Regional-specific changes in rat brain BDNF in a model of methamphetamine abuse.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays key roles in neuronal protection and synaptic plasticity. Changes in BDNF are associated with various pathological conditions, including methamphetamine (meth) addiction, although the effects of meth on BDNF expression are not always consistent. We have previously demonstrated region-specific effects of a chronic meth regime on BDNF methylation and expression in the rat brain. This study aims to determine the effect of chronic meth administration on the expression of BDNF protein using immunohistochemistry in the rat frontal cortex and hippocampus. Novel object recognition (NOR) as a measure of cognitive function was also determined. Male Sprague Dawley rats were administered a chronic escalating dose (0.1-4 mg/kg over 14 days) (ED) of meth or vehicle; a subgroup of animals receiving meth were also given an acute "binge" (4x6mg) dose on the final day before NOR testing. The results showed that hippocampal CA1 BDNF protein was significantly increased by 72 % above control values in the ED-binge rats, while other hippocampal regions and frontal cortex were not significantly affected. Meth-administered animals also demonstrated deficits in NOR after 24 h delay. No significant effect of the additional binge dose on BDNF protein or NOR findings was apparent. This finding is consistent with our previous results of reduced DNA methylation and increased expression of the BDNF gene in this region. The hippocampal BDNF increase may reflect an initial increase in a protective factor produced in response to elevated glutamate release resulting in neurodegenerative excitotoxicity.

Proteomic association with age-dependent sex differences in Wisconsin Card Sorting Test performance in healthy Thai subjects.

Sex differences in cognitive function exist, but they are not stable and undergo dynamic change during the lifespan. However, our understanding of how sex-related neural information transmission evolves with age is still in its infancy. This study utilized the Wisconsin Card Sorting Test (WCST) and the label-free proteomics method with bioinformatic analysis to investigate the molecular mechanisms underlying age-related sex differences in cognitive performance in 199 healthy Thai subjects (aged 20-70 years), as well as explore the sex-dependent protein complexes for predicting cognitive aging. The results showed that males outperformed females in two of the five WCST sub-scores: %Corrects and %Errors. Sex differences in these scores were related to aging, becoming noticeable in those over 60. At the molecular level, differently expressed individual proteins and protein complexes between both sexes are associated with the potential N-methyl-D-aspartate type glutamate receptor (NMDAR)-mediated excitotoxicity, with the NMDAR complex being enriched exclusively in elderly female samples. These findings provided a preliminary indication that healthy Thai females might be more susceptible to such neurotoxicity, as evidenced by their cognitive performance. NMDAR protein complex enrichment in serum could be proposed as a potential indication for predicting cognitive aging in healthy Thai females.

A solid lipid particle formulation of long pepper extract reduces pain and astrocyte activation in a rat model of neuropathic pain.

OBJECTIVES: To investigate the effects of solid lipid microparticle (SLM) creams containing a long pepper extract (LPE) or piperine on neuropathy-related pain and the expression of glial fibrillary acidic protein (GFAP) as a measure of astrogliosis. METHODS: Neuropathic pain in male Spraque Dawley rats was induced by sciatic nerve ligation (SNL) and followed by treatment with LPE-SLM, piperine-SLM, capsaicin or vehicle creams. The pain score was assessed by thermal hyperalgesia test. The GFAP expression in the spinal cord was determined by immunohistochemistry. RESULTS: Pain scores were significantly increased after SNL and decreased when treated by LPE-SLM. The number of GFAP immunopositive cells was significantly increased in the SNL rats. Treated by LPE-SLM and capsaicin creams resulted in a significant reduction of the number of GFAP immunopositive cells. The LPE-SLM treated rats showed greater effects than the piperine and capsaicin preparations. CONCLUSIONS: The LPE-SLM cream has a potential effect on pain attenuation via a decrease of spinal astrocyte activation-related mechanism. The LPE in SLM preparation could provide an alternative therapeutic strategy for treating neuropathic pain.

