The decanoate esters of nandrolone, testosterone, and trenbolone induce steroid specific memory impairment and somatic effects in the male rat.

Long-term use of anabolic androgenic steroids (AAS) in supratherapeutic doses is associated with severe adverse effects, including physical, mental, and behavioral alterations. When used for recreational purposes several AAS are often combined, and in scientific studies of the physiological impact of AAS either a single compound or a cocktail of several steroids is often used. Because of this, steroid-specific effects have been difficult to define and are not fully elucidated. The present study used male Wistar rats to evaluate potential somatic and behavioral effects of three different AAS; the decanoate esters of nandrolone, testosterone, and trenbolone. The rats were exposed to 15 mg/kg of nandrolone decanoate, testosterone decanoate, or trenbolone decanoate every third day for 24 days. Body weight gain and organ weights (thymus, liver, kidney, testis, and heart) were measured together with the corticosterone plasma levels. Behavioral effects were studied in the novel object recognition-test (NOR-test) and the multivariate concentric square field-test (MCSF-test). The results conclude that nandrolone decanoate, but neither testosterone decanoate nor trenbolone decanoate, caused impaired recognition memory in the NOR-test, indicating an altered cognitive function. The behavioral profile and stress hormone level of the rats were not affected by the AAS treatments. Furthermore, the study revealed diverse AAS-induced somatic effects i.e., reduced body weight development and changes in organ weights. Of the three AAS included in the study, nandrolone decanoate was identified to cause the most prominent impact on the male rat, as it affected body weight development, the weights of multiple organs, and caused an impaired memory function.

N-(Heteroaryl)thiophene sulfonamides as angiotensin AT2 receptor ligands.

Two series of N-(heteroaryl)thiophene sulfonamides, encompassing either a methylene imidazole group or a tert-butylimidazolylacetyl group in the meta position of the benzene ring, have been synthesized. An AT2R selective ligand with a Ki of 42 nM was identified in the first series and in the second series, six AT2R selective ligands with significantly improved binding affinities and Ki values of <5 nM were discovered. The binding modes to AT2R were explored by docking calculations combined with molecular dynamics simulations. Although some of the high affinity ligands exhibited fair stability in human liver microsomes, comparable to that observed with C21 undergoing clinical trials, most ligands displayed a very low metabolic stability with t½ of less than 10 min in human liver microsomes. The most promising ligand, with an AT2R Ki value of 4.9 nM and with intermediate stability in human hepatocytes (t½ = 77 min) caused a concentration-dependent vasorelaxation of pre-contracted mouse aorta.

Acute low dose caffeine affects behavior profile and activity, an examination of male rats with high or low anxiety-like behavior.

Anxiety disorders affect up to one third of the population. Caffeine, an adenosine receptor antagonist, is thought to have a dose-dependent effect on anxiety. We recently showed that a high dose of caffeine (50 mg/kg) differentially affected anxiety-like behavior in rats with high or low baseline anxiety-like behavior, replicating findings using relatively high doses in human patient samples. It is not known if low doses of caffeine have similar effects. The elevated plus maze (EPM) was used to categorize male Wistar rats (13 weeks of age) into groups of high or low anxiety-like behavior. Behavior was evaluated using the multivariate concentric square field (MCSF) test and the EPM after a low 10 mg/kg dose of caffeine. Multivariate data analysis demonstrated that caffeine decreased the differences between the high and low anxiety group, whereas the separation remained for the high and low control groups. For the caffeine treated rats, univariate statistics showed an increase in parameters regarding activity in the EPM and duration in the slope of the MCSF. Regarding risk-taking, shelter-seeking, and exploratory behavior, caffeine did not affect the groups differently. In conclusion, these results demonstrate increased activity in the caffeine-treated rats, together with a potentially anxiolytic effect and increased impulsivity that did not differ between the baseline anxiety groups. In contrast to high caffeine doses, a low dose does not generally affect rats with high anxiety at baseline differently than rats with low anxiety-like behavior. Further studies are warranted to fully elucidate the effects of caffeine in anxiety.

Acute caffeine differently affects risk-taking and the expression of BDNF and of adenosine and opioid receptors in rats with high or low anxiety-like behavior.

