The Role of HPA Axis and Allopregnanolone on the Neurobiology of Major Depressive Disorders and PTSD.

Under stressful conditions, the hypothalamic-pituitary-adrenal (HPA) axis acts to promote transitory physiological adaptations that are often resolved after the stressful stimulus is no longer present. In addition to corticosteroids (e.g., cortisol), the neurosteroid allopregnanolone (3α,5α-tetrahydroprogesterone, 3α-hydroxy-5α-pregnan-20-one) participates in negative feedback mechanisms that restore homeostasis. Chronic, repeated exposure to stress impairs the responsivity of the HPA axis and dampens allopregnanolone levels, participating in the etiopathology of psychiatric disorders, such as major depressive disorder (MDD) and post-traumatic stress disorder (PTSD). MDD and PTSD patients present abnormalities in the HPA axis regulation, such as altered cortisol levels or failure to suppress cortisol release in the dexamethasone suppression test. Herein, we review the neurophysiological role of allopregnanolone both as a potent and positive GABAergic neuromodulator but also in its capacity of inhibiting the HPA axis. The allopregnanolone function in the mechanisms that recapitulate stress-induced pathophysiology, including MDD and PTSD, and its potential as both a treatment target and as a biomarker for these disorders is discussed.

Neurosteroids and Neurotrophic Factors: What Is Their Promise as Biomarkers for Major Depression and PTSD?

Even though major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) are among the most prevalent and incapacitating mental illnesses in the world, their diagnosis still relies solely on the characterization of subjective symptoms (many of which are shared by multiple disorders) self-reported by patients. Thus, the need for objective measures that aid in the detection of and differentiation between psychiatric disorders becomes urgent. In this paper, we explore the potential of neurosteroids and neurotrophic proteins as biomarkers for MDD and PTSD. Circulating levels of the GABAergic neuroactive steroid, allopregnanolone, are diminished in MDD and PTSD patients, which corroborates the finding of depleted neurosteroid levels observed in animal models of these disorders. The neurotrophic protein, brain-derived neurotropic factor (BDNF), is also reduced in the periphery and in the brain of MDD patients and depressed-like animals that express lower neurosteroid levels. Although the role of BDNF in PTSD psychopathology seems less clear and merits more research, we propose a causal link between allopregnanolone levels and BDNF expression that could function as a biomarker axis for the diagnosis of both MDD and PTSD.

Effects of neonatal dopaminergic lesion on oral cocaine self-administration in rats: Higher female vulnerability to cocaine consumption.

The dopaminergic system is associated with cocaine-seeking behaviors, being influenced by other neurotransmitters such as GABA and deregulated by chronic cocaine self-administration. Administration of 6-hydroxydopamine (6-OHDA) to neonatal rats produces a depletion of brain dopamine, mainly, that results in behavioral alterations in adulthood. This model can be applied to better understanding of the role of the dopaminergic system in cocaine use and how its behavioral effects can modulate drug intake. Though there are well-established sex differences in the pattern of drug use, there are no published studies investigating sex-dependent effects of neonatal lesions with 6-OHDA on cocaine self-administration nor regarding GABAA receptor (GABAAR) subunits expression. Herein, neurotoxic lesion was induced in male and female neonatal rats by intracisternal injection of 6-OHDA at PND 4, and locomotion was evaluated before and after cocaine self-administration. Cocaine was diluted in a sweet solution (sucrose 1.5%) and offered for 27 consecutive 3-h daily sessions via a dispenser for oral intake, in an operant chamber under a fixed-ratio 1 (FR1) schedule. The 6-OHDA lesion reduced oral cocaine self-administration in male and female rats. Female rats, independent of dopaminergic condition, consumed more cocaine-containing solution than sucrose-only solution. Furthermore, as expected, 6-OHDA-lesioned animals presented a higher basal locomotor activity when compared to sham rats. We evaluated GABAAR subunit expression and found no statistically significant differences between rats that self-administered a sucrose-only solution and those that self-administered a cocaine-containing solution. Even when the reward system is depleted, some behavioral differences remain in females, providing more data that highlight the female vulnerability to cocaine consumption.

Low-cost apparatus for cigarette smoke exposure in rats.

