Targeting PI3K by Natural Products: A Potential Therapeutic Strategy for Attention-deficit Hyperactivity Disorder.

Attention-Deficit Hyperactivity Disorder (ADHD) is a highly prevalent childhood psychiatric disorder. In general, a child with ADHD has significant attention problems with difficulty concentrating on a subject and is generally associated with impulsivity and excessive activity. The etiology of ADHD in most patients is unknown, although it is considered to be a multifactorial disease caused by a combination of genetics and environmental factors. Diverse factors, such as the existence of mental, nutritional, or general health problems during childhood, as well as smoking and alcohol drinking during pregnancy, are related to an increased risk of ADHD. Behavioral and psychological characteristics of ADHD include anxiety, mood disorders, behavioral disorders, language disorders, and learning disabilities. These symptoms affect individuals, families, and communities, negatively altering educational and social results, strained parent-child relationships, and increased use of health services. ADHD may be associated with deficits in inhibitory frontostriatal noradrenergic neurons on lower striatal structures that are predominantly driven by dopaminergic neurons. Phosphoinositide 3-kinases (PI3Ks) are a conserved family of lipid kinases that control a number of cellular processes, including cell proliferation, differentiation, migration, insulin metabolism, and apoptosis. Since PI3K plays an important role in controlling the noradrenergic neuron, it opens up new insights into research on ADHD and other developmental brain diseases. This review presents evidence for the potential usefulness of PI3K and its modulators as a potential treatment for ADHD.

Metformin loaded phosphatidylserine nanoliposomes improve memory deficit and reduce neuroinflammation in streptozotocin-induced Alzheimer's disease model.

Alzheimer's disease (AD) is closely associated with neuroinflammation development in the brain. Co-delivery of metformin (MET) with phosphatidylserine liposomes neuroprotectant may be beneficial in ameliorating AD-related symptoms like memory impairment and inflammation. Therefore, we aimed to prepare metformin containing phosphatidylserine nanoliposomes formulation (MET-PSL) and to evaluate its effect on rats subjected to AD. Alzheimer's disease model was induced by bilateral intracerebroventricular injection of streptozotocin (3 mg/kg) into rat brains using the stereotactic technique. MET-PSL, MET, and PSL alone were administered intraperitoneally to AD-induced animals and factors including learning and memory storage in addition to cytokine and tissue inflammatory changes were evaluated after a 22-day experiment period. The learning and memory parameters significantly (P < 0.05) improved in AD-rats treated with MET-PSL. Moreover, MET-PSL administration significantly (P < 0.05) decreased cytokine levels of IL1-ß, TNF-α, and TGF-ß in hippocampal tissues of rats with AD. Histological results indicated a considerable reduction in inflammatory and necrotic neural cells along with significantly (P < 0.05) increased neurogenesis in MET-PSL treated rats. Furthermore, our results showed that MET-PSL formulation could potentially act better than the free form of MET and PSL alone in the recovery process of rats with AD. In general, our data suggest that combination therapy of metformin loaded phosphatidylserine liposomes may enhance the therapeutic performance in AD patients of a clinical study.

Effect of pretreatment with intracerebroventricular injection of minocycline on morphine-induced memory impairment in passive avoidance test: Role of P-CREB and c-Fos expression in the dorsal hippocampus and basolateral amygdala regions.

