Conceptual foundations of acetylcarnitine supplementation in neuropsychiatric long COVID syndrome: a narrative review.

Post-acute sequelae of COVID-19 can present as multi-organ pathology, with neuropsychiatric symptoms being the most common symptom complex, characterizing long COVID as a syndrome with a significant disease burden for affected individuals. Several typical symptoms of long COVID, such as fatigue, depressive symptoms and cognitive impairment, are also key features of other psychiatric disorders such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and major depressive disorder (MDD). However, clinically successful treatment strategies are still lacking and are often inspired by treatment options for diseases with similar clinical presentations, such as ME/CFS. Acetylcarnitine, the shortest metabolite of a class of fatty acid metabolites called acylcarnitines and one of the most abundant blood metabolites in humans can be used as a dietary/nutritional supplement with proven clinical efficacy in the treatment of MDD, ME/CFS and other neuropsychiatric disorders. Basic research in recent decades has established acylcarnitines in general, and acetylcarnitine in particular, as important regulators and indicators of mitochondrial function and other physiological processes such as neuroinflammation and energy production pathways. In this review, we will compare the clinical basis of neuropsychiatric long COVID with other fatigue-associated diseases. We will also review common molecular disease mechanisms associated with altered acetylcarnitine metabolism and the potential of acetylcarnitine to interfere with these as a therapeutic agent. Finally, we will review the current evidence for acetylcarnitine as a supplement in the treatment of fatigue-associated diseases and propose future research strategies to investigate the potential of acetylcarnitine as a treatment option for long COVID.

Sigma-1 receptor and seizures.

Over the last decade, sigma-1 receptor (Sig1R) has been recognized as a valid target for the treatment of seizure disorders and seizure-related comorbidities. Clinical trials with Sig1R ligands are underway testing therapies for the treatment of drug-resistant seizures, developmental and epileptic encephalopathies, and photosensitive epilepsy. However, the direct molecular mechanism by which Sig1R modulates seizures and the balance between excitatory and inhibitory pathways has not been fully elucidated. This review article aims to summarize existing knowledge of Sig1R and its involvement in seizures by focusing on the evidence obtained from Sig1R knockout animals and the anti-seizure effects of Sig1R ligands. In addition, this review article includes a discussion of the advantages and disadvantages of the use of existing compounds and describes the challenges and future perspectives on the use of Sig1R as a target for the treatment of seizure disorders.

Genetic inactivation of the sigma-1 chaperone protein results in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures.

There is a growing body of evidence demonstrating the significant involvement of the sigma-1 chaperone protein in the modulation of seizures. Several sigma-1 receptor (Sig1R) ligands have been demonstrated to regulate the seizure threshold in acute and chronic seizure models. However, the mechanism by which Sig1R modulates the excitatory and inhibitory pathways in the brain has not been elucidated. The aim of this study was to compare the susceptibility to seizures of wild type (WT) and Sig1R knockout (Sig1R-/-) mice in intravenous pentylenetetrazol (PTZ) and (+)-bicuculline (BIC) infusion-induced acute seizure and Sig1R antagonist NE-100-induced seizure models. To determine possible molecular mechanisms, we used quantitative PCR, Western blotting and immunohistochemistry to assess the possible involvement of several seizure-related genes and proteins. Peripheral tissue contractile response of WT and Sig1R-/- mice was studied in an isolated vasa deferentia model. The most important finding was the significantly decreased expression of the R2 subunit of the GABA-B receptor in the hippocampus and habenula of Sig1R-/- mice. Our results demonstrated that Sig1R-/- mice have decreased thresholds for PTZ- and BIC-induced tonic seizures. In the NE-100-induced seizure model, Sig1R-/- animals demonstrated lower seizure scores, shorter durations and increased latency times of seizures compared to WT mice. Sig1R-independent activities of NE-100 included downregulation of the gene expression of iNOS and GABA-A γ2 and inhibition of KCl-induced depolarization in both WT and Sig1R-/- animals. In conclusion, the results of this study indicate that the lack of Sig1R resulted in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures. Our results confirm that Sig1R is a significant molecular target for seizure modulation and warrants further investigation for the development of novel anti-seizure drugs.