BACKGROUND: The effects of tobacco smoke on the central nervous system are usually studied with isolated nicotine, ignoring other compounds present in cigarette smoke. The few studies that use in vivo whole-body cigarette smoke exposure are usually performed in expensive commercial apparatus. NEW METHOD: We presented a feasible, safe, and low-cost apparatus for cigarette smoke exposure in rodents. RESULTS: Rats exposed to cigarette smoke in this apparatus showed cotinine levels similar to human active smokers. Additional results showed that cigarette smoke exposure increased glutamate and aspartic acid levels and decreased leucine, isoleucine, ornithine, phenylalanine, and tryptophan levels in the cerebrospinal fluid of rats. COMPARISON WITH EXISTING METHOD(S): Our apparatus is feasible, safe, and costs 67-fold less than a commercial automatized smoking machine. Beyond the low cost, it does not require specialized knowledge for building or maintenance. CONCLUSIONS: We concluded that our low-cost apparatus is reliable and reproduces cigarette smoke use in humans.

The role of allopregnanolone in depressive-like behaviors: Focus on neurotrophic proteins.

Allopregnanolone (3α,5α-tetrahydroprogesterone; pharmaceutical formulation: brexanolone) is a neurosteroid that has recently been approved for the treatment of postpartum depression, promising to fill part of a long-lasting gap in the effectiveness of pharmacotherapies for depressive disorders. In this review, we explore the experimental research that characterized the antidepressant-like effects of allopregnanolone, with a particular focus on the neurotrophic adaptations induced by this neurosteroid in preclinical studies. We demonstrate that there is a consistent decrease in allopregnanolone levels in limbic brain areas in rodents submitted to stress-induced models of depression, such as social isolation and chronic unpredictable stress. Further, both the drug-induced upregulation of allopregnanolone or its direct administration reduce depressive-like behaviors in models such as the forced swim test. The main drugs of interest that upregulate allopregnanolone levels are selective serotonin reuptake inhibitors (SSRIs), which present the neurosteroidogenic property even in lower, non-SSRI doses. Finally, we explore how these antidepressant-like behaviors are related to neurogenesis, particularly in the hippocampus. The protagonist in this mechanism is likely the brain-derived neurotrophic factor (BFNF), which is decreased in animal models of depression and may be restored by the normalization of allopregnanolone levels. The role of an interaction between GABA and the neurotrophic mechanisms needs to be further investigated.

The effects of caffeine on alcohol oral self-administration behavior in rats.

Caffeine and alcohol are some of the most commonly used psychoactive substances in the world, and are often used concomitantly. However, little is known about the effect of caffeine on alcohol consumption. Here, our aim was to investigate the co-exposure of alcohol mixed with caffeine in self-administration. Thirty-two male and thirty-two female Wistar rats were randomly divided into the following groups: control, caffeine (0.25 mg/mL), alcohol (10% v/v) and alcohol mixed with caffeine. After one week of training, the animals underwent self-administration for 21 days (1 h per day) in a fixed ratio of 1 (FR1). The forced swimming test (FST) was performed before the training phase and 24 h after the last self-administration session to verify abstinence-induced depressive-like behaviors. Our results showed that all rats consumed a lower volume of alcohol-containing solution than control solution, and that the presence of caffeine did not influence this parameter. Females consumed less volume of alcohol solution than males but the average dose was similar. Females that self-administered alcohol mixed with caffeine presented a higher immobility in the FST than males that self-administered the same solution. These results support the conclusion that moderate doses of caffeine such as the ones from our study (approximately 7-8 mg/kg/day) do not influence alcohol consumption. Additionally, females might be more susceptible than males to depressive-like effects caused by the abstinence of the use of these substances in combination.

Correlations between subunits of GABAA and NMDA receptors after chronic alcohol treatment or withdrawal, and the effect of taurine in the hippocampus of rats.