Minocycline as a member of the tetracycline family is a lipophilic broad-spectrum antibiotic, which can display some non-antibiotic properties such as antioxidant, antiapoptosis, neuroprotection and modulation of pharmacological traits of drugs of abuse (ie, reward, sensitization and/or analgesia). Thus, the aim of the present study was to investigate the effect of intracerebroventricular (ICV) injection of minocycline on morphine-induced memory impairment and motor function in male Wistar rats. The behavioural responses were measured by a passive avoidance test for evaluating memory, and in the open field for studying motor function. Furthermore, the expression of Phospho-cAMP response element-binding protein (P-CREB) and c-Fos were assessed using immunohistochemistry in the dorsal hippocampus and basolateral amygdala (BLA). Our results showed that morphine dose-dependently impairs memory consolidation, but not motor function. Maximum effect was achieved with morphine at dose of 5 mg/kg. Pretreatment with ICV injection of minocycline (50 µg/rat) prevented morphine-induced memory impairment, but there was no effect on motor function. The results of immunohistochemistry analysis demonstrated that morphine decreased expression of P-CREB positive cells compared to saline control group in the BLA, but not in the dorsal hippocampus. On the other hand, pretreatment of animals with ICV injection of minocycline increased the expression of P-CREB in both brain areas. Moreover, there was no significant change in the expression of c-Fos positive cells in above-mentioned regions. In summary, our results indicated that pretreatment with ICV injection of minocycline prevented morphine-induced memory impairment and increased P-CREB expression in the dorsal hippocampus and BLA, which may explain its memory improvement property.

In vitro evaluation of effects of metformin on morphine and methadone tolerance through mammalian target of rapamycin signaling pathway.

The chronic use of opioids leads to tolerance, psychological, and physical dependence that limits their use as an effective long-term pain control. Several studies have shown that mammalian target of rapamycin (mTOR) plays a crucial role in the development of opioid tolerance. Metformin activates 5' adenosine monophosphate-activated protein kinase (AMPK) which directly suppresses the mTOR complex 1 signaling pathway. On the other hand, metformin can also inhibit mTOR directly and in an AMPK-independent manner. Thus, in the current study, we aimed to investigate the effects of metformin on the development of morphine and/or methadone-induced tolerance in human glioblastoma (T98G) cell line. We examined the effects of chronic treatment of morphine and/or methadone in the presence or absence of metformin with or without AMPK inhibitor (dorsomorphin hydrochloride) on levels of nitric oxide and cyclic adenosine monophosphate (cAMP), phosphorylated and dephosphorylated ribosomal protein S6 kinase ß-1 (S6K1) and 4E-binding protein 1 (4E-BP1) in T98G cells. Pretreatment of cells with metformin (40 µM) with or without AMPK inhibitor (dorsomorphin hydrochloride; 1 µM) before adding of morphine (2.5 µM) or methadone (1 µM) revealed a protective effects on the development of opioid tolerance. Prior administration of metformin reversed the elevation of nitric oxide levels induced by morphine (p < 0.001) and methadone (p < 0.001) and also prevented the raise of cAMP levels induced by morphine in T98G cells (p < 0.05). Contribution of mTOR signaling pathway in metformin-induced effect was shown by the inhibition of phosphorylation of S6K1 and 4E-BP1, the downstream targets of mTOR. mTOR activation suppresses opioid-induced antinociception, and its activity has also been increased during opioid tolerance.

Activation of TRPV1 receptors affects memory function and hippocampal TRPV1 and CREB mRNA expression in a rat model of biliary cirrhosis.

OBJECTIVE: Memory impairment induced by biliary cirrhosis is associated with abnormalities in the function of different neurotransmitter systems. However, the exact molecular mechanisms involved in the learning and memory dysfunctions following biliary cirrhosis is largely unknown. This study set out to determine whether activation of transient receptor potential vanilloid type 1 (TRPV1) in the CA1 area of the hippocampus in rats improve memory impairment induced by biliary cirrhosis. METHODS: To assess learning and memory, passive avoidance task was carried out using a shuttle box. The mRNA expression of TRPV1 and cAMP response element binding (CREB) protein in the hippocampus were also evaluated by qT-PCR. RESULTS: Our results indicated that activation of TRPV1 channels by capsaicin significantly decreased memory impairment and increased mRNA expression of the TRPV1 and CREB in the hippocampus of rats with biliary cirrhosis. Our findings also demonstrated that a positive correlation existed between mRNA expression of TRPV1 and CREB, and between memory function and TRPV1 expression. DISCUSSION: Taken together, the results of this study support the view that TRPV1 receptor may play an important role in the regulation of learning and memory functions, and suggest that activation of TRPV1 channels seems to be a promising therapeutic target for learning and memory impairments following biliary cirrhosis.

