Sex Differences and Role of Gonadal Hormones on Glutamate LevelAfter Spinal Cord Injury in Rats: A Microdialysis Study.

INTRODUCTION: Sex differences in outcomes of Spinal Cord Injury (SCI) suggest a sex-hormone-mediated effect on post-SCI pathological events, including glutamate excitotoxicity. This study aimed to investigate the importance of gonadal hormones on glutamate release subsequent to SCI in rats. METHODS: After laminectomy at T8-T9, an electrolytic lesion was applied to the spinothalamic tracts of male and female rats. Using spinal microdialysis, we assessed glutamate levels at the site of lesion in both intact and gonadectomized rats for 4 hours. In this way, we examined the sex differences in the glutamate concentrations. RESULTS: The peak retention time of glutamate level was 10.6 min and spinal glutamate concentration reached a maximum level 40 min following SCI. In male SCI rats, gonadectomy caused a significant elevation of glutamate level (P<0.001) following injury which was maximum 40 min post-SCI as well. However, no significant alterations were seen in gonadectomized female rats. CONCLUSION: The significant differences in glutamate levels between both intact and gonadectomized SCI male and female rats show the sex-hormone-related mechanisms underlying the molecular events in the second phase of SCI. It seems that the role of male gonadal hormones to prevent glutamate excitotoxicity is more prominent. The exact mechanisms of these hormones on the functional recovery after SCI should be clarified in further studies.

Cross State-dependent Learning Interaction Between Scopolamine and Morphine in Mice: The Role of Dorsal Hippocampus.

INTRODUCTION: The current study aimed at investigating the existence of the cross state-dependent learning between morphine and scopolamine (SCO) in mice by passive avoidance method, pointing to the role of CA1 area. METHODS: The effects of pre-training SCO (0.75, 1.5, and 3 µg, Intra-CA1), or morphine (1, 3, and 6 mg/kg, intraperitoneal (i.p.) was evaluated on the retrieval of passive avoidance learning using step-down task in mice (n=10). Then, the effect of pretest administration of morphine (1.5, 3, and 6 mg/kg, i.p.) was examined on passive avoidance retrieval impairment induced by pre-training SCO (3 µg/mice, Intra-CA1). Next, the effect of pretest Intra-CA1 injection of scopolamine (0.75, 1.5, and 3 µg/mice) was evaluated on morphine (6 mg/kg, i.p.) pre-training deficits in this task in mice. RESULTS: The pre-training Intra-CA1 injection of scopolamine (1.5 and 3 µg/mouse), or morphine (3 and 6 mg/kg, i.p.) impaired the avoidance memory retrieval when it was tested 24 hours later. Pretest injection of both drugs improved its pre-training impairing effects on mice memory. Moreover, the amnesia induced by the pre-training injections of scopolamine (3 µg/mice) was restored significantly (P<0.01) by pretest injections of morphine (3 and 6 mg/kg, i.p.). Similarly, pretest injection of scopolamine (3 µg/mice) restored amnesia induced by the pre-training injections of morphine (6 mg/kg, i.p.), significantly (P<0.01). CONCLUSION: The current study findings indicated a cross state-dependent learning between SCO and morphine at CA1 level. Therefore, it seems that muscarinic and opioid receptors may act reciprocally on modulation of passive avoidance memory retrieval, at the level of dorsal hippocampus, in mice.

Dendrosomal nanocurcumin prevents morphine self-administration behavior in rats despite CA1 damage.

Dendrosomal nanocurcumin (DNC) is fabricated from esterification of oleic acid and polyethylene glycol residues with curcumin. DNC has shown antioxidant, neuroprotective, and neurogenesis-enhancing effects. In addition, it can attenuate morphine tolerance. Morphine self-administration is associated with neurodegenerative changes of CA1 neurons in the adult hippocampus. The present study evaluated the effect of DNC pretreatment on morphine self-administration and hippocampal damage. Rats were pretreated with DNC (5 and 10 mg/kg, intraperitoneally) 30 min before a morphine self-administration paradigm performed in 2-h/sessions for 12 days under a FR-1 schedule. Pretreatment with both doses of DNC markedly suppressed morphine intake. Morphine self-administration resulted in a 71% reduction in the number of hippocampal CA1 neurons. DNC (5 mg/kg) pretreatment only marginally improved (by 22%) neuronal loss in this area. The data suggest that the effect of DNC on morphine self-administration is largely independent of the CA1 area. A functional restoration and regulation of reward circuit activity by DNC may reduce the motivation for morphine despite CA1 damage.