Reduced GFAP Expression in Bergmann Glial Cells in the Cerebellum of Sigma-1 Receptor Knockout Mice Determines the Neurobehavioral Outcomes after Traumatic Brain Injury.

Neuroprotective effects of Sigma-1 receptor (S1R) ligands have been observed in multiple animal models of neurodegenerative diseases. Traumatic brain injury (TBI)-related neurodegeneration can induce long-lasting physical, cognitive, and behavioral disabilities. The aim of our study was to evaluate the role of S1R in the development of neurological deficits after TBI. Adult male wild-type CD-1 (WT) and S1R knockout (S1R-/-) mice were subjected to lateral fluid percussion injury, and behavioral and histological outcomes were assessed for up to 12 months postinjury. Neurological deficits and motor coordination impairment were less pronounced in S1R-/- mice with TBI than in WT mice with TBI 24 h after injury. TBI-induced short-term memory impairments were present in WT but not S1R-/- mice 7 months after injury. Compared to WT animals, S1R-/- mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury. TBI induced astrocyte activation in the cortex of WT but not S1R-/- mice. S1R-/- mice presented a significantly reduced GFAP expression in Bergmann glial cells in the molecular layer of the cerebellum compared to WT mice. Our findings suggest that S1R deficiency reduces TBI-induced motor coordination impairments by reducing GFAP expression in Bergmann glial cells in the cerebellum.

Safety and Tolerability of the Anxiolytic and Nootropic Drug Phenibut: A Systematic Review of Clinical Trials and Case Reports.

Phenibut is a nootropic drug that exerts anxiolytic and antinociceptive effects by acting on the GABAB receptor and the α2-δ subunit of voltage-dependent calcium channels. An increased number of reports of dependence to and intoxication by phenibut purchased online on the one hand and the wide prescription of phenibut in Eastern Europe for more than half a century on the other hand have resulted in a number of controversies regarding its use. In this review, we have summarized currently available information from case reports of phenibut dependence and intoxication and safety data from clinical trials. We included 14 dependence and intoxication case reports (16 patients) and reviewed 11 phenibut clinical trials (583 patients). The clinical symptoms in the case reports included cardiovascular effects, insomnia, anxiety and agitation, hallucinations, and depressed level of consciousness. In addition, the doses used (0.5-100 g/day) were much higher than the recommended daily dose (0.25-2 g/day). An analysis of phenibut side effects described in the clinical trials showed adverse events in only 5.66% of patients, and the most reported side effect was somnolence (1.89%). There are discrepancies in the reported side effects of phenibut in clinical trials compared to those reported in cases of online-purchased phenibut dependence and intoxication. The current systematic review provides evidence that, at therapeutic doses, phenibut is safe and well tolerated with minor adverse effects, but questions regarding the quality of phenibut obtained online and the contribution of alcohol and other drug abuse to phenibut dependence and intoxication remain open.

Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice.

R-phenylpiracetam (R-PhP, (4R)-2-(4-phenyl-2-oxopyrrolidin-1-yl)acetamide) is an optical isomer of phenotropil, a clinically-used nootropic drug that improves physical condition and cognition. Recently, R-PhP was shown to bind to the dopamine transporter (DAT). Since growing evidence suggests that dysfunction of the dopaminergic system is associated with persistent neuroinflammation, the aim of this study was to determine whether R-PhP, an inhibitor of DAT, has neuroprotective and anti-inflammatory effects in male mice. The pharmacokinetic profiles of R-PhP in mouse plasma and its bioavailability in brain tissue were assessed. To study possible molecular mechanisms involved in the anti-inflammatory activity of R-PhP, target profiling was performed using radioligand binding and enzymatic activity assays. To clarify the neuroprotective and anti-inflammatory effects of R-PhP, we used a lipopolysaccharide (LPS)-induced endotoxaemia model characterized by reduced body temperature and overexpression of inflammatory genes in the brain. In addition, the antinociceptive and anti-inflammatory effects of R-PhP were tested using carrageenan-induced paw oedema and formalin-induced paw-licking tests. R-PhP (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (ip) and peroral (po) injections. The maximal concentration of R-PhP in the brain tissues was 28 µg/g and 18 µg/g tissue after ip and po administration, respectively. In radioligand binding assays, DAT was the only significant molecular target found for R-PhP. A single ip injection of R-PhP significantly attenuated the LPS-induced body temperature reduction and the overexpression of inflammatory genes, such as tumour necrosis factor-α (TNF-α), interleukin 1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS). Seven-day po pretreatment with R-PhP dose-dependently reduced paw oedema and the antinociceptive response, as shown by the carrageenan-induced paw oedema test. In addition, R-PhP decreased the nociceptive response during the inflammatory phase in the formalin-induced paw-licking test. Our study showed that R-PhP possesses neuroprotective and anti-inflammatory effects, demonstrating the potential of DAT inhibitors as effective therapeutics.