Anxiety disorders are common psychiatric conditions with a partially elucidated neurobiology. Caffeine, an unspecific adenosine receptor antagonist, is a common psychostimulant with anxiogenic effects in sensitive individuals. High doses of caffeine produce anxiety-like behavior in rats but it is not known if this is specific for rats with high baseline anxiety-like behavior. Thus, the aim of this study was to investigate general behavior, risk-taking, and anxiety-like behavior, as well as mRNA expression (adenosine A2A and A1, dopamine D2, and, µ, κ, δ opioid, receptors, BDNF, c-fos, IGF-1) in amygdala, caudate putamen, frontal cortex, hippocampus, hypothalamus, after an acute dose of caffeine. Untreated rats were screened using the elevated plus maze (EPM), giving each rat a score on anxiety-like behavior based on their time spent in the open arms, and categorized into a high or low anxiety-like behavior group accordingly. Three weeks after categorization, the rats were treated with 50 mg/kg caffeine and their behavior profile was studied in the multivariate concentric square field (MCSF) test, and one week later in the EPM. qPCR was performed on selected genes and corticosterone plasma levels were measured using ELISA. The results demonstrated that the high anxiety-like behavior rats treated with caffeine spent less time in risk areas of the MCSF and resituated towards the sheltered areas, a behavior accompanied by lower mRNA expression of adenosine A2A receptors in caudate putamen and increased BDNF expression in hippocampus. These results support the hypothesis that caffeine affects individuals differently depending on their baseline anxiety-like behavior, possibly involving adenosine receptors. This highlights the importance of adenosine receptors as a possible drug target for anxiety disorders, although further research is needed to fully elucidate the neurobiological mechanisms of caffeine on anxiety disorders.

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures.

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

Nandrolone decanoate and testosterone undecanoate differently affect stress hormones, neurotransmitter systems, and general activity in the male rat.

Anabolic androgenic steroids (AAS) are frequently used to improve physical appearance and strength. AAS are known to affect muscle growth, but many AAS-users also experience psychiatric and behavioral changes after long-term use. The AAS-induced effects on the brain seem to depend on the type of steroid used, but the rationale behind the observed effect is still not clear. The present study investigated and compared the impact of nandrolone decanoate and testosterone undecanoate on body weight gain, levels of stress hormones, brain gene expression, and behavioral profiles in the male rat. The behavioral profile was determined using the multivariate concentric squared field test (MCSF-test). Blood plasma and brains were collected for further analysis using ELISA and qPCR. Nandrolone decanoate caused a reduction in body weight gain in comparison with both testosterone undecanoate and control. Rats receiving nandrolone decanoate also demonstrated decreased general activity in the MCSF. In addition, nandrolone decanoate reduced the plasma levels of ACTH in comparison with the control and increased the levels of corticosterone in comparison with testosterone undecanoate. The qPCR analysis revealed brain region-dependent changes in mRNA expression, where the hypothalamus was identified as the region most affected by the AAS. Alterations in neurotransmitter systems and stress hormones may contribute to the changes in behavior detected in the MCSF. In conclusion, both AAS affect the male rat, although, nandrolone decanoate has more pronounced impact on the physiological and the behavioral parameters measured.

Structurally different anabolic androgenic steroids reduce neurite outgrowth and neuronal viability in primary rat cortical cell cultures.

The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.

The effects of morphine, methadone, and fentanyl on mitochondria: A live cell imaging study.

The important role of mitochondria in maintaining normal brain cell function has been demonstrated in several neurodegenerative diseases where mitochondrial dysfunction is a prominent feature. Accumulating evidence indicates that opioids may induce neuronal cell death and inhibit neurogenesis, two factors that are dependent on normal mitochondrial function. The aim of the present study was to examine the effects of morphine, methadone, and fentanyl on MitoTracker-stained mitochondria. Cells from the neuroblastoma/glioma hybrid cell-line NG108-15 were seeded on 96-well cell culture plates and treated with MitoTracker for 30 min prior to opioid treatment. Morphine, methadone, and fentanyl were added at various concentrations and images of mitochondria were acquired every 30 min for four hours using a high-content imaging device. The parameters total mitochondrial area, mitochondrial network, as well as the number and mean area of mitochondrial objects were analyzed using automated image analysis. Methadone and fentanyl, but not morphine, decreased the mitochondrial network, the number of mitochondrial objects, and increased the mean area of mitochondrial objects. Both methadone and fentanyl altered mitochondrial morphology with no effects seen from morphine treatment. These data suggest that methadone and fentanyl impact mitochondrial morphology negatively, which may be associated with neuronal cell death.

GTPγS-Autoradiography for Studies of Opioid Receptor Functionality.