Comparison of acute effects of heroin and Kerack on sensory and motor activity of honey bees (Apis mellifera).

OBJECTIVES: Previous studies demonstrated a functional similarity between vertebrate and honey bee nervous systems. The aim of the present study was to compare the effects of heroin and Iranian street Kerack, a combination of heroin and caffeine, on sensory threshold and locomotor activity in honey bees. MATERIALS AND METHODS: All drugs were given orally to honey bees 30 min before each experiment. The levels of these drugs and their metabolites in brain samples of honey bees were determined by GC/MS. The sucrose sensitivity test was used for evaluation of changes in honey bees' sensory threshold. Following the administration of both drugs, the honey bees' locomotor activity changes were evaluated in open fields. RESULTS: 6-acetylmorphine had a higher concentration in comparison with other heroin metabolites in honey bees' brains. Concentration of the compound in the brain was directly proportional to the amount ingested. Heroin reduced the sensory threshold of honey bees, but Kerack increased it in the same doses. Locomotor activity of honey bee in open field was enhanced after the administration of both drugs. However, immobility time of honey bees was only affected by high doses of heroin. CONCLUSION: Acute effects of heroin andKerack on the sensory and motor functions of honey bees were different. Findings of this research suggest that these differences originated from the activation of different neurotransmitter systems by caffeine together with activation of opioid receptors by heroin.

Epidural administration of neostigmine-loaded nanofibers provides extended analgesia in rats.

BACKGROUND: In this study, neostigmine-loaded electrospun nanofibers were prepared and then their efficacy and duration of analgesic action were studied after epidural administration in rats by repeated tail flick and formalin tests. METHODS: The neostigmine poly vinyl alcohol (PVA) nanofibers were fabricated by electrospinning methods. The nanofibers (1 mg) were injected into the lumbar epidural space (L5-L6) of rats (n = 6). Cerebrospinal fluid samples of rats were collected 1, 5 and 24 hours after injection and then were sampled once weekly for 4 weeks. Free-neostigmine concentration was measured in the samples spectrophotometrically. Rat nociceptive responses were evaluated by repeated tail-flick and formalin tests for 5 weeks after the nanofibers (1 mg) injection. Locomotor activity of rats was measured in the open-field at the same period. RESULTS: The cerebrospinal fluid concentration of free neostigmine reached 5 µg/ml five hours after injection and remained constant until the end of the experiments. The tail-flick latency of treated rats was significantly (p < 0.01) increased and remained constant up to 4 weeks. Pain scores of the rats in both phases of formalin test were significantly (p < 0.01) reduced during the same periods, Epidural injection of the nanofibers had no effect on locomotor activity of rats in an open-field. CONCLUSIONS: Our results indicate that the neostigmine nanofibers can provide sustained release of neostigmine for induction of prolonged analgesia after epidural administration. High tissue distribution and penetration of the nanofibers in dorsal horn can increase thermal and chemical analgesia duration without altering locomotor activity in rats for 4 weeks.

Improvement of Lidocaine Local Anesthetic Action Using Lallemantia royleana Seed Mucilage as an Excipient.

Lallemantia royleana (Balangu) is a well known Iranian medicinal plant that its seed mucilage has many applications in modern pharmacology. Plant mucilage traditionally was used as a gel supplement, and natural matrix for sustained release of drugs. But it seems that these compounds are not a simple additive and also have many undiscovered pharmacological properties. In this research, the anesthetic action of gel prepared from Balangu mucilage alone and its mixture with lidocaine hydrochloride are compared with the effect of commercial 2% lidocaine gel by rat tail flick test. Mucilage of Balangu seed alone showed analgesic effect. Duration and potency of anesthesia induced by gel containing mucilage alone (0.01 g/mL) were identical to commercial 2% lidocaine gel. But, local anesthetic potency and duration of gel made from 2% lidocaine-mucilage gel mixture was significantly higher than commercial 2% lidocaine gel. The gel prepared from mucilage causes a good analgesia with unknown mechanism. Besides, mixture of Balangu mucilage prepared gel with lidocaine improves lidocaine anesthesia. The increase in potency of lidocaine action results from mucilage dermal penetration enhancing effects; and longer anesthetic duration of this mixture are related to the capability of mucilage based gel for sustained drug release.