Cholinergic-estrogen interaction is associated with the effect of education on attenuating cognitive sex differences in a Thai healthy population.

The development of human brain is shaped by both genetic and environmental factors. Sex differences in cognitive function have been found in humans as a result of sexual dimorphism in neural information transmission. Numerous studies have reported the positive effects of education on cognitive functions. However, little work has investigated the effect of education on attenuating cognitive sex differences and the neural mechanisms behind it based on healthy population. In this study, the Wisconsin Card Sorting Test (WCST) was employed to examine sex differences in cognitive function in 135 Thai healthy subjects, and label-free quantitative proteomic method and bioinformatic analysis were used to study sex-specific neurotransmission-related protein expression profiles. The results showed sex differences in two WCST sub-scores: percentage of Total corrects and Total errors in the primary education group (Bayes factor>100) with males performed better, while such differences eliminated in secondary and tertiary education levels. Moreover, 11 differentially expressed proteins (DEPs) between men and women (FDR<0.1) were presented in both education groups, with majority of them upregulated in females. Half of those DEPs interacted directly with nAChR3, whereas the other DEPs were indirectly connected to the cholinergic pathways through interaction with estrogen. These findings provided a preliminary indication that a cholinergic-estrogen interaction relates to, and might underpin, the effect of education on attenuating cognitive sex differences in a Thai healthy population.

Differential protein expression of GABA A receptor alpha 1 subunit and calbindin in rat spermatozoa associated with proteomic analysis in testis following methamphetamine administration.

Methamphetamine (METH) can induce spermatogenesis impairment, testicular apoptosis, and abnormal sperm quality. It also promotes changes in the expression of receptors for sex hormones and neurotransmitters, including GABA receptors in the testis. Proteomic assessment focusing on proteins involved in the calcium signalling pathway in the testis can facilitate diagnostic factors contributing to testicular and sperm functions, especially those related to spermatogenesis and fertilisation. In this study, we proposed to determine the localisation and differential expression of GABA A receptor alpha 1 subunit (GABA A-α1) in the spermatozoa of METH-administered rats. The differential proteomic profile of the testis was also observed by focusing on proteins in the KEGG pathways belonging to the calcium signalling pathway. There were 212 differentially expressed proteins in the rat testis, based on the cut-off value of 1.2-fold change. Most of those proteins, 13 proteins, were classified in the calcium signalling pathway, including 4 down-regulated and 9 up-regulated proteins. An immunolocalisation study of the GABA A-α1 receptor and calbindin revealed their localisation in the equatorial segment of the head in the rat spermatozoa. The expression of calbindin is also found in the middle piece of sperm. An increase in GABA A-α1 receptor in rat spermatozoa was correlated with an increase in abnormal sperm motility and morphology after methamphetamine exposure. Moreover, calbindin expression in sperm decreased in METH-administered rats. All our findings demonstrate that METH influences intracellular calcium homeostasis by acting through the calcium signalling pathway-associated proteins. Moreover, it might disrupt ion homeostasis in sperm through the GABA A-α1 receptor and calbindin, triggering a change in intracellular calcium and chloride ions. These changes may cause abnormalities in spermatogenesis, testicular apoptosis, and sperm quality impairment.

Changes of BDNF exon IV DNA methylation are associated with methamphetamine dependence.

Aim: We investigated DNA methylation of BDNF in methamphetamine (METH) dependence in humans and an animal model. Materials & methods:BDNF methylation at exon IV was determined by pyrosequencing of blood DNA from METH-dependent and control subjects, and from rat brain following an escalating dose of METH or vehicle. Bdnf expression was determined in rat brain. Results:BDNF methylation was increased in human METH dependence, greatest in subjects with psychosis and in prefrontal cortex of METH-administered rats; rat hippocampus showed reduced Bdnf methylation and increased gene expression. Conclusion:BDNF methylation is abnormal in human METH dependence, especially METH-dependent psychosis, and in METH-administered rats. This may influence BDNF expression and contribute to the neurotoxic effects of METH exposure.