Foeniculum vulgare essential oil ameliorates acetic acid-induced colitis in rats through the inhibition of NF-kB pathway.

AIM: The aim of the present study is to investigate the protective effects of Foeniculum vulgare essential oil on intestinal inflammation through the inhibition of NF-kB pathway in acetic acid-induced rat colitis. METHODS: Acute colitis was induced by intra-rectal administration of 2 mL of diluted acetic acid (4%) solution. Two hours after the induction of colitis, 0.2% tween 80 in normal saline, dexamethasone (2 mg/kg) and F. vulgare essential oil (100, 200, 400 mg/kg) were administered to the animals by oral gavage and continued for 5 consecutive days. Assessment of macroscopic and microscopic lesions was done. MPO activity was evaluated by biochemical method. Furthermore, TNF-α activity was detected by immunohistochemistry (IHC) and the expression level of p-NF-kB p65 protein was measured by western blot analysis. RESULTS: Dexamethasone and F. vulgare essential oil (200, 400 mg/kg) reduced the macroscopic and microscopic lesions compared to the acetic acid group (p < 0.01, p < 0.001). In addition, these agents decreased the activity of MPO (p < 0.01, p < 0.001) and the expression of TNF-α positive cells (p < 0.05, p < 0.01, p < 0.001) in the colon tissue compared to acetic acid group. Furthermore, they inhibited acetic acid-induced expression of p-NF-kB p65 protein (p < 0.05, p < 0.001). CONCLUSION: It is proposed that the anti-inflammatory activity of F. vulgare essential oil on acetic acid-induced colitis in rats may involve the inhibition of NF-kB pathway.

Therapeutic Effect of Scrophularia striata Ethanolic Extract against Localized Cutaneous Leishmaniasis Caused by Leishmania major (MRHO/IR/75/ER).

BACKGROUND: We evaluated the effect of the ethanolic extract of Scrophularia striata on the Iranian strain of Leishmania major (MRHO/IR/75/ER) both in vitro and in vivo conditions. METHODS: The effective dose (ED) of ethanolic extract of S. striata were determined using MTT assay on the growth of promastigote forms of L. major in axenic culture media. Then, the ED50 of S. striata on mice peritoneal macrophages was determined using calculation of amastigote forms on mice peritoneal macrophages. For in vivo experiments, the therapeutic effects of various concentrations of S. striata on infected BALB/c mice was studied. A total of 75 infected mice were randomly divided into five groups: two groups (10% and 50% of S. striata) as experimental and three as control (ethanol 50%, Glucantime® and no treatment). The efficacy were determined by comparing the diameters of lesions and the microscopically examinations. RESULTS: The effect of S. striata extract (0/625%, 1/25%, 2/5%, 5%, 10%, 20% and 50%) on peritoneal macrophages of Balb/c mice infected with L.major in tissue-culture slides was assessed. S. striata extract (10%) removed the L.major amastigotes-infected macrophages significantly after 24 h (P < 0.05). The higher concentrations of S. striata ethanolic extract (20%, and 50%) had highly toxic effects on macrophages, resulted in the disintegration of the cytoplasm of macrophages after 48 and 72 h. In concentration 10% of S. striata, more than 85% of L. major amastigotes-infected macrophages were damaged without cytotoxicity effects on macrophages. The higher concentrations had toxic effects on cultured macrophages. CONCLUSION: S. striata ethanolic extract 10% had anti leishmanial effects in both in vivo and in vitro.

Modulatory effects of the basolateral amygdala α2-adrenoceptors on nicotine-induced anxiogenic-like behaviours of rats in the elevated plus maze.