Blockade of D1/D2 dopamine receptors within the nucleus accumbens attenuated the antinociceptive effect of cannabinoid receptor agonist in the basolateral amygdala.

Previous studies showed the role of basolateral amygdala (BLA) in cannabinoid-induced antinociception. Furthermore, the nucleus accumbens (NAc) plays an important role in mediating the suppression of pain in animal models. The present study extended the role of dopamine receptors within the NAc in antinociceptive effect of cannabinoid receptor agonist, WIN55,212-2, microinjected into the BLA following the tail-flick and formalin tests in rats. In this study, 174 adult male albino Wistar rats were unilaterally implanted by two separate cannulae into the BLA and NAc. In two separated groups, rats received intra-NAc infusions of the D1 receptor antagonist, SCH-23390 (0.25, 1 and 4 µg/0.5 µl saline) or D2 receptor antagonist, sulpiride (0.25, 1 and 4 µg/0.5 DMSO), and just 2 min later, WIN55,212-2 (15 µg/rat) was microinjected into the BLA. In the tail-flick test, antinociceptive responses of drugs represented as maximal possible effect (%MPE) in 5, 15, 30, 45 and 60min after their administrations. Moreover, in the formalin test, pain related behaviors were monitored in 5-min blocks for 60 min test period. Our findings showed that intra-accumbal SCH-233909 dose-dependently prevented antinociception induced by intra-BLA administration of WIN55,212-2 (15 µg/rat) in time set intervals in formalin, but not tail-flick test. Besides, administration of sulpiride in the NAc could affect WIN-induced analgesia in both models of pain. In conclusion, it seems that D2 receptors located in the NAc, in part, mediate the antinociceptive responses of cannabinoid within the BLA, while D1 receptors only are involved in modulation of persistent inflammatory model of pain.

The effect of aligned and random electrospun fibrous scaffolds on rat mesenchymal stem cell proliferation.

OBJECTIVE: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared, and then p-PCL scaffolds were evaluated for MSC culture. MATERIALS AND METHODS: Aligned and random PCL nanofibrus scaffolds were fabricated by electrospining and their surface modified with O2 plasma treatment to enhance MSC proliferation, adhesion and interaction. Chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance (ANOVA). RESULTS: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber. CONCLUSION: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.

Intra-accumbal NMDA but not AMPA/kainate receptor antagonist attenuates WIN55,212-2 cannabinoid receptor agonist-induced antinociception in the basolateral amygdala in a rat model of acute pain.

Previous studies showed the role of basolateral amygdala (BLA) in cannabinoid-induced antinociception. Several lines of evidence indicated that the nucleus accumbens (NAc) receives excitatory glutamatergic inputs primarily from limbic-related structures, including the hippocampus, BLA, and various thalamic nuclei. Additionally, it has been shown that the NAc plays an important role in mediating the suppression of animal models of pain. In the present study, we examined the role of NMDA and AMPA/kainate receptors within the NAc in antinociception induced by intra-BLA cannabinoid receptor agonist WIN55,212-2 in rats. 126 adult male albino Wistar rats weighing 230-280 g were unilaterally implanted by two separate cannulae into the BLA and NAc. Dose-response antinociceptive effects of different doses of intra-BLA WIN55,212-2 (5, 10 and 15 µg/0.3 µl/rat) were evaluated in this study. Moreover, animals received intra-accumbal infusions of either NMDA receptor antagonist, AP5 (0.5, 2.5 and 5 µg/0.5 µl saline) or AMPA/kainate receptor antagonist, CNQX (0.1, 0.5 and 2.5 µg/0.5 µl DMSO), just 2 min before microinjection of WIN55,212-2 into the BLA. Antinociceptive responses of drugs were obtained by tail-flick analgesiometer and represented as maximal possible effect (%MPE) at 5, 15, 30, 45 and 60 min after their administrations. Results showed that intra-accumbal AP5 dose-dependently prevented antinociception induced by intra-BLA administration of WIN55,212-2 (15 µg/rat) in time set intervals. Nonetheless, administration of AMPA/kainate receptor antagonist, CNQX, could not affect WIN-induced analgesia. Additionally, solely administration of intra-accumbal injection of CNQX (2.5 µg/0.5 µl DMSO), but not AP5 (5 µg/0.5 µl saline), could significantly change the baseline tail-flick latencies in the rats. It seems that NMDA receptors located in the NAc, in part, mediate the antinociceptive responses of cannabinoid within the BLA in acute model of pain.