Chronic use of alcohol and its withdrawal impairs the delicate balance between GABAergic and glutamatergic systems. This imbalance includes changes in GABA receptors - importantly in GABAA subtypes - and glutamate receptors, especially in NMDA subtypes. A better comprehension of the different roles of GABAAR and NMDAR subunits could be helpful to define new strategies to counteract the deleterious effects observed during alcohol withdrawal. Taurine, a sulfonated amino acid, has been proposed to attenuate alcohol withdrawal symptoms due to its neuromodulatory properties. In this study, we evaluated the correlations between GABAAR and NMDAR subunits in the hippocampus of rats chronically treated with alcohol or in alcohol withdrawal, and the effects of taurine treatment on these parameters. Male Wistar rats received alcohol (2 g/kg) or water by oral gavage (control), 2 × /day, for 28 days. From day 29 to day 33, withdrawal rats received water instead of alcohol and all groups were reallocated to receive 100 mg/kg taurine or saline intraperitoneally (i.p.), once a day. On day 34, rats were euthanized and the hippocampus was dissected for GABAAR α1, α4, δ, and γ2 and NMDAR GluN2A and GluN2B subunits mRNA expression determination by RT qPCR. There were no differences between groups in the studied GABAAR and NMDA subunits. However, we observed a correlation of α1 and γ2 subunits induced by taurine, while in the alcohol group there was a correlation between α4 and GluN2A. In the group treated with alcohol and taurine, we observed an extra correlation, between α1 and GluN2A. After 5 days of withdrawal, a correlation observed in the control group, between δ and GluN2A, was reestablished. The correlation found between subunits suggests a neuroadaptation of GABAergic and glutamatergic systems in withdrawal rats. Results from this study contribute to the elucidation of the mechanisms beyond neuroadaptations observed in alcohol use and withdrawal.

Combined use of alcohol and cigarette increases locomotion and glutamate levels in the cerebrospinal fluid without changes on GABAA or NMDA receptor subunit mRNA expression in the hippocampus of rats.

Interactions on neurotransmitter systems in the reward pathways may explain the high frequency of combined use of alcohol and cigarettes in humans. In this study, we evaluated some behavioral and neurochemical changes promoted by chronic exposure to alcohol and cigarette smoke in rats. Adult rats were administered with 2 g/kg alcohol (v.o.) or/and inhaled the smoke from 6 cigarettes, twice/day, for 30 days. Behavioral tests were performed 3 h after the alcohol administration and 1 h after the last exposure to cigarette smoke in the morning. Cerebrospinal fluid was collected for glutamate determination and the hippocampus was dissected for GABAA and NMDA receptor subunits mRNA expression determination. Results showed that the combined use of alcohol and cigarette smoke (ALTB) in rats increased the locomotor activity and all interventions decreased anxiety-like behaviors. Despite being on a short-term withdrawal, the cigarette smoke exposure decreased the percentage of open arm entries in the elevated plus maze test, which was prevented by combined use with alcohol. Even though GABAA and glutamate receptor subunits expression did not change in the hippocampus, glutamate levels were significantly higher in the cerebrospinal fluid from ALTB rats. Therefore, we showed that the combined use of alcohol and cigarette maintained a psychostimulant effect after a short-term withdrawal that was associated with the elevated glutamatergic activity. The combined use also prevented anxiety-like signs in cigarette smoke exposure rats, decreasing an adverse effect caused by nicotine withdrawal. These results could explain, in part, the elevated frequency of combined use of these two drugs of abuse in humans.

Hemisphere-dependent Changes in mRNA Expression of GABAA Receptor Subunits and BDNF after Intra-prefrontal Cortex Allopregnanolone Infusion in Rats.

Allopregnanolone is a neurosteroid implicated in mood disorders such as depression and anxiety. It acts as a GABAA receptor (GABAAR)-positive allosteric modulator and changes the expression of GABAAR subunits and of brain-derived neurotrophic factor (BDNF) in different brain regions. It has been demonstrated that such neurochemical changes may have an asymmetrical pattern regarding brain hemispheres. The aim of this study was to verify the behavioral and hemisphere-specific neurochemical effects of the bilateral intra-prefrontal cortex (intra-PFC) infusion of allopregnanolone in rats. Rats were exposed to the forced swim test and to the grooming microstructure test, followed by the right and left hemisphere-specific quantification of mRNA expression by Real-Time PCR of δ and γ2 GABAAR subunits and BDNF in the PFC and in the hippocampus. Though we did not observe any significant effects in the behavioral tests, intra-PFC allopregnanolone infusion bilaterally increased the mRNA expression of the δ subunit in the same area and of BDNF in the hippocampus. Both mRNA expressions of the γ2 subunit and BDNF were higher in the right than in the left PFC of control animals, and the hemisphere differences were not seen after allopregnanolone infusion. Overall hippocampal BDNF expression was also higher in the right hemisphere, but this asymmetry was not normalized by allopregnanolone. No asymmetries or changes were observed in the hippocampal mRNA expression of GABAAR subunits. These results point to a hemisphere-dependent regulation of GABAAR subunits and BDNF that can be modulated by intra-PFC allopregnanolone infusion, even in the absence of associated behavioral effects.