Lay abstract The effects of methamphetamine (METH), an addictive psychostimulant drug, on changes of DNA methylation of an important regulator of neuronal survival, BDNF, were examined in blood of METH-dependent patients and in the brain of METH-administered rats. BDNF methylation was increased in patients and in the prefrontal cortex of METH-administered rats, while rat hippocampus showed a reduction of Bdnf methylation, with an equivalent increase in gene expression. The methylation increases in humans were greatest in those with a METH-induced psychosis. Although a relationship between Bdnf methylation and its expression has not been proven, changes of BDNF DNA methylation are associated with METH dependence, especially METH-dependent psychosis, suggesting that METH neurotoxicity may relate to the effects of changes in BDNF methylation.

High mRNA expression of GABA receptors in human sperm with oligoasthenoteratozoospermia and teratozoospermia and its association with sperm parameters and intracytoplasmic sperm injection outcomes.

OBJECTIVE: This study investigated the mRNA expression of gamma-aminobutyric acid (GABA) receptors in the sperm of oligoasthenoteratozoospermic (OAT) and teratozoospermic (TER) men compared to normozoospermic (NOR) men, as well as the relationships between GABA receptor expression and sperm parameters, fertilization rate, and embryo quality. METHODS: The mRNA expression of GABA A-α1 and GABA B-R2 receptors in sperm was examined using reverse transcription-polymerase chain reaction in three groups of patients: NOR (n=32), OAT (n=22), and TER (n=45). The fertilization rate and embryo quality were assessed in 35 patients undergoing Intracytoplasmic sperm injection (ICSI; 10 NOR, 10 OAT, and 15 TER men). RESULTS: OAT men had significantly higher mRNA expression of GABA A-α1 and GABA B-R2 receptors in sperm than NOR men; however, the difference between TER and NOR men was not significant. High levels of these receptors were significantly correlated with low sperm concentration, motility, and morphology, as well as the rate of good-quality embryos (GQEs) at the cleavage stage after ICSI. Patients whose female partners had a >50% GQE rate at the cleavage stage had significantly lower levels of GABA A-α1 receptor expression than those whose partners had a ≤50% GQE rate. CONCLUSION: Our findings indicate that mRNA levels of GABA receptors in human sperm are correlated with poor sperm quality and associated with embryo development after ICSI treatment. The GABA A-α1 receptor in sperm has a stronger relationship with embryo quality at the cleavage stage than the GABA B-R2 receptor.

Pharmacogenetics of drug dependence: Polymorphisms of genes involved in GABA neurotransmission.

GABA plays a critical role in brain reward pathways via projecting signals from the ventral tegmental area to the nucleus accumbens. Activation of the reward circuitry by abused drugs induces abnormalities of GABA neurotransmission. Recent studies have indicated the involvement of GABAergic genes in the mechanism of drug dependence and its consequences. The aim of this paper is to provide a brief review of association studies of GABA-related genes with drug dependence. Single nucleotide polymorphisms (SNPs) in genes involved in GABA neurotransmission such as GABA receptor genes (GABR, GABBR), and glutamic acid decarboxylase genes (GAD) are the focus of this review as potential risk factors for drug dependence and its consequence psychosis.

Pharmacogenetics of drug dependence: Polymorphisms of genes involved in glutamate neurotransmission.

Multiple studies provide evidence to support dysfunction of glutamate neurotransmission in the pathogenesis of drug dependence. Pharmacogenetic investigation of glutamate-related genes has provided further support for the involvement of this neurotransmitter in the risk of, and consequences of, drug abuse and dependence. This paper aims to provide a brief review of these association studies. Findings involving single nucleotide polymorphisms (SNPs) in glutamate receptor genes (GRIN, GRIA) and glutamate transporter genes (SLC1A, SLC17A) are reviewed as potential risk factors. As yet a clear perspective of the functional consequences and interactions of the various reported findings is lacking.