The present study was designed to clarify whether α2-adrenoceptors of the basolateral amygdala (BLA) are involved in nicotine-induced anxiogenic-like behaviours. Adult male Wistar rats were bilaterally cannulated in the BLA and anxiety-like behaviours were assessed in an elevated plus maze (EPM) task. Systemic intraperitoneal (i.p.) administration of nicotine (0.3, 0.5 and 0.7 mg/kg) dose-dependently decreased open arm time (%OAT) and open arm entry (%OAE), indicating the anxiogenic-like effect of nicotine. The activation of the BLA α2-adrenoceptors by the injection of α2-receptor agonist, clonidine (0.1, 0.3 and 0.5 µg/rat) into the BLA (intra-BLA) reversed nicotine-induced anxiogenic-like behaviours. It is important to note that intra-BLA injection of a higher dose of clonidine (0.5 µg/rat) by itself increased %OAT, but not %OAE which showed an anxiolytic effect of the agonist. On the other hand, intra-BLA injection of different doses of α2-adrenoceptor antagonist, yohimbine (1, 3 and 5 µg/rat) in combination with an ineffective dose of nicotine (0.3 mg/kg) decreased %OAT and %OAE, suggesting a potentiative effect of the antagonist on nicotine response. In addition, intra-BLA injection of the same doses of yohimbine did not alter %OAT and %OAE. Interestingly, intra-BLA injection of yohimbine (0.5 and 1 µg/rat) significantly reversed the inhibitory effect of clonidine on nicotine-induced anxiogenic-like behaviours. It should be considered that the drug treatments had no effect on locomotor activity in all experiments. Taken together, it can be concluded that nicotine produces anxiogenic-like behaviours which may be mediated through the BLA α2-adrenoceptor mechanism.

Anti-inflammatory effects of eugenol nanoemulsion as a topical delivery system.

Eugenol is the main constituent of clove oil with anti-inflammatory properties. In this work, for the first time, O/W nanoemulsion of eugenol was designed for the evaluation of anti-inflammatory effects as a topical delivery system. Topical formulations containing 1%, 2% and 4% of eugenol as well as a nanoemulsion system containing 4% eugenol and 0.5% piroxicam were prepared. Further to physicochemical examinations, such as determination of particle size, polydispersity index, zeta potential and physical stability, anti-inflammatory activity was examined in carrageenan-induced paw edema in rats. The optimum formulation was found to contain 2% eugenol (oil phase), 14% Tween 20 (surfactant) and 14% isopropyl alcohol (co-surfactant) in water. Nanoemulsion with polydispersity index of 0.3 and median droplet diameter of 24.4 nm (d50) was obtained. Animal studies revealed that the nanoemulsions exhibited significantly improved anti-inflammatory activity after 1.5 h, compared with marketed piroxicam gel. Additionally, it was shown that increasing the concentration of eugenol did not show higher inhibition of inflammation. Also, the nanoemulsion having piroxicam showed less anti-inflammatory properties compared with the nanoemulsion without piroxicam.

Cyclic AMP pathway modifies memory through neural cell adhesion molecule alterations in the rat hippocampus.

Neural Cell Adhesion Molecules (NCAMs) are known to influence memory by affecting neural cell-cell and cell-extracellular matrix junctions. This study investigated the possible role of cAMP pathway in the expression of hippocampal NCAM and its polysialylated derivative (PSA-NCAM). The following pharmacological tools were employed for manipulation of cAMP pathway: a) forskolin; the activator of adenylyl cyclase (AC), b) 8-Br-cAMP; a protein kinase A (PKA) agonist, c) 8-pCPT-2'-O-Me-cAMP; a selective enhancer of exchange protein activated by cAMP (Epac) and d) Rp-cAMP; a PKA inhibitor. Memory acquisition was tested by passive avoidance paradigm after injecting the above compounds for three consecutive days into the CA1 region of dorsal hippocampus of rats. Forskolin and 8-Br-cAMP enhanced memory retrieval while Rp-cAMP significantly reduced memory and NCAM levels. 8-pCPT-2'-O-Me-cAMP failed to alter memory performance or NCAM levels as compared to vehicle. We observed no significant changes in PSA-NCAM, however the expression of St8sia4 and St8sia2 (the polysialyltransferase isoforms) were altered. The mRNA levels of St8sia4 was down-regulated by 8-Br-cAMP, Rp-cAMP and 8-pCPT while forskolin led to almost 3 and 5 fold increase in mRNAs of St8sia2 and St8sia4, respectively. The current insight might endorse the predominant role of PKA as compared to Epac in cAMP pathway in expression of NCAM and memory function.

