyperlocomotion evoked by mephedrone. The study was carried out in male albino Swiss mice. The tested mice received mephedrone (2.5 mg/kg) for 5 consecutive days and on the 20th day of the experiment (the 'challenge' day) animals received both mephedrone (2.5 mg/kg) and a given substance that affects the L-arginine-NO-cGMP signalling, that is, L-arginine hydrochloride (125 or 250 mg/kg), 7-nitroindazole (10 or 20 mg/kg), L-NAME (25 or 50 mg/kg) or methylene blue (5 or 10 mg/kg). We observed that 7-nitroindazole, L-NAME and methylene blue inhibited the expression of sensitization to the mephedrone-induced hyperlocomotion. Moreover, we demonstrated that the mephedrone-induced sensitization is accompanied by lowered levels of D1 receptors and NR2B subunits in the hippocampus, whereas a concurrent administration of L-arginine hydrochloride, 7-nitroindazole and L-NAME with the mephedrone challenge dose reversed these effects. Methylene blue only reversed the mephedrone-induced effects on hippocampal levels of the NR2B subunit. Our study confirms that the L-arginine-NO-cGMP pathway contributes to mechanisms underlying the expression of sensitization to the mephedrone-evoked hyperlocomotion.

Impact of Mephedrone on Fear Memory in Adolescent Rats: Involvement of Matrix Metalloproteinase-9 (MMP-9) and N-Methyl-D-aspartate (NMDA) Receptor.

Treatment of Post-Traumatic Stress Disorder (PTSD) is complicated by the presence of drug use disorder comorbidity. Here, we examine whether conditioned fear (PTSD model) modifies the rewarding effect of mephedrone and if repeated mephedrone injections have impact on trauma-related behaviors (fear sensitization, extinction, and recall of the fear reaction). We also analyzed whether these trauma-induced changes were associated with exacerbation in metalloproteinase-9 (MMP-9) and the GluN2A and GluN2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptor expression in such brain structures as the hippocampus and basolateral amygdala. Male adolescent rats underwent trauma exposure (1.5 mA footshock), followed 7 days later by a conditioned place preference training with mephedrone. Next, the post-conditioning test was performed. Fear sensitization, conditioned fear, anxiety-like behavior, extinction acquisition and relapse were then assessed to evaluate behavioral changes. MMP-9, GluN2A and GluN2B were subsequently measured. Trauma-exposed rats subjected to mephedrone treatment acquired a strong place preference and exhibited impairment in fear extinction and reinstatement. Mephedrone had no effect on trauma-induced MMP-9 level in the basolateral amygdala, but decreased it in the hippocampus. GluN2B expression was decreased in the hippocampus, but increased in the basolateral amygdala of mephedrone-treated stressed rats. These data suggest that the modification of the hippocampus and basolateral amygdala due to mephedrone use can induce fear memory impairment and drug seeking behavior in adolescent male rats.

Linagliptin, a Selective Dipeptidyl Peptidase-4 Inhibitor, Reduces Physical and Behavioral Effects of Morphine Withdrawal.

(1) Background: Recent data indicate that receptors for GLP-1 peptide are involved in the activity of the mesolimbic system. Thus, the purpose of the present study was to examine the effect of the selective dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on morphine dependence in mice. (2) Methods: Morphine dependence in mice was obtained by administration of increasing doses of morphine for eight consecutive days, twice a day. On the 9th day of the experiment, the naloxone-induced (2 mg/kg, ip) morphine withdrawal signs (jumping) were assessed. Moreover, behavioral effects of short-term (60 h after morphine discontinuation) and long-term (14 days after morphine discontinuation) morphine withdrawal were observed. In terms of behavioral effects, the depressive effect in the forced swim test and anxiety in the elevated plus maze test were investigated. Locomotor activity of mice was also studied. (3) Results: The administration of linagliptin (10 and 20 mg/kg, ip) for 8 consecutive days before morphine injections significantly diminished the number of naloxone-induced morphine withdrawal signs (jumping) in mice. In addition, the cessation of morphine administration induced depressive behavior in mice which were observed during short- and long-term morphine withdrawal. Linagliptin administered during morphine withdrawal significantly reduced the depressive behavior in studied mice. Furthermore, the short-term morphine withdrawal evoked anxiety which also was reduced by linagliptin in mice. (4) Conclusions: The present study reveals that GLP-1 receptors are involved in morphine dependence. What is more, linagliptin might be a valuable drug in attenuating the physical symptoms of morphine dependence. It might be also a useful drug in reducing emotional disturbances which may develop during the morphine withdrawal period.

Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9).

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.

Chronic and Cycling Hypoxia: Drivers of Cancer Chronic Inflammation through HIF-1 and NF-κB Activation: A Review of the Molecular Mechanisms.

Chronic (continuous, non-interrupted) hypoxia and cycling (intermittent, transient) hypoxia are two types of hypoxia occurring in malignant tumors. They are both associated with the activation of hypoxia-inducible factor-1 (HIF-1) and nuclear factor κB (NF-κB), which induce changes in gene expression. This paper discusses in detail the mechanisms of activation of these two transcription factors in chronic and cycling hypoxia and the crosstalk between both signaling pathways. In particular, it focuses on the importance of reactive oxygen species (ROS), reactive nitrogen species (RNS) together with nitric oxide synthase, acetylation of HIF-1, and the action of MAPK cascades. The paper also discusses the importance of hypoxia in the formation of chronic low-grade inflammation in cancerous tumors. Finally, we discuss the effects of cycling hypoxia on the tumor microenvironment, in particular on the expression of VEGF-A, CCL2/MCP-1, CXCL1/GRO-α, CXCL8/IL-8, and COX-2 together with PGE2. These factors induce angiogenesis and recruit various cells into the tumor niche, including neutrophils and monocytes which, in the tumor, are transformed into tumor-associated neutrophils (TAN) and tumor-associated macrophages (TAM) that participate in tumorigenesis.

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60-70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit's up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.

Effects of Mephedrone and Amphetamine Exposure during Adolescence on Spatial Memory in Adulthood: Behavioral and Neurochemical Analysis.

A synthetic cathinone, mephedrone is widely abused by adolescents and young adults. Despite its widespread use, little is known regarding its long-term effects on cognitive function. Therefore, we assessed, for the first time, whether (A) repeated mephedrone (30 mg/kg, i.p., 10 days, once a day) exposure during adolescence (PND 40) induces deleterious effects on spatial memory and reversal learning (Barnes maze task) in adult (PND 71-84) rats and whether (B) these effects were comparable to amphetamine (2.5 mg/kg, i.p.). Furthermore, the influence of these drugs on MMP-9, NMDA receptor subunits (GluN1, GluN2A/2B) and PSD-95 protein expression were assessed in adult rats. The drug effects were evaluated at doses that per se induce rewarding/reinforcing effects in rats. Our results showed deficits in spatial memory (delayed effect of amphetamine) and reversal learning in adult rats that received mephedrone/amphetamine in adolescence. However, the reversal learning impairment may actually have been due to spatial learning rather than cognitive flexibility impairments. Furthermore, mephedrone, but not amphetamine, enhanced with delayed onset, MMP-9 levels in the prefrontal cortex and the hippocampus. Mephedrone given during adolescence induced changes in MMP-9 level and up-regulation of the GluN2B-containing NMDA receptor (prefrontal cortex and hippocampus) in young adult (PND 63) and adult (PND 87) rats. Finally, in adult rats, PSD-95 expression was increased in the prefrontal cortex and decreased in the hippocampus. In contrast, in adult rats exposed to amphetamine in adolescence, GluN2A subunit and PSD-95 expression were decreased (down-regulated) in the hippocampus. Thus, in mephedrone-but not amphetamine-treated rats, the deleterious effects on spatial memory were associated with changes in MMP-9 level. Because the GluN2B-containing NMDA receptor dominates in adolescence, mephedrone seems to induce more harmful effects on cognition than amphetamine does during this period of life.

