New amide and diterpene alkaloids with anticholinesterase activity from Delphinium cyphoplectrum roots.

BACKGROUND: The cholinergic hypothesis posits a robust correlation between the onset of Alzheimer's disease and a pronounced deficit in acetylcholine, a pivotal neurotransmitter crucial for the central cholinergic nervous system's function, pivotal for memory and learning. Diterpene alkaloids exhibit intricate and distinctive chemical structures that facilitate their passage through the blood-brain barrier. Moreover, their potent pharmacological attributes render them promising candidates for addressing central nervous system disorders. OBJECTIVES: This investigation aims to scrutinize the alkaloidal composition of Delphinium cyphoplectrum (Ranunculaceae) roots, further exploring their anticholinesterase inhibitory activity and mode of inhibition. METHOD: Innovative chromatography techniques were repetitively employed to purify the alkaloids. Acetylcholinesterase (AChE) inhibition assays were conducted using Ellman's tests. The mode of inhibition was meticulously characterized through Michaelis-Menten, and Lineweaver-Burk plots. Conducting molecular docking studies, we employed the AUTO DOCK 4.2 software package. RESULTS: Eight alkaloids were identified including five C19-diterpene alkaloids (6,14,16,18-tetramethoxy-1,7,8-trihydroxy-4-methylaconitane (1), 6,16,18-trimethoxy-1,7,8,14-tetrahydroxy-4-methylaconitane (2), 6,8,16,18-tetramethoxy-1,7,14-trihydroxy-4-methylaconitane (3), 6,14,16-trimethoxy-1,7,8,18-tetrahydroxy-4-methylaconitane (4), and 14-O-acetyl-8,16-dimethoxy-1,6,7,18-tetrahydroxy-4-methylaconitane (5)), an epoxy C18-diterpene alkaloid (6,8,16-trimethoxy-1,7,14-trihydroxy-3,4-epoxyaconitane (6)), a known (pyrrolidin-2-one (7) and an undescribed amide alkaloid (1-(2'-hydroxylethylamine)-3,5,5,-trimethyl-1,5-dihydro-2H-pyrrol-2-one (8). All diterpene alkaloids underwent assessment for acetylcholinesterase (AChE) inhibition assay and displayed noteworthy AChE activity, surpassing that of the reference drug (with IC50 values of 13.7, 21.8, 23.4, 28.2, 40.4, and 23.9 for compounds 1-6, respectively, in comparison to 98.4 for Rivastigmine). Analysis of Michaelis-Menten and Lineweaver-Burk plots represents an uncompetitive mode of inhibition for compound 1 on AChE. Notably, computational docking simulations indicated that all diterpene alkaloids were accommodated within the same enzymatic cleft as the reference ligand, and displaying superior free binding energy values (from - 10.32 to -8.59 Kcal.mol-1) in contrast to Rivastigmine (-6.31 Kcal.mol-1). CONCLUSION: The phytochemical analysis conducted on the roots of Delphinium cyphoplectrum yielded the identification of eight alkaloidal compounds including one C18-diterpene, five C19-diterpene, one pyrrolidine and one amide alkaloids. AChE inhibition assay and molecular simulations unveiled remarkable significant potency attributed to the C19-diterpene alkaloids by the order of 1 > 2 > 3,6 > 4 > 5. Presence of hydroxyl group on C-1, C-7, C-8, C-14, and C-18 increased the effect. The best in vitro activity was recorded for compound 1 able to bind to Asp72 in the narrow region of PAS, while interacting by pi-sigma with Phe330 at the hydrophobic region of the gorge involving the acyl and choline binding site. This observation underscores the substantial promise of this category of natural products in the realm of drug discovery for Alzheimer's Disease, offering a compelling avenue for further research and therapeutic development.

Step-by-step approach: Stereotaxic surgery for in vivo extracellular field potential recording at the rat Schaffer collateral-CA1 synapse using the eLab system.