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR ) that are Gi coupled which make them suitable for studying the receptor functionality. The [35S]GTP γS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the a-subunit of the G-protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.

Assessing Cell Viability Effects of Opioids in Primary Cortical Cells from Rat.

Although the number of studies that have examined the impact of opioids on cell viability is very limited, it has clearly shown that opioids commonly used in the clinic can both decrease neurogenesis and induce cell death. These negative effects induced by opioids are worrying and there is a need for further in-depth investigations addressing the impact of opioids on cell function and cell viability. A useful in vitro approach for studying the effects of opioids on cellular function and viability is using primary cortical cell cultures obtained from embryonic day 17 (E17) rat embryos. These cell cultures contain both neurons and glial cells that provide a more physiologically relevant culture condition when compared to the use of various commercially available cell lines. The primary cortical cells can be cultivated in 96-well plates, treated with various concentrations of opioids, and cell viability functions such as mitochondrial function and membrane integrity can easily be assessed using specific colorimetric assays.

N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor ligands.

A series of meta-substituted acetophenone derivatives, encompassing N-(alkyloxycarbonyl)thiophene sulfonamide fragments have been synthesized. Several selective AT2 receptor ligands were identified, among those a tert-butylimidazole derivative (20) with a Ki of 9.3 nM, that demonstrates a high stability in human liver microsomes (t½ = 62 min) and in human hepatocytes (t½ = 194 min). This methyloxycarbonylthiophene sulfonamide is a 20-fold more potent binder to the AT2 receptor and is considerably more stable in human liver microsomes, than a previously reported and broadly studied structurally related AT2R prototype antagonist 3 (C38). Ligand 20 acts as an AT2R agonist and caused an AT2R mediated concentration-dependent vasorelaxation of pre-contracted mouse aorta. Furthermore, in contrast to imidazole derivative C38, the tert-butylimidazole derivative 20 is a poor inhibitor of CYP3A4, CYP2D6 and CYP2C9. It is demonstrated herein that smaller alkyloxycarbonyl groups make the ligands in this series of AT2R selective compounds less prone to degradation and that a high AT2 receptor affinity can be retained after truncation of the alkyloxycarbonyl group. Binding modes of the most potent AT2R ligands were explored by docking calculations combined with molecular dynamics simulations.

GABAB receptor intracellular signaling: novel pathways for depressive disorder treatment?

Affecting over 320 million people around the world, depression has become a formidable challenge for modern medicine. In addition, an increasing number of studies cast doubt on the monoamine theory of depressive disorder and, worryingly, antidepressant medications only significantly benefit patients with severe depression. Thus, it is not surprising that researchers have shown an increased interest in new theories attempting to explain the pathogenesis of this disease. One example is the excitatory/inhibitory transmission imbalance theory. These abnormalities involve glutamate and γ-aminobutyric acid (GABA) signaling. Studies on GABAB receptors and their antagonists are particularly promising for the treatment of depressive disorders. In this paper, intracellular pathways controlled by GABAB receptors and their links to depression are described, including the impact of ketamine on GABAergic synaptic transmission.

Anabolic androgenic steroids exert a selective remodeling of the plasma lipidome that mirrors the decrease of the de novo lipogenesis in the liver.

INTRODUCTION: The abuse of anabolic androgenic steroids (AASs) is a source of public concern because of their adverse effects. Supratherapeutic doses of AASs are known to be hepatotoxic and regulate the lipoproteins in plasma by modifying the metabolism of lipids in the liver, which is associated with metabolic diseases. However, the effect of AASs on the profile of lipids in plasma is unknown. OBJECTIVES: To describe the changes in the plasma lipidome exerted by AASs and to discuss these changes in the light of previous research about AASs and de novo lipogenesis in the liver. METHODS: We treated male Wistar rats with supratherapeutic doses of nandrolone decanoate and testosterone undecanoate. Subsequently, we isolated the blood plasma and performed lipidomics analysis by liquid chromatography-high resolution mass spectrometry. RESULTS: Lipid profiling revealed a decrease of sphingolipids and glycerolipids with palmitic, palmitoleic, stearic, and oleic acids. In addition, lipid profiling revealed an increase in free fatty acids and glycerophospholipids with odd-numbered chain fatty acids and/or arachidonic acid. CONCLUSION: The lipid profile presented herein reports the imprint of AASs on the plasma lipidome, which mirrors the downregulation of de novo lipogenesis in the liver. In a broader perspective, this profile will help to understand the influence of androgens on the lipid metabolism in future studies of diseases with dysregulated lipogenesis (e.g. type 2 diabetes, fatty liver disease, and hepatocellular carcinoma).