Effects of the Positive Allosteric Modulator of Metabotropic Glutamate Receptor 5, VU-29, on Maintenance Association between Environmental Cues and Rewarding Properties of Ethanol in Rats.

: Metabotropic glutamate subtype 5 (mGlu5) receptors are implicated in various forms of synaptic plasticity, including drugs of abuse. In drug-addicted individuals, associative memories can drive relapse to drug use. The present study investigated the potential of the mGlu5 receptor positive allosteric modulator (PAM), VU-29 (30 mg/kg, i.p.), to inhibit the maintenance of a learned association between ethanol and environmental context by using conditioned place preference (CPP) in rats. The ethanol-CPP was established by the administration of ethanol (1.0 g/kg, i.p. × 10 days) using an unbiased procedure. Following ethanol conditioning, VU-29 was administered at various post-conditioning times (ethanol free state at the home cage) to ascertain if there was a temporal window during which VU-29 would be effective. Our experiments indicated that VU-29 did not affect the expression of ethanol-induced CPP when it was given over two post-conditioning days. However, the expression of ethanol-CPP was inhibited by 10-day home cage administration of VU-29, but not by first 2-day or last 2-day injection of VU-29 during the 10-day period. These findings reveal that VU-29 can inhibit the maintenance of ethanol-induced CPP, and that treatment duration contributes to this effect of VU-29. Furthermore, VU-29 effect was reversed by pretreatment with either MTEP (the mGlu5 receptor antagonist), or MK-801 (the N-methyl-D-aspartate-NMDA receptor antagonist). Thus, the inhibitory effect of VU-29 is dependent on the functional interaction between mGlu5 and NMDA receptors. Because a reduction in ethanol-associated cues can reduce relapse, mGlu5 receptor PAM would be useful for therapy of alcoholism. Future research is required to confirm the current findings.

Fluoride Affects Dopamine Metabolism and Causes Changes in the Expression of Dopamine Receptors (D1R and D2R) in Chosen Brain Structures of Morphine-Dependent Rats.

Disturbances caused by excess or shortages of certain elements can affect the cerebral reward system and may therefore modulate the processes associated with the development of dependence as was confirmed by behavioural studies on animals addicted to morphine. Earlier publications demonstrated and proved the neurodegenerative properties of both low and high doses of fluoride ions in animal experiments and in epidemiological and clinical studies. The aim of the experiments conducted in the course of the present study was to analyse the effect of pre- and postnatal exposure to 50 ppm F- on the initiation/development of morphine dependence. For this purpose, the following were conducted: behavioural studies, the analysis of concentrations of dopamine and its metabolites, and the analyses of mRNA expression and dopamine receptor proteins D1 and D2 in the prefrontal cortex, striatum, hippocampus, and cerebellum of rats. In this study, it was observed for the first time that pre- and postnatal exposure to fluoride ions influenced the phenomenon of morphine dependence in a model expressing withdrawal symptoms. Behavioural, molecular, and neurochemical studies demonstrated that the degenerative changes caused by toxic activity of fluoride ions during the developmental period of the nervous system may impair the functioning of the dopaminergic pathway due to changes in dopamine concentration and in dopamine receptors. Moreover, the dopaminergic disturbances within the striatum and the cerebellum played a predominant role as both alterations of dopamine metabolism and profound alterations in striatal D1 and D2 receptors were discovered in these structures. The present study provides a new insight into a global problem showing direct associations between environmental factors and addictive disorders.

Modification of NO-cGMP Pathway Differentially Affects Diazepam- and Flunitrazepam-Induced Spatial and Recognition Memory Impairments in Rodents.