In vivo extracellular field potential recording is a commonly used technique in modern neuroscience research. The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. However, there is limited use of visually guided stereotaxic neurosurgery and the application of the eLab/ePulse electrophysiology system in rodent models. This study presents a practical and functional manual guide for surgical electrode implantation in rodent models using the eLab/ePulse electrophysiology system for recording and stimulation purposes to assess neuronal functionality and synaptic plasticity. The evaluation parameters included the input/output function (IO), paired-pulse facilitation or depression (PPF/PPD), long-term potentiation (LTP), and long-term depression (LTD).•Provides a detailed picture-guided procedure for conducting in vivo stereotaxic neurosurgery.•Specifically covers the insertion of hippocampal electrodes and the recording of evoked extracellular field potentials.

Low Current Intensity Plus an Ineffective Dose of Morphine Affect Conditioning Place Preference Through Different Pathways in the Lateral Habenula.

Background: The involvement of lateral habenula and the ineffective dose of morphine on reward-related learning and memory is less well-known. This study looked into the effects of electrical stimulation, GABAB receptor blockade, and a combination of both with morphine on conditioned place preference. Materials and Methods: In this experiment, male rats were anesthetized with ketamine/xylazine (six rats in each group). A 5-day biased conditioned place preference paradigm was used for the behavioral test. The effects of electrical stimulation and phaclofen plus a low dose of morphine on the acquisition and expression phases were examined during conditioning sessions and before the test phase, respectively. Results: The conditioning scores were reduced by antagonist injection during the acquisition phase. Interestingly, different intensities exhibited opposite effects on the acquisition phase. Conditioned place preference scores during the acquisition phase were significantly induced by 25 µA electrical stimulation, while conditioning scores were suppressed by electrical stimulation at 150 µA. Phaclofen (2 µg/rat) combined with high intensity induced aversion during the acquisition phase, while inhibiting expression. In contrast, high intensity with phaclofen (1 µg/rat) inhibited only the acquisition session. However, low intensity during the acquisition phase had an additive effect that was prevented by pretreatment with phaclofen (2 µg/rat), but this response was modified by the antagonist's low dose. Conclusions: A behavioral technique called conditioned place preference is frequently used to evaluate learning that is related to rewards. Therefore, lateral habenula electrical stimulation and phaclofen plus morphine could affect place preference through the involvement of the reward system.

In vivo synaptic potency, short-term and long-term plasticity at the hippocampal Schaffer collateral-CA1 synapses: Role of different light-dark cycles in male rats.

The changes in the light-dark(L/D) cycle could modify cellular mechanisms in some brain regions. The present study compared the effects of various L/D cycles on invivo synaptic potency, short-term and long-term plasticity in the hippocampal CA1 area, adrenal glands weight(AGWs), corticosterone (CORT) levels, and body weight differences(BWD) in male rats. Male rats were assigned into different L/D cycle groups: L4/D20, L8/D16, L12/D12(control), L16/D8, and L20/D4. The slope, amplitude, and the area under curve(AUC) related to the field excitatory postsynaptic potentials(fEPSPs) were assessed, using the input-output(I/O) functions, paired-pulse(PP) responses at different interpulse intervals, and after the induction of long-term potentiation(LTP) in the hippocampal CA1 area. Also, the CORT levels, AGWs, and BWDs were measured in all groups. The slope, amplitude, and AUC of fEPSP in the I/O functions, all three phases of PP, before and after the LTP induction, were significantly decreased in all experimental groups, especially in the L20/D4 and L4/D20 groups. As such, the CORT levels and AGWs were significantly increased in all experimental groups, especially in the L20/D4 group. Overall, the uncommon L/D cycles (minimum and particularly maximum durations of light) significantly reduced the cellular mechanism of learning and memory. Also, downtrends were observed in synaptic potency, as well as short-term and long-term plasticity. The changes in PP with high interpulse intervals, or activity of GABAB receptors, were more significant than the changes in other PP phases with different L/D durations. Additionally, the CORT levels, adrenal glands, and body weight gain occurred time-independently concerning different L/D lengths.