Glucose concentration in culture medium affects mRNA expression of TRPV1 and CB1 receptors and changes capsaicin toxicity in PC12 cells.

OBJECTIVE S: Hyperglycemia is widely recognized as the underlying cause for some debilitating conditions in diabetic patients. The role of cannabinoid CB1 and vanilloid TRPV1 receptors and their endogenous agonists, endovanilloids, in diabetic neuropathy is shown in many studies. Here we have used PC12 cell line to investigate the possible influence of glucose concentration in culture medium on cytoprotective or toxic effects of a CB1 [WIN55 212-2 (WIN)], or TRPV1 [Capsaicin (CAS)] agonist. MATERIALS AND METHODS: Cell viability was tested using the MTT assay. We have also measured TRPV1 and CB1 transcripts by real time reverse transcription-polymerase chain reaction while cells were grown in low (5.5 mM) and high (50 mM) glucose concentrations. RESULTS: Real time PCR results indicated that high glucose medium increased (P<0.01) TRPV1 mRNA and decreased (P <0.001) that of CB1. Cell culture tests show that hyperglycemic cells are more vulnerable (Dose × Medium, F (3,63)=41.5, P<0.001) to the toxic effects of capsaicin compared to those grown in low glucose medium. CONCLUSION: These findings propose that hyperglycemic conditions may result in neuronal cell death because of inducing a counterbalance between cytotoxic TRPV1 and cytoprotective CB1 receptors.

EPAC-STX interaction may play a role in neurodevelopment/neurogenesis.

During embryonic life a group of cells become proliferated, migrated and differentiated to develop central nervous system. Migration has been suggested to be due to accumulation of polysialic acid (PSA), a negatively-charged glycoside, on the outer cell membrane. The same event happens to PSA in a tumor mass as well. Polysialylation is the product of polysialyl transfrase isozymes; STX (ST8SIA2), the embryonic active isoform, and PST (ST8SIA4), expressed in adults CNS. Additionally, cAMP concludes to activation of PKA and EPAC resulting to the initiation of gene expressions which are highly required during development. EPAC, the latter known target of cAMP in mammalian nervous system, has proliferative properties in the developing CNS. We propose for the proper action of EPAC, namely CNS development, the presence of STX and its elevation after EPAC activation is mandatory. This hypothesis is put forward after observing, in a preliminary experiment, a relationship between EPAC activation and STX mRNA expression levels in rat hippocampus. The interaction between EPAC and STX may be suggested to be through EPAC-induced gene expression of the latter. From the above assumptions one may suggest the use of EPAC activators as neurogenesis inducers and its inhibitors as tumor modulators.

Diabetic thermal hyperalgesia: role of TRPV1 and CB1 receptors of periaqueductal gray.