This study investigated the influence of sildenafil and methylene blue (MB), two modulators of the nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway on amnesic effects of two benzodiazepines (BZs) (diazepam (DZ) and flunitrazepam (FNZ)), in rodents-mice and rats. In the modified elevated plus maze (mEPM) and novel object recognition (NOR) tests, MB given ip at a dose of 5 mg/kg 5 min prior to DZ administration (0.25 or 1 mg/kg, sc) enhanced/induced memory impairment caused by DZ. When MB (2.5, 5, and 10 mg/kg) was applied 5 min prior to FNZ administration (0.05 and 0.1 mg/kg), an effect was opposite and memory impairment induced by FNZ was reduced. When sildenafil (2.5 and 5 mg/kg, ip) was applied 5 min prior to DZ, we observed a reduction of DZ-induced memory deficiency in the mEPM test. A similar effect of sildenafil was shown in the NOR test when the drug was applied at doses of 1.25, 2.5, and 5 mg/kg prior to DZ. In the mEPM test, sildenafil at abovementioned doses had no effects on FNZ-induced memory impairment. In turns, sildenafil administered at doses of 2.5 and 5 mg/kg increased the effect of FNZ on memory impairment in the NOR test. In conclusion, the NO-cGMP pathway is involved differentially into BZs-induced spatial and recognition memory impairments assessed using the NOR and mEPM tests. Modulators of the NO-cGMP pathway affect animal behavior in these tests in a different way depending on what benzodiazepine is applied.

The role of linagliptin, a selective dipeptidyl peptidase-4 inhibitor, in the morphine rewarding effects in rats.

Linagliptin is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor which suppresses the rapid degradation of endogenous glucagon-like peptide-1 (GLP-1). In clinical practice, it is used as an antidiabetic drug, but recent studies have confirmed its role in the activity of the central nervous system (CNS). The reported study focused on the role of linagliptin (10 and 20 mg/kg, ip) in the morphine rewarding effect, analyzing how the agent had influenced the conditioned place preference (CPP) in rats via the expression, acquisition, extinction and reinstatement of the morphine rewarding effect. The obtained results clearly demonstrated linagliptin to inhibit the expression and acquisition, to accelerate the extinction and, eventually, to reduce the reinstatement of morphine-induced CPP. The undertaken experiments significantly extended our knowledge on the mechanisms behind the morphine rewarding effect.

Phosphodiesterase inhibitors say NO to Alzheimer's disease.

Phosphodiesterases (PDEs) consisted of 11 subtypes (PDE1 to PDE11) and over 40 isoforms that regulate levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), the second messengers in cell functions. PDE inhibitors (PDEIs) have been attractive therapeutic targets due to their involvement in diverse medical conditions, e.g. cardiovascular diseases, autoimmune diseases, Alzheimer's disease (AD), etc. Among them; AD with a complex pathology is a progressive neurodegenerative disorder which affect mostly senile people in the world and only symptomatic treatment particularly using cholinesterase inhibitors in clinic is available at the moment for AD. Consequently, novel treatment strategies towards AD are still searched extensively. Since PDEs are broadly expressed in the brain, PDEIs are considered to modulate neurodegenerative conditions through regulating cAMP and cGMP in the brain. In this sense, several synthetic or natural molecules inhibiting various PDE subtypes such as rolipram and roflumilast (PDE4 inhibitors), vinpocetine (PDE1 inhibitor), cilostazol and milrinone (PDE3 inhibitors), sildenafil and tadalafil (PDE5 inhibitors), etc have been reported showing encouraging results for the treatment of AD. In this review, PDE superfamily will be scrutinized from the view point of structural features, isoforms, functions and pharmacology particularly attributed to PDEs as target for AD therapy.

The Mechanisms Involved in Morphine Addiction: An Overview.

Opioid use disorder is classified as a chronic recurrent disease of the central nervous system (CNS) which leads to personality disorders, co-morbidities and premature death. It develops as a result of long-term administration of various abused substances, along with morphine. The pharmacological action of morphine is associated with its stimulation of opioid receptors. Opioid receptors are a group of G protein-coupled receptors and activation of these receptors by ligands induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or ß-arrestin. The present review focuses on major mechanisms which currently are considered as essential in morphine activity and dependence and may be important for further studies.

Impact of the metabotropic glutamate receptor7 (mGlu7) allosteric agonist, AMN082, on fear learning and memory and anxiety-like behavior.