Neuropharmacological Potential of Diterpenoid Alkaloids.

This study provides a narrative review of diterpenoid alkaloids (DAs), a family of extremely important natural products found predominantly in some species of Aconitum and Delphinium (Ranunculaceae). DAs have long been a focus of research attention due to their numerous intricate structures and diverse biological activities, especially in the central nervous system (CNS). These alkaloids originate through the amination reaction of tetra or pentacyclic diterpenoids, which are classified into three categories and 46 types based on the number of carbon atoms in the backbone structure and structural differences. The main chemical characteristics of DAs are their heterocyclic systems containing ß-aminoethanol, methylamine, or ethylamine functionality. Although the role of tertiary nitrogen in ring A and the polycyclic complex structure are of great importance in drug-receptor affinity, in silico studies have emphasized the role of certain sidechains in C13, C14, and C8. DAs showed antiepileptic effects in preclinical studies mostly through Na+ channels. Aconitine (1) and 3-acetyl aconitine (2) can desensitize Na+ channels after persistent activation. Lappaconitine (3), N-deacetyllapaconitine (4), 6-benzoylheteratisine (5), and 1-benzoylnapelline (6) deactivate these channels. Methyllycaconitine (16), mainly found in Delphinium species, possesses an extreme affinity for the binding sites of α7 nicotinic acetylcholine receptors (nAChR) and contributes to a wide range of neurologic functions and the release of neurotransmitters. Several DAs such as bulleyaconitine A (17), (3), and mesaconitine (8) from Aconitum species have a drastic analgesic effect. Among them, compound 17 has been used in China for decades. Their effect is explained by increasing the release of dynorphin A, activating the inhibitory noradrenergic neurons in the ß-adrenergic system, and preventing the transmission of pain messages by inactivating the Na+ channels that have been stressed. Acetylcholinesterase inhibitory, neuroprotective, antidepressant, and anxiolytic activities are other CNS effects that have been investigated for certain DAs. However, despite various CNS effects, recent advances in developing new drugs from DAs were insignificant due to their neurotoxicity.

The role of NMDA glutamate receptors in the lateral habenula on morphine-induced conditioned place preference in rats.

The lateral habenula (LHb) has received special attention due to its role in modulating motivated behavior, stress response, and rewarding and aversive stimuli through monoamine transmission. In the present study, the involvement of the N-methyl-d-aspartate (NMDA) receptors of the LHb in the expression and acquisition phases of morphine-induced conditioned place preference (CPP) was studied in male rats. Bilateral injections of agonist/antagonist (MK-801) of NMDA receptor were performed during the conditioning sessions of the acquisition phase. In other separate groups, drugs were also injected into the LHb before the test session during the expression phase of CPP. A 5-day CPP bias paradigm was used to study the effect of injections of NMDA and MK-801 into the LHb on morphine reward-related behavior. Different doses of NMDA plus morphine reduced the CPP score during the acquisition phase, whereas MK-801 significantly increased conditioning scores during the acquisition phase of CPP. The injection of agonists and antagonists of NMDA receptors in LHb had no significant effect on CPP scores and locomotion during the expression phase of CPP, whereas the motor activity in the acquisition phase was affected by the drugs. The reduction effect of NMDA on the CPP scores during the acquisition phase was blocked by pretreatment with MK-801. Our findings also suggest that NMDA receptors in the LHb may be involved in the acquisition phase of morphine-induced CPP.

Effects of GABAB receptor blockade on lateral habenula glutamatergic neuron activity following morphine injection in the rat: an electrophysiological study.