Hyperalgesia is one of the debilitating complications of diabetes. This condition might be caused by defects in central or peripheral processing of pain signals. In the present study we aim to see if diabetic hyperalgesia is related to changes in Transient Receptor Potential Vanilloid 1 (TRPV1) or Cannabinoid CB1 receptors of periaqueductal gray (PAG). Activation of glutamatergic projecting neurons in midbrain ventrolateral periaqueductal gray (VL-PAG) induces antinociception. Agonists of TRPV1 in VL-PAG increase firing of these glutamatergic neurons. CB1 receptor agonists also cause antinociception by decreasing Gamma Aminobutyric Acid (GABA) release in PAG and disinhibiting these glutamatergic neurons. In the present study antinociceptive effect of intra VL-PAG microinjections of CB1 and TRPV1 agonists [WIN55,212-2 (WIN) and capsaicin respectively] were compared in diabetic vs. non-diabetic rats, meanwhile mRNA expression of these receptors in PAG of diabetic and non-diabetic rats were evaluated by real time polymerase chain reaction (real time PCR) assay. Our results showed an attenuation of capsaicin antinociceptive effect (P<0.05) and TRPV1 receptor expression (P=0.023) but an increase in WIN antinociceptive effect (P<0.05) and CB1 receptor expression (P<0.001) in PAG of diabetic vs. non-diabetic rats. It is concluded that down-regulation of TRPV1 receptors in PAG is responsible for reduced antinociceptive effect of TRPV1 agonist. This finding may be an underlying cause of diabetic hyperalgesia. Up-regulation of CB1 receptors might be a compensatory mechanism but the precise elucidation of the effects of CB1 changes on disinhibition needs further studies.

Effects of cannabinoids infused into the dorsal hippocampus upon memory formation in 3-days apomorphine-treated rats.

In the present study, the effects of intra-dorsal hippocampus (intra-CA1) injection of cannabinoid receptor agents on memory formation have been investigated in 3-days apomorphine-treated rats. Step-through inhibitory avoidance task of memory has been used to examine retrieval of memory formation, 24h after training. Apomorphine was injected subcutaneously (S.C.), once daily for 3-days followed by 5 days free of the apomorphine before training. Bilateral post-training intra-CA1 infusions of the non selective CB1-CB2 receptor agonist, WIN55,212-2 (0.1, 0.25 and 0.5 microg/rat), dose-dependently shortened the step-through latency, suggesting amnesia by the drug, whereas bilateral post-training intra-CA1 micro-injections of the selective CB1 receptor antagonist, AM251 (25, 50 and 100 ng/rat), did not affect memory formation. Intra-CA1 infusions of AM251 and WIN55,212-2, two min apart, modify the WIN55,212-2-induced reduction of step-through latency. Furthermore, the deleterious effect of WIN55,212-2 (0.25 microg/rat) was completely abolished in rats previously given apomorphine (0.5 and 1 mg/kg/day, S.C.) for 3 days. This prevention of WIN55,212-2-induced amnestic-like effect was counteracted by the dopamine D2 receptor antagonist, sulpiride (0.25, 0.5 and 1 mg/kg/day x 3-days, S.C.), administered 30 min before each injection of apomorphine (0.5 mg/kg/day x 3-days, S.C.). The D1 receptor antagonist, SCH 23390 (0.01, 0.02, 0.07 and 0.1 mg/kg/day x 3-days, S.C.), was ineffective in this respect. The results suggest that subchronic apomorphine treatment may induce dopamine D2 receptor sensitization, which in turn prevented amnesia induced by WIN55,212-2.

Intra-dorsal hippocampal microinjections of lithium and scopolamine induce a cross state-dependent learning in mice.