Growth hormone increases dendritic spine density in primary hippocampal cell cultures.

OBJECTIVE: Growth hormone (GH) is widely known for its peripheral effects during growth and development. However, numerous reports also suggest that GH exert pro-cognitive, restorative, and protective properties in the brain. In in vitro studies, the detection of dendritic spines, small protrusions extending from axons, can act as a marker for cognition-related function as spine formation is considered to be associated with learning and memory. Here we show that an acute 24-hour treatment of GH can increase dendritic spine density in primary hippocampal cell cultures. DESIGN: Primary hippocampal cells were harvested from embryonic Wistar rats and cultured for 14 days. Cells were treated with supra-physiological doses of GH (10-1000 nM) and subjected to a high-throughput screening protocol. Images were acquired and analyzed using automated image analysis and the number of spines, spines per neurite length, neurite length, and mean area of spines, was reported. RESULTS: GH treatment (1000 nM) increased the number of dendritic spines by 83% and spines per neurite length by 82% when compared to control. For comparison BDNF, a known inducer of spine densities, produced statistically non-significant increase in this setting. CONCLUSION: The results was found significant using the highest supra-physiological dose of GH, and the present study further confirms a potential role of the hormone in the treatment of cognitive dysfunction.

Toxic Impact of Anabolic Androgenic Steroids in Primary Rat Cortical Cell Cultures.

The use of anabolic androgenic steroids (AASs) among non-athletes is a public health-problem, as abusers underestimate the negative effects associated with these drugs. The present study investigated the toxic effects of testosterone, nandrolone, stanozolol, and trenbolone, and aimed to understand how AAS abuse affects the brain. Mixed cortical cultures from embryonic rats were grown in vitro for 7 days and thereafter treated with increasing concentrations of AASs for 24 h (single-dose) or 3 days (repeated exposure). Cells were co-treated with the androgen-receptor (AR) antagonist flutamide, to determine whether the potential adverse effects observed were mediated by the AR. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase (LDH) release, and caspase-3/7 activity. Nandrolone, unlike the other AASs studied, indicated an effect on mitochondrial activity after 24 h. Furthermore, single-dose exposure with testosterone, nandrolone and trenbolone increased LDH release, while no effect was detected with stanozolol. However, all of the four steroids negatively affected mitochondrial function and resulted in LDH release after repeated exposure. Testosterone, nandrolone, and trenbolone caused their toxic effects by induction of apoptosis, unlike stanozolol that seemed to induce necrosis. Flutamide almost completely prevented AAS-induced toxicity by maintaining mitochondrial function, cellular integrity, and inhibition of apoptosis. Overall, we found that supra-physiological concentrations of AASs induce cell death in mixed primary cortical cultures, but to different extents, and possibly through various mechanisms. The data presented herein suggest that the molecular interactions of the AASs with the AR are primarily responsible for the toxic outcomes observed.

The Protective and Restorative Effects of Growth Hormone and Insulin-Like Growth Factor-1 on Methadone-Induced Toxicity In Vitro.

Evidence to date suggests that opioids such as methadone may be associated with cognitive impairment. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are suggested to be neuroprotective and procognitive in the brain and may therefore counteract these effects. This study aims to explore the protective and restorative effects of GH and IGF-1 in methadone-treated cell cultures. Primary cortical cell cultures were harvested from rat fetuses and grown for seven days in vitro. To examine the protective effects, methadone was co-treated with or without GH or IGF-1 for three consecutive days. To examine the restorative effects, methadone was added for the first 24 h, washed, and later treated with GH or IGF-1 for 48 h. At the end of each experiment, mitochondrial function and membrane integrity were evaluated. The results revealed that GH had protective effects in the membrane integrity assay and that both GH and IGF-1 effectively recovered mitochondrial function and membrane integrity in cells pretreated with methadone. The overall conclusion of the present study is that GH, but not IGF-1, protects primary cortical cells against methadone-induced toxicity, and that both GH and IGF-1 have a restorative effect on cells pretreated with methadone.

Chronic administration of morphine using mini-osmotic pumps affects spatial memory in the male rat.