Background and purpose: The lateral habenula (LHb), a key area in the regulation of the reward system, exerts a major influence on midbrain neurons. It has been shown that the gamma-aminobutyric acid (GABA)- ergic system plays the main role in morphine dependency. The role of GABA type B receptors (GABABRs) in the regulation of LHb neural activity in response to morphine, remains unknown. In this study, the effect of GABABRs blockade in response to morphine was assessed on the neuronal activity in the LHb. Experimental approach: The baseline firing rate was recorded for 15 min, then morphine (5 mg/kg; s.c) and phaclofen (0, 0.5, 1, and 2 µg/rat), a GABABRs' antagonist, were microinjected into the LHb. Their effects on firing LHb neurons were investigated using an extracellular single-unit recording in male rats. Findings/Results: The results revealed that morphine decreased neuronal activity, and GABABRs blockade alone did not have any effect on the neuronal activity of the LHb. A low dose of the antagonist had no significant effect on neuronal firing rate, while blockade with doses of 1 and 2 µg/rat of the antagonist could significantly prevent the inhibitory effects of morphine on the LHb neuronal activity. Conclusion and implications: This result indicated that GABABRs have a potential modulator effect, in response to morphine in the LHb.

Involvement of AMPA receptors of lateral habenula in the expression and acquisition phases of morphine-induced place preference.

The lateral habenula (LHb), known as the brain structure of the epithalamic, plays the main role in depression and drug addiction. The glutamatergic system influences morphine reward. The effect of activation/inhibition of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) in the LHb on different phases of morphine-induced conditioned place preference (CPP) remains unknown. In this research, the effect of bilateral intra-LHb microinjection of AMPARs agonist and antagonist on the acquisition and expression phases of CPP in male rats has been investigated. Different doses of NBQX, the antagonist of AMPARs, in combination with the effective dose of morphine, increased the CPP score during the acquisition phase. While AMPA, the agonist of AMPARs, significantly reduced the conditioning scores in the acquisition phase. Pretreatment with NBQX (0.5 and 1 µg/rat) reversed the inhibitory effect of AMPA (1 µg/rat) on the acquisition phase of morphine-induced CPP. The antagonist (1 µg/rat) increased the effect of a high dose of agonist (2 µg/rat) on CPP. On the other hand, NBQX significantly increased CPP scores during the expression phase. AMPA did not significantly affect CPP scores in the expression phase, but significantly reduced locomotor activity in the test phase. These results confirmed the importance of AMPARs in the LHb in morphine reward. Our data also suggest that injection of an AMPARs antagonist into the LHb may alter the AMPA-induced morphine response in a dose-dependent manner.

The impact of different dark chocolate dietary patterns on synaptic potency and plasticity in the hippocampal CA1 area of the rats under chronic isolation stress.

INTRODUCTION: Although, stress causes brain dysfunction, consumption of dark chocolate (DC) has positive effects on brain functions. The current study investigated the impact of different DC dietary patterns on synaptic potency and plasticity in the hippocampal CA1 area, as well as food intake and body weight in rats under chronic isolation stress. METHODS: Thirty-five rats were allocated into five groups of control, stress, and stress accompanied by three DC dietary patterns (stress-compulsory, -optional, and -restricted DC). The stressed rats on a compulsory diet only received DC and the ones on an optional diet received unlimited chow and/or DC. Also, the stressed rats on a restricted diet each received chow freely and only 4 g DC daily. Subsequently, the slope and amplitude of field excitatory postsynaptic potentials (fEPSPs) were assessed based on the Input-Output (I/O) curves and after the longterm potentiation (LTP). Moreover, food intake and body weight were measured for all groups. RESULTS: The fEPSP slope and amplitude in the I/O curves and after LTP decreased significantly in the stress group compared to the control group. Although the slope and amplitude both enhanced non-significantly in the optional DC diet, these parameters changed significantly in both compulsory and restricted DC dietary patterns compared to the stress group. Also, food intake and body weight decreased significantly in all DC groups. CONCLUSION: The compulsory and restricted DC dietary patterns reversed the harmful effects of chronic isolation stress on the hippocampal synaptic potency, plasticity, learning, and memory. All DC diets, especially compulsory and restricted ones, reduced food intake and body weight.