The neuroprotective effects of R-phenibut after focal cerebral ischemia.

R-phenibut is a γ-aminobutyric acid (GABA)-B receptor and α2-δ subunit of the voltage-dependent calcium channel (VDCC) ligand. The aim of the present study was to test the effects of R-phenibut on the motor, sensory and tactile functions and histological outcomes in rats following transient middle cerebral artery occlusion (MCAO). In this study, MCAO was induced by filament insertion (f-MCAO) or endothelin-1 (ET1) microinjection (ET1-MCAO) in male Wistar or CD rats, respectively. R-phenibut was administrated at doses of 10 and 50mg/kg for 14 days in the f-MCAO or 7 days in the ET1-MCAO. The vibrissae-evoked forelimb-placing and limb-placing tests were used to assess sensorimotor, tactile and proprioceptive function. Quantitative reverse transcriptase-PCR was used to detect brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) gene expression in the damaged brain hemisphere. Both f-MCAO and ET1-MCAO resulted in statistically significant impairment of sensorimotor function and brain infarction. R-phenibut at a dose of 10mg/kg significantly improved histological outcome at day 7 in the ET1-MCAO. R-phenibut treatment at a dose of 50mg/kg significantly alleviated reduction of brain volume in damaged hemisphere in both f-MCAO and ET1-MCAO. In R-phenibut treated animals a trend of recovery of tactile and proprioceptive stimulation in the vibrissae-evoked forelimb-placing test was observed. After R-phenibut treatment at a dose of 50mg/kg statistically significant increase of BDNF and VEGF gene expression was found in damaged brain hemisphere. Taken together, obtained results provide evidence for the neuroprotective activity of R-phenibut in experimental models of stroke. These effects might be related to the modulatory effects of the drug on the GABA-B receptor and α2-δ subunit of VDCC.

Hydroxymethylglutaryl-CoA reductase activity is essential for mitochondrial ß-oxidation of fatty acids to prevent lethal accumulation of long-chain acylcarnitines in the mouse liver.

BACKGROUND AND PURPOSE: Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), and exert adverse effects on mitochondrial function, although the mechanisms underlying these effects remain unclear. We used a tamoxifen-induced Hmgcr-knockout (KO) mouse model, a multi-omics approach and mitochondrial function assessments to investigate whether decreased HMGCR activity impacts key liver energy metabolism pathways. EXPERIMENTAL APPROACH: We established a new mouse strain using the Cre/loxP system, which enabled whole-body deletion of Hmgcr expression. These mice were crossed with Rosa26Cre mice and treated with tamoxifen to delete Hmgcr in all cells. We performed transcriptomic and metabolomic analyses and thus evaluated time-dependent changes in metabolic functions to identify the pathways leading to cell death in Hmgcr-KO mice. KEY RESULTS: Lack of Hmgcr expression resulted in lethality, due to acute liver damage caused by rapid disruption of mitochondrial fatty acid ß-oxidation and very high accumulation of long-chain (LC) acylcarnitines in both male and female mice. Gene expression and KO-related phenotype changes were not observed in other tissues. The progression to liver failure was driven by diminished peroxisome formation, which resulted in impaired mitochondrial and peroxisomal fatty acid metabolism, enhanced glucose utilization and whole-body hypoglycaemia. CONCLUSION AND IMPLICATIONS: Our findings suggest that HMGCR is crucial for maintaining energy metabolism balance, and its activity is necessary for functional mitochondrial ß-oxidation. Moreover, statin-induced adverse reactions might be rescued by the prevention of LC acylcarnitine accumulation.

Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor.

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.

Knockout of Tmlhe in mice is not associated with autism spectrum disorder phenotypes or motor dysfunction despite low carnitine levels.

Deletion of exon 2 of the trimethyllysine hydroxylase epsilon (TMLHE) gene was identified in probands with autism spectrum disorder (ASD). TMLHE encodes the first enzyme in carnitine biosynthesis, N6-trimethyllysine dioxygenase (TMLD). Researchers have suggested that carnitine depletion could be important for the development of ASD and cognitive, locomotor and social dysfunctions, but previous findings have been inconclusive regarding the specific role of endogenous carnitine. We developed a mouse knockout model with constitutive TMLD enzyme inactivation that exhibited a significant decrease in the carnitine by more than 90% compared to wild-type (WT) mice. However, we did not observe any significant social, cognitive, or repetitive-behavior changes associated with ASD in the knockout mice; muscle strength and coordination were also not affected. In addition, the life expectancy of knockout mice was similar to that of WT mice. In conclusion, knockout of Tmlh in mice does not induce an ASD phenotype or motor dysfunction despite extremely low carnitine and gamma-butyrobetaine concentrations. Moreover, inactivation of TMLD does not induce a phenotype similar to previously described primary carnitine deficiency; indeed, our results showed that low levels of carnitine sustained adequate energy production, muscle function and social behavior in mice.

Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats.

Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia-reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion.

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy.

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

Moderate traumatic brain injury triggers long-term risks for the development of peripheral pain sensitivity and depressive-like behavior in mice.

As traumatic brain injury (TBI) is one of the major causes of permanent disability, there is increasing interest in the long-term outcome of TBI. While motor deficits, cognitive impairment and longer-term risks of neurodegenerative disease are well-established consequences in animal models of TBI, pain is discussed less often despite its high prevalence. The current study addresses the need to characterize the extent of chronic pain and long-term behavioral impairments induced by moderate lateral fluid percussion injury (latFPI) in mice up to 12 months post-TBI and evaluates the validity of the model. Adult male BALB/c mice were subjected to latFPI, and the results were compared with outcomes in sham-operated mice. Mouse behavior was assessed at 1 and 7 days and 1, 3, 6, 9, and 12 months post-injury using sensory-motor (neurological severity score, NSS), cold (acetone) and mechanical sensitivity (von Frey), depressive-like behavior (tail suspension), locomotor (open field), motor coordination (rotarod) and cognitive (Morris water maze, y-maze, passive avoidance) tests. Animals with TBI demonstrated significantly higher NSS than the sham-operated group for up to 9 months after the injury. Cold sensitization was significantly increased in the contralateral hind paw in the TBI group compared to that of the sham group at 3, 6, and 9 months after TBI. In the von Frey test, the withdrawal threshold of the contralateral and ipsilateral hind paws was reduced at 6 months after TBI and lasted for up to 12 months post-injury. latFPI induced progressive depressive-like behavior starting at 6 months post-injury. No significant deficits were observed in memory, motor coordination or locomotion over the 12-month assessment period. The present study demonstrates that moderate TBI in mice elicits long-lasting impairment of sensory-motor function, results in progressive depression and potentiates peripheral pain. Hence, the latFPI model provides a relevant preclinical setting for the study of the link between brain injury and chronic sequelae such as depression and peripheral pain.

Antidepressive-like Behavior-Related Metabolomic Signatures of Sigma-1 Receptor Knockout Mice.