The present study was conducted to evaluate the influence of AMN082, the metabotropic glutamate receptor subtype 7 (mGlu7) allosteric agonist on different stages of memory processes connected with fear conditioning in the passive avoidance (PA) learning task in mice and negative emotional state (anxiety-like) induced by ethanol- and morphine-withdrawal in the elevated plus maze (EPM) test in rats. To perform the PA test, AMN082 (1.25, 2.5 and 5 mg/kg, i. p.) was injected to interfere with (or inhibit) acquisition, consolidation, and retrieval processes. The retention latency in each group was recorded using a step-through passive avoidance task 24 h after training. In turn, in ethanol- and morphine-withdrawal rats, the influence of AMN082 on anxiety-like behavior was estimated in the EPM test 24 h- (ethanol) and 72- h (morphine) after the last dose of repeated drug administrations. In all experimental groups, AMN082 at the dose of 5 mg/kg significantly decreased the step-through latency of long-term memory in the PA task. These AMN082 effects were reversed by MMPIP (10 mg/kg), the antagonist of mGlu7 receptor. AMN082 (2.5 and 5 mg/kg) also decreased ethanol- and morphine withdrawal-induced anxiety-like behavior in the EPM test, and this AMN082 (5 mg/kg) effect was counteracted by MMPIP pretreatment. Taken together, the results show that mGlu7 is involved in fear learning to the context and anxiety-like state connected with unpleasant experiences after ethanol- and morphine withdrawal in rodents. However, it appears that functional dissociation exists between these two AMN082 effects.

‬The expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures during morphine withdrawal in rats.

Morphine is one of the most potent analgesics used in medicine and it's long-term use is associated with the risk of the state of dependence. The cessation of chronic morphine administration leads to withdrawal signs which are associated with neurotransmitter dysregulations within mesolimbic system. Adenosine 5'-triphosphate (ATP) and purinergic system play an important role in the activity of central nervous system (CNS). Purinergic receptors are widely distributed in neurons and glial cells throughout the CNS taking part in integration of functional activity between neurons, glial and vascular cells. In the present study the mRNA and protein expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures (striatum, hippocampus and prefrontal cortex) during morphine withdrawal in rats was investigated by RT-PCR and Western Blot analysis. Two experimental models of morphine withdrawal were studied: single and repeated morphine withdrawal. We demonstrated that expression of P2X4 and P2X7 receptors was altered during morphine withdrawal period in rats. These alterations were varied in particular mesolimbic areas depending on the scheme of morphine administration. Our results extend the current knowledge on morphine withdrawal and for the first time high-light interactions between purinergic system and morphine withdrawal. It seems, the purinergic system may be a new, valuable tool in searching for a new strategy of management of opioid dependence.

SB-334867 (an Orexin-1 Receptor Antagonist) Effects on Morphine-Induced Sensitization in Mice-a View on Receptor Mechanisms.

The present study focused upon the role of SB-334867, an orexin-1 receptor antagonist, in the acquisition of morphine-induced sensitization to locomotor activity in mice. Behavioral sensitization is an enhanced systemic reaction to the same dose of an addictive substance, which assumingly increases both the desire for the drug and the risk of relapse to addiction. Morphine-induced sensitization in mice was achieved by sporadic doses (five injections every 3 days) of morphine (10 mg/kg, i.p.), while a challenge dose of morphine (10 mg/kg) was injected 7 days later. In order to assess the impact of orexin system blockade on the acquisition of sensitization, SB-334867 was administered before each morphine injection, except the morphine challenge dose. The locomotor activity test was performed on each day of morphine administration. Brain structures (striatum, hippocampus, and prefrontal cortex) were collected after behavioral tests for molecular experiments in which mRNA expression of orexin, dopamine, and adenosine receptors was explored by the qRT-PCR technique. Additionally, the mRNA expression of markers, such as GFAP and Iba-1, was also analyzed by the same technique. SB-334867 inhibited the acquisition of morphine-induced sensitization to locomotor activity of mice. Significant alterations were observed in mRNA expression of orexin, dopamine, and adenosine receptors and in the expression of GFAP and Iba-1, showing a broad range of interactions in the mesolimbic system among orexin, dopamine, adenosine, and glial cells during behavioral sensitization. Summing up, the orexin system may be an effective measure to inhibit morphine-induced behavioral sensitization.