Effects of treadmill exercise and chronic stress on anxiety-like behavior, neuronal activity, and oxidative stress in basolateral amygdala in morphine-treated rats.

The basolateral amygdala (BLA), which is sensitive to stress, is necessary for reward-seeking behavior and addiction. Regular exercise can produce various positive effects by affecting the BLA. Therefore, we aimed to investigate the effects of chronic stress and treadmill running (TR) on anxiety-like behavior, neuronal activity, lipid peroxidation (measured by malondialdehyde (MDA) levels, a marker for oxidative stress), and total thiol in BLA, in morphine-treated rats. Male Wistar rats were restricted in restraint stress and/or ran on the treadmill and treated with morphine (5 mg/kg) for 21 days. Anxiety-like behavior was evaluated using an elevated plus maze (EPM) and open field tests (OFTs), on day 22. On day 23, neuronal activity in BLA was assessed via single-unit recording. Finally, MDA and total thiol were assessed in BLA. Our results showed that chronic administration of morphine (5 mg/kg) did not affect anxiety-like behavior. However, the morphine-treated rats, subjected to chronic stress and exercise, showed fewer anxiety-like behaviors. Morphine increased BLA's MDA levels but it was prevented by TR. Glutamatergic and GABAergic basal neuronal activities were low in morphine-treated rats but after acute morphine application, there was a significant decrease in GABAergic neuronal activities in the morphine-exercise-stress (Mor-Exe-St) group. The results of this study showed that in morphine-treated rats, stress and exercise or their combination could have either co-directional or opposite effects to the chronic effects of morphine. These results indicate the existence of common pathways similar to endogenous opioids.

Therapeutic effects of exercise-accompanied escitalopram on synaptic potency and long-term plasticity in the hippocampal CA1 area in rats under chronic restraint stress.

Objectives: Administration of antidepressants and exercise are among the therapeutic approaches to chronic stress. Therefore, this study compared the therapeutic effects of different doses of escitalopram, exercise, and exercise-accompanied escitalopram on synaptic potency and long-term plasticity in the hippocampal CA1 area in rats under chronic restraint stress. Materials and Methods: The rats were allocated to different groups. The chronic restraint stress (6 hr/day) continued for 14 days. Injection of escitalopram (10 and 20 mg/kg) and treadmill running (1 hr/day) were performed after the stress induction. The input/output (I/O) functions and LTP induction were evaluated in the hippocampal CA1 area. Results: The fEPSP slope and amplitude after the LTP induction significantly decreased in the chronically stressed group. However, the serum corticosterone levels had significant enhancement in this group. In addition to serum corticosterone levels, the fEPSP slope and amplitude after the LTP induction were enhanced by exercise, escitalopram 20 mg/kg alone, and exercise-accompanied escitalopram 10 and/or 20 mg/kg in chronically stressed groups. Conclusion: Overall, chronic stress impaired synaptic potency and long-term plasticity. These impairments were effectively reversed by exercise, escitalopram 20 mg/kg alone, and exercise-accompanied escitalopram 10 and 20 mg/kg. However, escitalopram 10 mg/kg alone could not alleviate the memory deficits in chronically stressed subjects. Therefore, exercise with both doses of escitalopram seems to have had additive effects on chronic stress conditions.

Involvement of GABAA receptors of lateral habenula in the acquisition and expression phases of morphine-induced place preference in male rats.