Association study of the functional Catechol-O-Methyltranferase (COMT) Val158Met polymorphism on executive cognitive function in a Thai sample.

Catechol-O-Methyltranferase (COMT) plays a crucial role in the removal of cortical dopamine and is strongly implicated in human executive function. Numerous studies have reported associations of the COMT Val158Met (rs4680) polymorphism with executive function in healthy subjects. However, little work has investigated this in the Thai population and the relationship of age and education with this association remains unclear. Therefore, this study was designed to investigate the association of this polymorphism of the COMT gene with executive cognitive brain function in healthy subjects and the relationship with age and education. The Wisconsin Card Sorting Test (WCST) was performed to assess executive function in 254 healthy Thai subjects (aged 20-72 years). The results showed a significant association of rs4680 with executive function, in which Val/Met heterozygotes demonstrated better cognitive set shifting performance. Moreover, Met allele carriers showed a significantly stronger effect in the categories completed score than did Val homozygotes. Furthermore, age and education also showed a significant association with COMT genotype and WCST. These results revealed that executive cognitive function is associated with COMT genotype and influenced by age and/or education level in a Thai sample.

Parvalbumin Promoter Methylation Altered in Major Depressive Disorder.

Aims: To determine the extent of DNA methylation of parvalbumin gene (PVALB) promoter in major depressive disorder (MDD) patients with and without suicide attempt in comparison with healthy controls. Methods: The extracted DNA from dried blood spots of MDD patients (n = 92) including non-suicidal MDD and suicidal-MDD subgroups (n = 45 and n = 47, respectively) and age-matched control subjects (n = 95) was used for DNA methylation analysis at four CpG sites in the promoter sequence of PVALB by pyrosequencing. Results: The PVALB methylation was significantly increased at CpG2 and decreased at CpG4 in the MDD group compared to the control group, while there was no difference between non-suicidal MDD and suicidal-MDD subgroups. A significant inverse correlation of severity of MDD was indicated only for CpG4. Conclusion: This study provides the first evidence of abnormalities of PVALB promoter methylation in MDD and its correlation with MDD severity indicating a role for epigenetics in this psychiatric disorder.

GABAergic Alterations in the Rat Testis after Methamphetamine Exposure.

Gamma-aminobutyric acid (GABA), GABA-A receptors and GABA transporter 1 (GAT1) were reported to be involved in the proliferation of Leydig cells, testosterone production and spermatogenesis. Since methamphetamine (METH) has been reported to have adverse effects on testis and its functions, the aim of this study was therefore to determine the changes of GABAergic activity in testis after METH exposure. Male Sprague-Dawley rats were divided into control, acute binge (AB-METH), escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups. After sacrifice, rat testes were removed and used to estimate GABA concentration and the expression of GABA-A receptor, GAD1, GAD2 and GAT1 genes by using HPLC and RT-PCR, respectively. The GABA concentration was significantly increased in all METH-administrated groups. In addition, significant increases of GABA-A α1 receptor and GAD1 genes expression were found in the ED-binge METH group. Gene expressions of GAT1 were numerically decreased in all METH-administrated rats and reached significant in the ED METH group. These results indicated a compensatory upregulation of GABA production and its functions in testis after METH exposure. Thus, these changes might represent a homeostatic response of GABAergic to the adverse effects of METH.

Recovery effect of pre-germinated brown rice on the changes of sperm quality, testicular structure and androgen receptor expression in a rat model of drug addiction.