Effects of the Positive Allosteric Modulator of Metabotropic Glutamate Receptor 5, VU-29, on Impairment of Novel Object Recognition Induced by Acute Ethanol and Ethanol Withdrawal in Rats.

Glutamate is essential for learning and memory processes, and acute and chronic exposures to ethanol (or protracted abstinence) alter glutamatergic transmission. In the current study, we investigated the effects of VU-29, positive allosteric modulator of metabotropic glutamate 5 (mGlu5) receptor, on the acute ethanol- and ethanol withdrawal-induced impairment of novel object recognition (NOR) task in rats. The influence of VU-29 (30 mg/kg) on memory retrieval was measured (a) at 4-h delay after acute ethanol administration, as well as (b) after acute withdrawal (24 and 48 h) of repeated (2.0 g/kg, once daily for 7 days) ethanol administration. Additionally, the effects of VU-29 on expression of mGlu5 and mGlu2 receptor proteins in the hippocampus, prefrontal cortex, and striatum were determined 48 h after ethanol withdrawal. Our results indicated that VU-29, given before acute ethanol administration, prevented the ethanol-induced impairments in spatial memory retrieval. Furthermore, VU-29 given before the testing session on the first day of abstinence facilitated NOR performance in ethanol-withdrawn rats at 4- and 24-h delay after administration. Our ELISA results show that VU-29 normalized ethanol withdrawal induced increase in expression of mGlu5 receptor protein in the hippocampus, prefrontal cortex, and striatum, as well as expression of mGlu2 receptor protein in the hippocampus. Thus, results from our study indicate that positive modulation of mGlu5 receptor prevented and reversed ethanol-induced memory impairment. Moreover, mGlu5 (hippocampus, prefrontal cortex, and striatum) and mGlu2 (hippocampus) receptors play an important role in the ethanol-induced recognition memory impairment induced by ethanol withdrawal.

ADX-47273, a mGlu5 receptor positive allosteric modulator, attenuates deficits in cognitive flexibility induced by withdrawal from 'binge-like' ethanol exposure in rats.

Repeated exposure to and withdrawal from ethanol induces deficits in spatial reversal learning. Data indicate that metabotropic glutamate 5 (mGlu5) receptors are implicated in synaptic plasticity and learning and memory. These receptors functionally interact with N-methyl-d-aspartate (NMDA) receptors, and activation of one type results in the activation of the other. We examined whether (S)-(4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)-piperidin-1-yl (ADX-47273), a positive allosteric modulator (PAM) of mGlu5 receptor, attenuates deficits in reversal learning induced by withdrawal (11-13days) from 'binge-like' ethanol input (5.0g/kg, i.g. for 5days) in the Barnes maze (a spatial learning) task in rats. We additionally examined the effects of ADX-47273 on the expression of the NMDA receptors subunit, GluN2B, in the hippocampus and prefrontal cortex, on the 13th day of ethanol withdrawal. Herein, withdrawal from repeated ethanol administration impaired reversal learning, but not the probe trial. Moreover, ADX-47273 (30mg/kg, i.p.) given prior to the first reversal learning trial for 3days in the Barnes maze, significantly enhanced performance in the ethanol-treated group. The 13th day of ethanol abstinence decreased the expression of the GluN2B subunit in the selected brain regions, but ADX-47273 administration increased it. In conclusion, positive allosteric modulation of mGlu5 receptors recovered spatial reversal learning impairment induced by withdrawal from 'binge-like' ethanol exposure. Such effect seems to be correlated with the mGlu5 receptors mediated potentiation of GluN2B-NMDA receptor mediated responses in the hippocampus and prefrontal cortex. Thus, our results emphasize the role of mGlu5 receptor PAM in the adaptive learning impaired by ethanol exposure.