The lateral habenula (LHb) is a critical brain structure involved in the aversive response to drug abuse. It has been determined that the gamma-aminobutyric acid (GABA)-ergic system plays the main role in morphine dependency. The role of GABA type A receptors (GABAARs) in LHb on morphine-induced conditioned place preference (CPP) remains unknown. In this study, the effect of bilateral intra-LHb microinjection of GABAAR agonist and antagonist on the acquisition and expression phases of CPP, utilizing a 5-day CPP paradigm in male rats, was evaluated. Subcutaneous administration of different doses of morphine caused a dose-dependent CPP. Intra-LHb microinjection of the GABAAR agonist, muscimol, in combination with morphine (5 mg/kg; subcutaneously) enhanced CPP scores in the acquisition phase of morphine CPP, whereas the GABAAR antagonist, bicuculline, significantly reduced the conditioning scores in the acquisition phase. Furthermore, pretreatment with a high dose of bicuculline reversed the additive effect of muscimol during the acquisition phase, yet the low dose of antagonist had no significant effect on agonist-induced CPP scores. On the other hand, muscimol (3 µg/rat) significantly increased CPP scores in the expression phase but bicuculline did not induce a significant effect on CPP scores. Bicuculline and muscimol microinjections did not affect locomotor activity in the testing sessions. Our results confirm that GABAARs in LHb play an active role in morphine reward. In addition, microinjections of bicuculline/muscimol may alter the morphine response through the GABAergic system.

The Effect of Orexin-2 and Endocannabinoid-1 Antagonists on Neuronal Activity of Hippocampal CA1 Pyramidal Neurons in Response to Tramadol in Rats.

Background: CA1, as a major structure involved in learning and memory, has been shown to be affected by tramadol addiction. Both orexin and endocannabinoid receptors express in CA1 and play an important role in drug dependency. The aim of this study was to evaluate the modulatory effects of orexin-2 (OX2R) and endocannabinoid-1 (CB1R) receptors on neuronal activity in CA1, in response to tramadol in rats. Materials and Methods: Male Wistar rats were divided into 8 groups (n = 6-7); saline-dimethyl sulfoxide (DMSO), tramadol-DMSO, saline-TCS-OX2-29, saline-AM251, tramadol-TCS-OX2-29, tramadol-AM251, saline-TCS-OX2-29-AM251, tramadol-TCS-OX2-29-AM251. Tramadol was injected intraperitoneally, and then, AM251 (1 nmol/0.3 µL), CB1R antagonist and TCS-OX2-29 (1 nmol/0.3 µL), OX2R antagonist, were microinjected individually or concurrently into the CA1. Using in vivo extracellular single-unit recording, the firing of CA1 pyramidal neurons was investigated. Results: Tramadol decreased neuronal activity in CA1 (P < 0.01) but increased it after micro-injection of DMSO. TCS-OX2-29 increased neuronal activity in saline group (P < 0.05) but decreased it in tramadol group. AM251 had no effect on saline group but decreased neuronal activity in tramadol group (P < 0.05). Concurrent micro-injection of TCS-OX2-29 and AM251 had no effect on saline group but decreased neuronal activity in tramadol group (P < 0.05). Conclusions: Our findings suggest that neural activity in CA1 is rapidly affected by acute use of tramadol, and some of these effects may be induced through the endocannabinoid and orexin systems. Thus, the function of endocannabinoid and orexin systems in CA1 may play a role in tramadol addiction.

The Acute Effects of Different doses of Tramadol on Neuronal Activity of Medial Prefrontal Cortex in Rats.

Background: Tramadol is an opioid analgesic with monoamine reuptake inhibitory effects. Although tramadol has been widely used to control pain, there is controversy about the risk of abuse. Therefore, in the present study, the acute effects of tramadol on neuronal activity in the medial prefrontal cortex (mPFC), which is one of the important centers of the reward system, were investigated electrophysiologically. Materials and Methods: Tramadol was injected interperitoneally (12.5 and 25 mk/kg) or subcutaneously (40 mg/kg) and its effect on the firing of mPFC neurons was investigated, using in vivo extracellular single unit recording. Results: Tramadol could not significantly affect neural activity in mPFC, suggesting no acute and rapid effect on mPFC. Conclusions: The present results showed that neural activity in mPFC was not rapidly affected by acute application of tramadol. Since the role of mPFC in tramadol addiction has been elucidated, it can be concluded that these effects may be due to delayed responses or chronic use of tramadol.

Involvement of Basolateral Amygdala Dopamine D1 Receptors in the Acquisition and Expression of Morphine-Induced Place Preference in Rats.