Drug addiction is reported to have adverse effects in male reproduction. Dextromethorphan (DXM) administration was used in this study as a model of addiction in rats, and various treatments including the use of pre-germinated brown rice (PGBR) were investigated for their effects on the changes of sperm quality, testicular structure and androgen receptor (AR) expressions in rats receiving DXM. The results demonstrated that these animals showed significant reduction in all parameters of sperm quality, an increase in abnormal testicular structure and decreased androgen receptor expression in spermatogenic, Sertoli and Leydig cells. However, different effects of the treatments applied in this study were observed with the greatest recovery effect from treatment with PGBR. Sperm motility and sperm concentration reverted to normal after treatment with PGBR for 60 days. Moreover, all parameters of testicular structure also returned to normal after 60 days of PGBR treatment, as well as AR expression in Sertoli and Leydig cells. Therefore, we have demonstrated that PGBR treatment can reverse the changes in sperm quality, testicular structure and AR expression in addicted animals and PGBR may be a novel therapeutic strategy for the treatment of drug addiction.

Genetic variation of GRIA3 gene is associated with vulnerability to methamphetamine dependence and its associated psychosis.

Methamphetamine (METH) is an addictive psychostimulant drug commonly leading to schizophrenia-like psychotic symptoms. Disturbances in glutamatergic neurotransmission have been proposed as neurobiological mechanisms and the α-amino-3 hydroxy-5 methyl-4 isoxazole propionic acid (AMPA) glutamate receptor has been implicated in these processes. Moreover, genetic variants in GRIAs, genes encoding AMPA receptor subunits, have been observed in association with both drug dependence and psychosis. We hypothesized that variation of GRIA genes may be associated with METH dependence and METH-induced psychosis. Genotyping of GRIA1 rs1428920, GRIA2 rs3813296, GRIA3 rs3761554, rs502434 and rs989638 was performed in 102 male Thai controls and 100 METH-dependent subjects (53 with METH-dependent psychosis). We observed no evidence of association with METH dependence and METH-dependent psychosis in the GRIA1 and GRIA2 polymorphisms, nor with single polymorphisms rs3761554 and rs989638 in GRIA3. An association of GRIA3 rs502434 was identified with both METH dependence and METH-dependent psychosis, although this did not withstand correction for multiple testing. Combining the analysis of this site with the previously-demonstrated association with BDNF rs6265 resulted in a highly significant effect. These preliminary findings indicate that genetic variability in GRIA3 may interact with a functional BDNF polymorphism to provide a strong risk factor for the development of METH dependence in the Thai population.

Increased DNA methylation in the parvalbumin gene promoter is associated with methamphetamine dependence.

AIM: The parvalbumin (PV)-containing subgroup of GABAergic neurons is particularly affected in schizophrenia and animal models of psychosis, including after methamphetamine (METH) administration. We investigated whether METH dependence and METH-induced psychosis may involve an effect on DNA methylation of the PVALB promoter. MATERIALS & METHODS: The methylation of a PVALB promoter sequence was determined in 100 METH-dependent and 102 control subjects using pyrosequencing. RESULTS: A significant increase in PVALB methylation was observed in METH dependence and METH-induced psychosis. No significant effect on long interspersed nucleotide element-1 methylation, a measure of global DNA methylation, was observed. CONCLUSION: These results demonstrate a specific association between elevated PVALB methylation and METH-induced psychosis. This finding may contribute to the GABAergic deficits associated with METH dependence.

Association of polymorphisms in GAD1 and GAD2 genes with methamphetamine dependence.

AIM: Association between polymorphisms in GAD genes and methamphetamine (METH) dependence was investigated in the Thai population. MATERIALS & METHODS: Genotypes of rs769404 and rs701492 in GAD1 and rs2236418 in GAD2 polymorphisms were determined in 100 METH-dependent male subjects and 102 matched controls. RESULTS: The genotype and allele frequencies of rs2236418 (GAD2) were associated with METH dependence and METH with psychosis, in which the G allele was related to increased risk. The presence of the rs769404-rs701492 (GAD1) C-C haplotype was associated with METH psychosis. CONCLUSION: This study indicates that genetic variability in GAD1 and GAD2 contributes to risk of METH dependence and METH psychosis in the Thai population and indicates the role of the GABAergic system in these disorders.