Functional involvement of Ca(2+) and Ca(2+)-activated K(+) channels in anethol-induced changes in Ca(2+) dependent excitability of F1 neurons in Helix aspersa.

The effects of anethol, the major component of anise oil, on the Ca(2+)-dependent excitability and afterhyperpolarization (AHP) in snail neurons were examined using intracellular recording. Anethol (0.5%) significantly broadened the spike, reduced the firing frequency and enhanced the AHP amplitude. In contrast, anethol (2%) significantly increased the firing frequency and decreased the AHP. Blockade of Ca(2+) channels after anethol application depolarized the membrane potential and significantly reduced the firing rate. Furthermore, in the presence of anethol (0.5%) a significant decrease in the AHP was observed by Ca(2+) channels blockage. Here, anethol-induced functional modification of Ca(2+) and Ca(2+)-activated K(+) channels is suggested.

Changes in phosphorylation of CREB, ERK, and c-fos induction in rat ventral tegmental area, hippocampus and prefrontal cortex after conditioned place preference induced by chemical stimulation of lateral hypothalamus.

Experimental evidence indicates that chemical stimulation of lateral hypothalamus (LH) by carbachol can produce conditioned place preference (CPP) in rats. Several lines of evidence have shown that cAMP-response element binding protein (CREB), extracellular signal-regulated kinase (ERK), and c-fos have pivotal role in CPP induced by drugs of abuse, such as morphine, cocaine, nicotine, and alcohol. Therefore, in the present study, we investigated the changes in phosphorylated-CREB (p-CREB) and -ERK (p-ERK), and c-fos induction within ventral tegmental area (VTA), hippocampus and prefrontal cortex (PFC) after the acquisition of CPP induced by intra-LH administration of carbachol. Animals were unilaterally implanted by cannula into LH. For chemical stimulation of LH, carbachol (250 nmol/0.5 µl saline) was microinjected once each day, during 3-day conditioning phase (acquisition period) of CPP paradigm. After the acquisition period, the brains were removed, and p-CREB and p-ERK, and c-fos induction in the ipsilateral VTA, hippocampus and PFC were measured by Western blot analysis. The results indicated a significant increase in level of phosphorylated CREB (P<0.01) in VTA, and PFC (P<0.05), during LH stimulation-induced CPP, while its level decreased in hippocampus (P<0.05). Also, in aforementioned regions, an increase in c-fos level was observed, but this enhancement in PFC was not significant. Moreover, p-ERK changed in these areas, but not significantly. Our findings suggest that studying the intracellular signals and their changes, such as phosphorylated-CREB, can elucidate a functional relationship between LH and other brain structures involved in reward processing in rats.

Chemical stimulation of the lateral hypothalamus induces conditioned place preference in rats: Involvement of OX1 and CB1 receptors in the ventral tegmental area.

Orexinergic projection originated from the lateral hypothalamus (LH) to the ventral tegmental area (VTA) has an important role in the acquisition of morphine conditioned place preference (CPP). However, little if any is known about the function and/or effect of orexin on CPP in rats. In the present study, we investigated the direct effect of orexinergic neurons on acquisition of CPP by chemical stimulation of LH and involvement of orexin-A and CB1 receptors within the VTA in development of reward-related behaviors. 129 adult male albino Wistar rats weighing 220-320 g were unilaterally implanted by two separate cannulae into the LH and VTA. The CPP paradigm was done; conditioning score and locomotor activity were recorded by Ethovision software. The results showed that unilateral intra-LH administration of carbachol (62.5, 125 and 250 nmol/0.5 µl saline) as a cholinergic agonist, during conditioning phase, induced CPP in a dose-dependent manner. The maximal effect was shown at the dose of 250 nmol (P<0.001) compared to vehicle (saline) group. However, intra-VTA administration of SB334867 as a selective orexin-A receptor antagonist (0.1, 1 and 10 nmol/0.3 µl DMSO) and AM251 (5, 25 and 125 nmol/0.3 µl DMSO) as a CB1 receptor antagonist, just 5 min before carbachol during the 3-day conditioning phase, could dose-dependently inhibit the development of LH stimulation-induced CPP in the rats. It is supposed that the orexinergic projection from LH to VTA is involved in LH chemical stimulation-induced CPP and orexin-A receptor in the VTA has a substantial role in this phenomenon. Our findings also suggest the existence of cross-talk between cannabinoid and orexinergic systems within the VTA in conditioned place preference paradigm.