Background: In the present study, the effects of intra-basolateral amygdala (BLA) blockade of dopamine D1 receptor on morphine-induced conditioned place preference (CPP) were investigated in male Wistar rats. Materials and Methods: A 5-day CPP paradigm was used. Morphine was injected subsequently at effective (5 mg/kg) and ineffective (0.5 mg/kg) doses. SCH 23390 (0.5- µg/rat), as a selective D1 receptor antagonist, was microinjected bilaterally into the BLA. Results: Effective dose of morphine induced a significant CPP, and increased the locomotor activity during the testing phase. The results showed that morphine-induced CPP was significantly suppressed by D1 receptors antagonist in BLA in the acquisition phase and caused an aversion even at high doses. The antagonist also significantly prevented CPP expression. Morphine increased the motor activity, but the D1 receptors blockade, significantly reduced it. Conclusions: The findings of this study suggest a possible role for BLA dopamine D1 receptors in reward responses in morphine dependency.

Effects of selective orexin receptor-2 and cannabinoid receptor-1 antagonists on the response of medial prefrontal cortex neurons to tramadol.

Tramadol is widely used to control pain in various diseases, but the relevant mechanisms are less known despite the severe risks of abuse. The medial prefrontal cortex (mPFC) is one of the critical centers of the reward system. Studies have shown that orexins and endocannabinoids are likely to play an important role in addiction. In this study, the effect of orexin receptor-2 (OX2R) and endocannabinoid receptor-1 (CB1R) blockade on the neuronal activity of mPFC was investigated in response to tramadol in male rats. Tramadol was injected intraperitoneally, and its effects on the firing of mPFC pyramidal neurons were investigated using in vivo extracellular single-unit recording. Tramadol affected the pyramidal neuronal activity of the mPFC. AM251 (18 nmol/4 µl), as a selective CB1R antagonist, and TCS-OX2-29 (50 nmol/4 µl), as a selective OX2R antagonist, individually or simultaneously were microinjected into the lateral ventricle of the brain (intracerebroventricular, ICV). The results showed that the ratio of neurons with the excitatory/inhibitory or no responses was significantly changed by tramadol (p < .05). These changes were prevented by blockade of CB1Rs alone or blockade of OX2Rs and CB1Rs simultaneously (p < .05). However, blockade of these receptors in the vehicle group had no significant effect on neuronal activity. The findings of this study indicate the potential role of orexin and endocannabinoid systems in mediating the effects of tramadol in mPFC and the possible interaction between the two systems via OX2 and CB1 receptors. However, further studies are needed to identify these effects by examining intracellular signaling.

Effects of electrical stimulation and temporary inactivation of basolateral amygdala on morphine-induced conditioned place preference in rats.

In the present study, to evaluate the role of the basolateral amygdala (BLA) in morphine addiction, the BLA was stimulated electrically, or inactivated temporarily using lidocaine. The electrical stimulation (ES) was delivered to BLA with low or high intensities (LI or HI: 25 or 150 µA, respectively), and five minutes before morphine administration with effective or ineffective doses, lidocaine was microinjected into the BLA. Using a 5-day conditioned place preference (CPP) paradigm, the dependence on morphine was evaluated. The results showed that LI-ES of BLA induced CPP in both the acquisition and expression phases, in the control and the ineffective dose of morphine groups. HI-ES had no effect on CPP acquisition but induced aversion in the expression, with both effective and ineffective doses of morphine. Inactivation of BLA using lidocaine, inhibited morphine-induced CPP in both acquisition and expression phases. The results of the present study indicate the prominent role of BLA in morphine addiction and dependence. Considering the contradictory results of different intensities of ES, it can be inferred that there are different neural circuits in this area of the brain, in relation to the reward responses.

Wavelet-Based Biphase Analysis of Brain Rhythms in Automated Wake-Sleep Classification.