Effect of Methamphetamine Exposure on Expression of Calcium Binding Proteins in Rat Frontal Cortex and Hippocampus.

Methamphetamine (METH) is a psychostimulant drug with potent effects on the central nervous system that can cause psychotic symptoms similar to those of schizophrenia. Specific alterations in GABAergic neuronal markers have been reported in schizophrenia and animal models of psychotic illness. The aim of this study was to determine whether there are changes in subpopulations of GABAergic neurons, defined by the presence of calcium binding proteins (CBPs), in animal models of METH abuse. Rats received acute (Binge) doses of 4 × 6 mg/kg, a chronic escalating dose regime (0.1-4 mg/kg over 14 days) or a combination of the two and were compared with a vehicle-administered control group. Brains were taken and sections of frontal cortex (Cg1) and hippocampus (dentate gyrus and CA1-3 regions) underwent immunostaining for three CBPs [parvalbumin (PV), calbindin (CB), and calretinin (CR)]. Significant decreases in PV-immunoreactive (IR) neurons in each METH group and all regions were observed. Smaller METH-induced deficits in CB-IR cells were observed, reaching significance primarily following chronic METH regimes, while CR-IR was significantly reduced only in frontal cortex following chronic administration. These results suggest that METH regimes in rats can induce selective deficits in GABAergic neuronal subtypes similar to those seen in schizophrenia and may underlie the psychosis and/or cognitive impairment that can occur in METH abuse and dependence.

Does elevated peripheral benzodiazepine receptor gene expression relate to cognitive deficits in methamphetamine dependence?

BACKGROUND: Methamphetamine (METH) is a neurotoxin and psychostimulant drug with potent effects on the central nervous system. With chronic METH administration, an inflammatory glial response is observed as a result of METH-induced neurotoxicity. One inflammatory marker is the peripheral benzodiazepine receptor (PBR). OBJECTIVE: The purpose of the present study was to determine whether PBR expression is changed in METH dependence and whether the changes relate to cognitive deficits. METHODS: Reverse transcriptase-polymerase chain reaction was used to investigate PBR gene expression in blood samples taken from 14 male subjects with METH dependence and 14 controls. RESULTS: The results showed a significant increase in PBR gene expression in METH dependence, suggestive of a systemic inflammatory response. The increase remained elevated for more than 1 year following abstinence from METH use, but eventually returned to normal. Subjects with elevated PBR also exhibited a deficit in one domain of the Wisconsin Card Sorting Test. CONCLUSION: The results suggest that systemic inflammatory effects can be associated with chronic METH abuse, and this may relate to the cognitive deficits seen in METH dependence. Copyright © 2016 John Wiley & Sons, Ltd.

Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERß were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERß immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERß were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERß expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm.

BDNF (Val66Met) genetic polymorphism is associated with vulnerability for methamphetamine dependence.

AIM: Association of the brain-derived neurotrophic factor (BDNF) genetic polymorphism rs6265 (Val66Met) with methamphetamine (METH) dependence and METH-induced psychosis was investigated in the Thai population. MATERIALS & METHODS: The rs6265 genotype was determined in 100 male METH-dependent subjects and 102 controls using a real-time PCR high-resolution melt (RT-PCR-HRM) assay. RESULTS: The rs6265 genotype demonstrated significant differences in distribution between METH-dependent subjects and controls in which the frequency of GG genotype versus A-allele carriers was associated with METH dependence. Moreover, a significant effect of genotype on the occurrence of psychosis was found, with a lower frequency of GG genotype associated with METH-induced psychosis. CONCLUSION: The present findings indicate that rs6265 is associated with METH dependence in the Thai population, with the GG genotype greater in METH-dependent subjects but reducing the emergence of METH-dependent psychosis.