Sub-chronic administration of AM251, CB1 receptor antagonist, within the nucleus accumbens induced sensitization to morphine in the rat.

It seems that there is a cross-talk between the cannabinoid CB1 and opioid receptors in the process of sensitization to opiates. In present study, we tried to examine the effect of solely administration of AM251, a CB1 receptor antagonist, on conditioned place preference (CPP) by ineffective dose of morphine in the rat. 102 adult male albino Wistar rats were used in these experiments. Subcutaneous administration of morphine (0.5, 1, 2.5, 5, 7.5 and 10 mg/kg) induced CPP only at the doses of > or = 5 mg/kg. The dose of 0.5mg/kg of morphine was selected as the appropriate (ineffective) dose for induction of CPP in animals which were previously received AM251 (5, 25 and 125 ng/0.5 microl per side) once daily for three days as a sub-chronic administration or those that received a single dose on the test day. Bilateral intra-accumbal sub-chronic but not single administration of AM251 dose-dependently produced sensitization to morphine and induced CPP by ineffective dose of morphine (0.5 mg/kg) in the rat. Bilateral intra-accumbal administration of neither saline nor DMSO (0.5 microl/side) had effects on sensitization to morphine. Our findings indicated that CB1 receptors within the nucleus accumbens are involved in the sensitization to morphine in rats.

Effects of CB1 receptor antagonist within the nucleus accumbens on the acquisition and expression of morphine-induced conditioned place preference in morphine-sensitized rats.

It has been shown that cannabinoids interact with the opiate system in reward-related behaviors and in animal models of addiction. In the present study, the effects of bilateral intra-accumbal administration of AM251, a CB1 receptor antagonist, on the acquisition and expression of ineffective dose of morphine-induced conditioned place preference (CPP) in morphine-sensitized rats were investigated. 158 adult male albino Wistar rats were used in these experiments. Subcutaneous (s.c.) administration of morphine (0.25, 0.5, 0.75, 1, 2.5 and 5mg/kg) induced CPP only at the dose of 5mg/kg. In addition, repeated administration of morphine (5mg/kg; s.c.), once daily for 3 days followed by 5 days free of the opioid (sensitization period), increased conditioning response induced by ineffective doses of morphine (0.25, 0.5 and 0.75 mg/kg). Bilateral intra-accumbal administration of AM251 (5, 25 and 125 ng/0.5 microl per side) dose-dependently reduced the acquisition and expression of morphine-induced CPP in morphine-sensitized rats, while bilateral intra-accumbal administration of neither saline nor DMSO (0.5 microl/side) had effects on the acquisition and expression of morphine-induced CPP in sensitized rats. The results indicated that CB1 receptors within the nucleus accumbens are involved in the acquisition and expression of morphine-induced CPP in sensitized rats. Our findings also suggest the existence of cross-talk between cannabinoids and opiates on the sensitization to morphine and the implication of endocannabinoid system in the process of sensitization to opiates.

Effects of reversible inactivation of the ventral tegmental area on the acquisition and expression of morphine-induced conditioned place preference in the rat.

The mesolimbic dopaminergic system that projects from the ventral tegmental area (VTA) to the nucleus accumbens is critical for initiation of opioid reinforcement and reward-related effects of drugs of abuse. In the present study, the effects of reversible inactivation of VTA on acquisition and expression of morphine-induced conditioned place preference (CPP) were investigated in rats. One hundred and eighty-one adult male albino Wistar rats were used in these experiments. Reversible inactivation of VTA was done through the unilateral and bilateral intra-VTA microinjection of 2% lidocaine during the acquisition and expression of morphine (5 mg/kg; s.c.)-induced CPP. Animal displacement, conditioning score and locomotor activity were recorded by Ethovision software. The results showed that bilateral but not unilateral intra-VTA administration of lidocaine significantly decreases the acquisition (P<0.01) and expression (P<0.05) of morphine-induced CPP compared to their respective saline-microinjected groups. Moreover, intra-VTA administration of lidocaine had no effect on locomotor activity in these experiments. Our results further support the idea that VTA may play an important role in the acquisition of morphine-induced CPP. In addition, there is no functional lateralization in the VTA for acquisition and/or expression of morphine-induced CPP in the rat.