Sigma-1 receptor (Sig1R) has been proposed as a therapeutic target for neurological, neurodegenerative, and psychiatric disorders, including depression and anxiety. Identifying metabolites that are affected by Sig1R absence and cross-referencing them with specific mood-related behaviors would be helpful for the development of new therapies for Sig1R-associated disorders. Here, we examined metabolic profiles in the blood and brains of male CD-1 background Sig1R knockout (KO) mice in adulthood and old age and correlated them with the assessment of depression- and anxiety-related behaviors. The most pronounced changes in the metabolic profile were observed in the plasma of adult Sig1R KO mice. In adult mice, the absence of Sig1R significantly influenced the amino acid, sphingolipid (sphingomyelin and ceramide (18:1)), and serotonin metabolic pathways. There were higher serotonin levels in plasma and brain tissue and higher histamine levels in the plasma of Sig1R KO mice than in their age-matched wild-type counterparts. This increase correlated with the reduced behavioral despair in the tail suspension test and lack of anhedonia in the sucrose preference test. Overall, these results suggest that Sig1R regulates behavior by altering serotonergic and histaminergic systems and the sphingolipid metabolic pathway.

Total synthesis and in vivo evaluation of 8-deoxypumiliotoxin 193H.

The total synthesis of both the double bond isomers of indolizine alkaloid 8-deoxypumiliotoxin 193H has been accomplished. Both the double bond isomers Z-4 and E-4 induced convulsions and inhibited neuro-muscular activity at a dose of 25 mg/kg after intraperitoneal injection in mice. The lethal dose of Z-4 and E-4 was 100 mg/kg, indicating that 8-deoxypumiliotoxin 193H is 10-times less toxic than the known pumiliotoxin (+)-251 D.

Development of depression-like behavior and altered hippocampal neurogenesis in a mouse model of chronic neuropathic pain.

Chronic-pain patients often suffer from depression. In rodent models of neuropathic pain, animals develop depression-like and anxiety behaviors, indicating a relationship between chronic pain and affective disorders. However, the underlying neurobiological mechanisms linking chronic pain and depression are not yet fully understood. Neurogenesis in the hippocampus is a fundamental process related to brain plasticity. Reduced neurogenesis has been associated with the development of mood disorders and cognitive impairments. The current study aims to elucidate the underlying long-term changes in brain plasticity induced by neuropathic pain in mice at a time point when depression-like behavior has already developed. Furthermore, our focus is set on alterations in neurogenesis in the hippocampus. We found that manifestation of anxiety- and depressive-like behavior as well as cognitive impairment co-occur with decreased survival of newly generated cells but not with impaired proliferative activity or reduced number of immature neurons in the dentate gyrus area of the hippocampus. Moreover, we detected an impairment of differentiation of newly generated cells into mature calbindin-positive neurons, accompanied with a shift towards increased differentiation into astroglial cells. These findings indicate that a reduction in mature functional neurons, rather than reduced proliferation or neuronal progenitor cells, are the long-term changes in hippocampal plasticity that manifest in neuropathic pain conditions after depression-like behavior has developed.

Mildronate exerts acute anticonvulsant and antihypnotic effects.

The effects of mildronate [3-(2,2,2-trimethylhydrazinium) propionate], an inhibitor of L-carnitine biosynthesis and an anti-ischaemic drug, were examined in various in-vivo conditions to investigate the neuropharmacological profile after acute administration. Mildronate (200 mg/kg, acute intraperitoneal administration) exerted anticonvulsant activity in a chemoconvulsant pentylenetetrazole-induced clonic and tonic seizure test but did not change the effects of a convulsion-inducing dose of (+)-bicuculline, a gamma-aminobutyric acid receptor antagonist. Mildronate also dose-dependently inhibited the sleeping time in ethanol-induced loss of righting reflex test. However, in a pentylenetetrazole-induced seizure test, mildronate significantly stimulated the anticonvulsant activity of ethanol. The anticonvulsant activity of mildronate was completely blocked after pre-treatment with alpha2-adrenergic receptor antagonist yohimbine (2 mg/kg) and nitric oxide synthase inhibitor N(G)-nitro-L-arginine (10 mg/kg). These results show that the acute administration of mildronate induces anticonvulsant and antihypnotic effects, which involve alpha2-adrenergic receptor and nitric oxide -dependent mechanisms. These findings indicate that the acute administration of mildronate could be beneficial for the treatment of seizures and alcohol intoxication.