BACKGROUND: Lithium, a mood stabilizer, may exert adverse effects on memory. We have previously shown that lithium induces state-dependent learning. Cholinergic systems of the brain may play an important role in memory function and mood regulation. In the present study, the effects of intra-dorsal hippocampal (intra-CA1) injections of lithium and scopolamine on memory and cross state-dependent learning between the two drugs were investigated. METHODS: For memory assessment, a one-trial step-down inhibitory avoidance task was used in adult male NMRI mice. RESULTS: Intra-CA1 administration of lithium (0.5 and 1 microg/mouse) after training or injection of the drug (0.5microg/mouse) before testing impaired memory when retrieval was tested 24 hours later. The memory impairment by post-training lithium was reversed by pretest administration of the drug (0.5 microg/mouse, intra-CA1) suggesting lithium state-dependent learning. On the other hand, intra-CA1 administration of scopolamine (0.5, 1, and 2 microg/mouse) after training or injection of the drug (2 microg/mouse) before testing impaired memory when retrieval was tested 24 hours later. The impairment of memory by post-training injection of scopolamine (2 microg/mouse) was restored by the pretest injection of the drug (1 and 2 microg/mouse). Furthermore, memory impairment induced by post-training injection of lithium (0.5 microg/mouse) and scopolamine (2 microg/mouse) was reversed by pretest administration of scopolamine (0.5, 1, and 2 microg/mouse) and lithium (0.5 and 1 microg/mouse), respectively. The impairment by lithium was also reversed by physostigmine. CONCLUSION: The results suggest that microinjections of both lithium and scopolamine induce state-dependent memory and there may be a cross state-dependency between the two drugs.

Lithium chloride protects PC12 pheochromocytoma cell line from morphine-induced apoptosis.

BACKGROUND: Opioid drugs are considered as important members of drugs of abuse. Opioid abusers are more likely to be infected which may be due to apoptotic effects of the drugs on immune cells. Furthermore, there are some reports on the apoptotic effect of morphine on neural cells. In the present study, the effect of morphine and lithium on apoptosis in PC12 cell line (as a model of neural cells) was examined. METHODS: We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-fluorescein isothiocyanate test and quantitative real time RT-polymerase chain reaction for detection of necrosis and apoptosis (programmed cell death). RESULTS: PC12 cells were exposed to different concentrations of morphine for six, 12, 24, 48, and 96 hours. Quantitative real-time RT-polymerase chain reaction revealed that mRNA expression of BAX (proapoptotic element) increased while a decrement in the mRNA expression of BCL-2 (protective element) was observed after six hours (but not after 12 or 24 hours) exposure to morphine. Furthermore, the results of MTT assay and annexin V-fluorescein isothiocyanate test indicated that morphine exposure causes an increase in the percentage of apoptotic and necrotic cells, respectively. Interestingly, the results of MTT assay and annexin V-fluorescein isothiocyanate test were observed 12 and 24 hours after morphine exposure. Thus, it can be concluded that alteration in mRNA expression is an early event rather than as a consequence of apoptosis or necrosis. On the other hand, lower concentrations of lithium elicit protective effect against apoptosis in some of mammalian cells while the higher concentrations are toxic. Despite large body of evidences on the protective effect of lithium, elucidation of downstream events are still unknown. In the present study, 72-hour preincubation of PC12 cells with 1.2 mM lithium chloride reversed the effects of morphine on the mRNA expression of BAX and BCL-2. Furthermore, the results of real time RT-polymerase chain reaction were supported by annexin V-fluorescein isothiocyanate test and MTT assay. CONCLUSION: The protective effect of lithium on the morphine-induced cytotoxicity is mediated via down-regulation of BAX and up-regulation of BCL-2 mRNA expression.

Decreased AMPA GluR2, but not GluR3, mRNA expression in rat amygdala and dorsal hippocampus following morphine-induced behavioural sensitization.

1. Repeated administration of psychostimulants and micro-opioid receptor agonists elicits a progressive enhancement of drug-induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor function. 2. In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state-dependent memory in a passive avoidance test were measured in the presence or absence of 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine. 3. The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state-dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed. 4. These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium-permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.

Morphine-induced behavioral sensitization increased the mRNA expression of NMDA receptor subunits in the rat amygdala.