Many studies in the field of sleep have focused on connectivity and coherence. Still, the nonstationary nature of electroencephalography (EEG) makes many of the previous methods unsuitable for automatic sleep detection. Time-frequency representations and high-order spectra are applied to nonstationary signal analysis and nonlinearity investigation, respectively. Therefore, combining wavelet and bispectrum, wavelet-based bi-phase (Wbiph) was proposed and used as a novel feature for sleep-wake classification. The results of the statistical analysis with emphasis on the importance of the gamma rhythm in sleep detection show that the Wbiph is more potent than coherence in the wake-sleep classification. The Wbiph has not been used in sleep studies before. However, the results and inherent advantages, such as the use of wavelet and bispectrum in its definition, suggest it as an excellent alternative to coherence. In the next part of this paper, a convolutional neural network (CNN) classifier was applied for the sleep-wake classification by Wbiph. The classification accuracy was 97.17% in nonLOSO and 95.48% in LOSO cross-validation, which is the best among previous studies on sleep-wake classification.

Mutual assistance of nucleus accumbens cannabinoid receptor-1 and orexin receptor-2 in response to nicotine: a single-unit study.

BACKGROUND AND PURPOSE: The nucleus accumbens (NAc) express both orexin-2 receptor (OX2R) and cannabinoid receptor type 1 (CB1R). Orexin and cannabinoid regulate the addictive properties of nicotine. In this study, the effect of the CB1R blockade on the electrical activity of NAc neurons in response to nicotine, and its probable interaction with the OX2R in this event, within this area, were examined via the single-unit recording. EXPERIMENTAL APPROACH: The spontaneous firing rate of NAc was initially recorded for 15 min, and then 5 min before subcutaneous injection of nicotine (0.5 mg/kg)/saline, AM251 and TCS-OX2-29 were injected into the NAc. Neuronal responses were recorded for 70 min, after nicotine administration. FINDINGS/RESULTS: Nicotine excited the NAc neurons significantly and intra-NAc microinjection of AM251 (25 and 125 ng/rat), as a selective CB1R antagonist, prevented the nicotine-induced increases of NAc neuronal responses. Moreover, microinjection of AM251 (125 ng/rat), before saline injection, could not affect the percentage of change of the neuronal response. Finally, simultaneous intra-NAc administration of the effective or ineffective doses of AM251 and TCS-OX2-29 (a selective antagonist of OX2R) prevented the nicotine- induced increases of NAc neuronal responses, so that there was a significant difference between the group received ineffective doses of both antagonists and the AM251 ineffective dose. CONCLUSION AND IMPLICATIONS: The results suggest that the CB1R can modulate the NAc reaction to the nicotine, and it can be concluded that there is a potential interplay between the OX2R and CB1R in the NAc, in relation to nicotine.

Lateral habenula electrical stimulation with different intensities in combination with GABAB receptor antagonist reduces acquisition and expression phases of morphine-induced CPP.

The lateral habenula (LHb) plays a principal role in response to aversive stimuli and negative emotional states. In this study, we have evaluated the effects of unilateral electrical stimulation (e-stim) of the LHb on morphine-conditioned place preference (CPP), before or after bilateral injections of Gamma-aminobutyric acid-B receptor (GABABR) antagonist, phaclofen, in male rats. Morphine (5 mg/kg; s.c.) induced a significant CPP, using a 5-day CPP paradigm. Intra-LHb microinjection of phaclofen or the LHb e-stim decreased only the acquisition of CPP. The 150 µA stimulation plus phaclofen significantly suppressed the expression phase but induced aversion in the acquisition of CPP, and an e-stim of 25 µA in combination with the antagonist, significantly prevented only the acquisition phase. The findings of this study confirm the possible role of GABABRs in the LHb on the acquisition and the expression of CPP. These results show that e-stim of LHb alone or plus phaclofen may change the GABA transmission, involving into CPP. Therefore, the GABAergic system, especially through GABABRs, may play a prominent role in the behavioral responses to morphine-induced CPP by LHb stimulation.