Inhibition of sigma-1 receptors substantially modulates GABA and glutamate transport in presynaptic nerve terminals.

Sigma-1 receptors (Sig-1Rs) have been implicated in many neurological and psychiatric disorders and are a novel target for the treatment of such disorders. Sig-1R expression/activity deficits are linked to neurodegeneration, whereas the mechanisms mediated by Sig-1R are still unclear. Here, presynaptic [3H]GABA and L-[14C]glutamate transport was analysed in rat brain nerve terminals (synaptosomes) in the presence of the Sig-1R antagonist NE-100. NE-100 at doses of 1 and 10 µM increased the initial rate of synaptosomal [3H]GABA uptake, whereas 50 and 100 µM NE-100 decreased this rate, exerting a biphasic mode of action.Antagonists of GABAA and GABAB receptors, flumazenil and saclofen, respectively, prevented an increase in [3H]GABA uptake caused by 10 µM NE-100. L-[14C]glutamate uptake was decreased by 10-100 µM NE-100. A decrease in the uptake of both neurotransmitters mediated by NE-100 (50-100 µM) may have resulted from simultaneous antagonist-induced membrane depolarization, which was measured using the potential-sensitive fluorescent dye rhodamine 6G. The extracellular level of [3H]GABA was decreased by 1-10 µM NE-100, but that of L-[14C]glutamate remained unchanged. The tonic release of [3H]GABA measured in the presence of NO-711 was not changed by the antagonist, suggesting that NE-100 did not disrupt membrane integrity. The KCl- and FCCP-induced transporter-mediated release of L-[14C]glutamate was decreased by the antagonist; this may underlie the neuroprotective action of the antagonist in hypoxia/ischaemia. NE-100 (10-100 µM) decreased the KCl-evoked exocytotic release of [3H]GABA and L-[14C]glutamate, whereas the induction of the release of both neurotransmitters by the Ca2+ ionophore ionomycin was not affected by the antagonist; therefore, the mitigation of KCl-evoked exocytosis was associated with the NE-100-induced dysfunction of potential-dependent Ca2+ channels. Therefore, the Sig-1R antagonist can specifically act in an acute manner at the presynaptic level through the modulation of GABA and glutamate uptake, transporter-mediated release and exocytosis.

Mitochondrial-Protective Effects of R-Phenibut after Experimental Traumatic Brain Injury.

Altered neuronal Ca2+ homeostasis and mitochondrial dysfunction play a central role in the pathogenesis of traumatic brain injury (TBI). R-Phenibut ((3R)-phenyl-4-aminobutyric acid) is an antagonist of the α 2 δ subunit of voltage-dependent calcium channels (VDCC) and an agonist of gamma-aminobutyric acid B (GABA-B) receptors. The aim of this study was to evaluate the potential therapeutic effects of R-phenibut following the lateral fluid percussion injury (latFPI) model of TBI in mice and the impact of R- and S-phenibut on mitochondrial functionality in vitro. By determining the bioavailability of R-phenibut in the mouse brain tissue and plasma, we found that R-phenibut (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (i.p.) and peroral (p.o.) injections. The maximal concentration of R-phenibut in the brain tissues was 0.6 µg/g and 0.2 µg/g tissue after i.p. and p.o. administration, respectively. Male Swiss-Webster mice received i.p. injections of R-phenibut at doses of 10 or 50 mg/kg 2 h after TBI and then once daily for 7 days. R-Phenibut treatment at the dose of 50 mg/kg significantly ameliorated functional deficits after TBI on postinjury days 1, 4, and 7. Seven days after TBI, the number of Nissl-stained dark neurons (N-DNs) and interleukin-1beta (IL-1ß) expression in the cerebral neocortex in the area of cortical impact were reduced. Moreover, the addition of R- and S-phenibut at a concentration of 0.5 µg/ml inhibited calcium-induced mitochondrial swelling in the brain homogenate and prevented anoxia-reoxygenation-induced increases in mitochondrial H2O2 production and the H2O2/O ratio. Taken together, these results suggest that R-phenibut could serve as a neuroprotective agent and promising drug candidate for treating TBI.