This study was designed to evaluate the effect of repeated morphine treatment on rat behavioral responses. In the genetic section, the mRNA expression of NMDA receptor subunits (NR1 and NR2A) was measured in certain areas of the male rat brain (striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala). In the behavioral section, the effect of repeated morphine treatment on animal models such as locomotion, oral stereotypy, and state-dependent memory in a passive avoidance test was evaluated in the presence or absence of MK801 (NMDA receptor antagonist). Our results showed that chronic morphine treatment, followed by a 7-day (but not 24-hour) washout period, potentiated the effect of test doses of morphine, which is referred to as behavioral sensitization. Meanwhile, pretreatment of animals with MK801 (0.1 and 0.25 mg/kg), 30 min before a test dose of morphine (5 mg/kg), failed to attenuate the locomotion and oral stereotypy in the behavioral sensitization state. Interestingly, a higher dose of MK801 (0.25 mg/kg) decreased memory retrieval induced by morphine (2.5 mg/kg) in state-dependent memory. This effect may be due to the intrinsic motor enhancer property of higher doses of MK801, rather than the blockade of NMDA receptors. It can be concluded that MK801 does not affect morphine-induced behavioral sensitization in the expression phase. In the genetic section of the study, results of quantitative real-time RT-PCR clearly indicated that morphine sensitization increased the expression of NMDA receptor subunits mRNA in the amygdala (NR1 by 104% and NR2A by 85%), while the other areas of the brain were unaffected. Maenwhile, no change in the mRNA levels was observed in non-sensitized animals (chronic morphine treatment followed by a 24-hour washout period). In summary, the present study indicates that repeated morphine treatment followed by long-term (7-day washout) induces behavioral sensitization and causes a delayed increase in mRNA levels of NMDA receptor subunits in the rat amygdala. Meanwhile, it has previously been reported that the amygdala is involved in behavioral sensitization. Thus, it can be concluded that the increase in NMDA receptor expression is associated with morphine-induced behavioral sensitization.

Involvement of amlodipine, diazoxide, and glibenclamide in development of morphine tolerance in mice.

In this study, the effects of the calcium channel blocker (amlodipine), potassium channel opener (diazoxide), and potassium channel blocker (glibenclamide) on the development of morphine-induced tolerance in the formalin test was investigated. During development of tolerance to morphine, intraperitoneal (i.p.) administration of different doses of amlodipine (5, 7.5 and 10 mg/kg) or diazoxide (1, 5 and 10 mg/kg) in combination with morphine (20 and 30 mg/kg) increased tolerance in the first and second phase of the test. However, glibenclamide (2.5, 5, and 10 mg/kg) decreased morphine tolerance in the second phase of formalin test. It is concluded that calcium and potassium channel mechanisms may be involved in the morphine tolerance.

Morphine-induced antinociception in the formalin test: sensitization and interactions with D1 and D2 dopamine receptors and nitric oxide agents.

In this study, the effects of dopamine receptor antagonists and nitric oxide agents on morphine-induced sensitization in the formalin test in mice have been investigated. Repeated daily intraperitoneal administration of morphine (30 mg/kg for 3 days) followed by a 11-day wash out period increased morphine-induced antinociception in the formalin test, which may be due to sensitization. The antinociceptive response to higher doses of morphine (6 and 9 mg/kg) but not 3 mg/kg was significantly increased in sensitized animals compared with control groups. Pretreatment of animals with an opioid receptor antagonist, naloxone (4 mg/kg), during repeated administration of morphine, attenuated the morphine-induced sensitization. In the second part of the study, the animals received SCH23390 (D1 receptor antagonist), sulpiride (D2 receptor antagonist), L-Arg (nitric oxide precursor) and NG-nitro-L-Arg methylester (nitric oxide synthase inhibitor) during repeated morphine administration, to evaluate the role of dopamine receptor antagonists and nitric oxide agents in this phenomenon. Pretreatment of animals with NG-nitro-L-Arg methylester (20 mg/kg) and sulpiride (100 mg/kg) during morphine sensitization decreased the antinociceptive response to higher doses of morphine in the formalin test. It is concluded that D2 dopamine receptor and nitric oxide mechanisms may be involved at least partly in morphine-induced sensitization in the formalin test.