The use of opioid analgesics to treat non-cancer pain has increased over the years. Many chronic pain patients suffer from numerous adverse effects, such as reduced quality of life, development of dependence, and cognitive impairments. Cognitive processes are regulated by several systems, one of which involves growth hormone (GH) and its secondary mediator insulin-like growth factor-1 (IGF-1), but also glutamatergic transmission, including receptors such as the N-methyl-d-aspartate (NMDA)-receptor complex. In the laboratory, repeated injections are commonly used to establish animal models of long-term or chronic drug exposure. However, in the present study, we aimed to mimic a more human dose regimen using constant drug delivery provided by mini-osmotic pumps implanted subcutaneously in male Sprague Dawley rats. After developing opioid tolerance the cognitive function of rats was studied. Spatial learning and memory capabilities were evaluated using the rat Morris water maze (MWM). Moreover, gene expression related to the GH/IGF-1-axis and the NMDA-receptor system was analyzed using quantitative PCR (qPCR) and plasma levels of IGF-1 were assessed using the ELISA technique. Our results demonstrate that rats exposed to morphine for 27 days display memory impairments in the MWM probe trial. However, the behavioral effects of chronic morphine treatment were not accompanied by any significant differences in terms of mRNA expression or IGF-1 plasma concentration. The animal model used in this study provides a simple and suitable way to investigate the behavioral and neurochemical effects of chronic opioid treatment similar to the exposure seen in human pain patients.

Expression and regulation of CYP17A1 and 3ß-hydroxysteroid dehydrogenase in cells of the nervous system: Potential effects of vitamin D on brain steroidogenesis.

Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). 3ß-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3ß-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3ß-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3ß-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3ß-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3ß-HSD, particularly on the transcriptional level, may play a role in the nervous system.

The mRNA expression of insulin-like growth factor-1 (Igf1) is decreased in the rat frontal cortex following gamma-hydroxybutyrate (GHB) administration.

In recent years, growth hormone (GH), together with its secondary mediators insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2), have been highlighted for their beneficial effects in the central nervous system (CNS), in particular as cognitive enhancers. Cognitive processes, such as learning and memory, are known to be impaired in individuals suffering from substance abuse. In the present study, we investigated the effect of gamma-hydroxybuturate (GHB), an illicit drug used for its sedating and euphoric properties, on genes associated with the somatotrophic axis in regions of the brain important for cognitive function. Sprague Dawley rats (n=36) were divided into three groups and administered either saline, GHB 50mg/kg or GHB 300mg/kg orally for seven days. The levels of Ghr, Igf1 and Igf2 gene transcripts were analyzed using qPCR in brain regions involved in cognition and dependence. The levels of IGF-1 in blood plasma were also determined using ELISA. The results demonstrated a significant down-regulation of Igf1 mRNA expression in the frontal cortex in high-dose treated rats. Moreover, a significant correlation between Igf1 and Ghr mRNA expression was found in the hippocampus, the frontal cortex, and the caudate putamen, indicating local regulation of the GH/IGF-1 axis. To summarize, the current study concludes that chronic GHB treatment influences gene expression of Ghr and Igf1 in brain regions involved in cognitive function.

Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex.

Human growth hormone (GH) displays promising protective effects in the central nervous system after damage caused by various insults. Current evidence suggests that these effects may involve N-methyl-d-aspartate (NMDA) receptor function, a receptor that also is believed to play a role in opioid-induced neurotoxicity. The aims of the present study were to examine the acute toxic effects of methadone, an opioid receptor agonist and NMDA receptor antagonist, as well as to evaluate the protective properties of recombinant human GH (rhGH) on methadone-induced toxicity. Primary cortical cell cultures from embryonic day 17 rats were grown for 7days in vitro. Cells were treated with methadone for 24h and the 50% lethal dose was calculated and later used for protection studies with rhGH. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase release, and caspase activation. Furthermore, the mRNA expression levels of NMDA receptor subunits were investigated following methadone and rhGH treatment using quantitative PCR (qPCR) analysis. A significant protective effect was observed with rhGH treatment on methadone-induced mitochondrial dysfunction and in methadone-induced LDH release. Furthermore, methadone significantly increased caspase-3 and -7 activation but rhGH was unable to inhibit this effect. The mRNA expression of the NMDA receptor subunit GluN1, GluN2a, and GluN2b increased following methadone treatment, as assessed by qPCR, and rhGH treatment effectively normalized this expression to control levels. We have demonstrated that rhGH can rescue cells from methadone-induced toxicity by maintaining mitochondrial function, cellular integrity, and NMDA receptor complex expression.