The effect of intracerebroventricular allopregnanolone on depressive-like behaviors of rats selectively bred for high and low immobility in the forced swim test.

Depression is a highly incapacitating disorder known to have a multifactorial etiology, including a hereditary genetic background. The neurosteroid allopregnanolone (ALLO) is a positive allosteric modulator of the GABAA receptor and has been shown to have an antidepressant-like effect in animals. This study aimed to assess the behavioral effect of ALLO in animals with different backgrounds of depressive-like activity. An initial population (F0) of male and female Wistar rats was screened for immobility behavior utilizing the Forced Swim Test (FST). Rats with extreme immobility scores were selected for either the High Immobility (HI) group or the Low Immobility (LI) group for breeding, giving origin to the subsequent generations F1 and F2. Guide cannulas were implanted in the lateral ventricle of F2 males for intracerebroventricular infusions of 5 µg/rat of ALLO, 5 µg/rat of imipramine (IMI) or vehicle (CTR), which occurred 24, 5 and 1 h prior to the test session of the drug FST. In the pre-drug FST, a statistically significant difference was observed between the immobility scores from the HI and LI groups of F2 rats. HI rats from F2 also showed significantly higher immobility time when compared to F0. In these HI animals, both IMI and ALLO significantly reduced immobility when compared to the CTR group. IMI-treated rats also showed lower immobility than the ALLO group. In the LI rats, no difference in immobility was found between treatments. In conclusion, two strains of rats with significantly different immobility profiles in the FST were obtained in a relatively short time, after only two generations. Infusions of both ALLO and IMI showed a strain-dependent antidepressant-like effect, being detected in the HI animals but not in the LI animals, which is in line with the clinical understanding that antidepressants have higher efficacy in more severe forms of depression.

Environmental enrichment reduces cocaine neurotoxicity during cocaine-conditioned place preference in male rats.

Environmental enrichment (EE) has a neuroprotective role and prevents the development of cocaine addiction behavior in rats. Studies showing the role of EE in cocaine toxicity are nonexistent. We hypothesized that rats exposed to EE are protected from cocaine-induced changes in the redox profile and DNA damage after undergoing conditioned place preference (CPP). Ten male Wistar rats were placed in EE cages equipped with toys, a ladder and tunnels, and ten were provided clean, standard laboratory housing (non-EE). EE and non-EE rats were randomly allocated to the classical CPP cocaine vs. saline (COC/Saline) group, where cocaine (15 mg/kg; i.p.) was tested alternately with saline. Afterwards, intracellular reactive species and antioxidant enzymes were evaluated and the comet essay was performed in the prefrontal cortex and hippocampus of rats. As expected, EE rats spent less time in the cocaine-paired chamber, and as a new result, less cocaine-induced DNA damage was observed in the two brain structures. Altogether, our results demonstrate that EE decreases neurotoxicity in brain regions linked to cocaine addiction but does not extinguish it completely.

Taurine restores the exploratory behavior following alcohol withdrawal and decreases BDNF mRNA expression in the frontal cortex of chronic alcohol-treated rats.

Alcohol use disorder is an alarming health problem, and the withdrawal symptoms increase the risk of relapse. We have hypothesized that taurine, a multitarget substance acting as a gamma-aminobutyric acid A receptor (GABAAR) positive modulator and a partial inhibitor of N-methyl-d-aspartate (NMDA) glutamate receptors, may reduce the withdrawal symptoms or modify behaviors when combined with alcohol. Therefore, we investigated the effects of taurine on behavior in the open field test (OFT), the GABAAR α2 subunit and BDNF mRNA expression in the frontal cortex of rats after chronic alcohol treatment or upon withdrawal. Rats received alcohol 2g/kg (alcohol and withdrawal groups) or water (control group) twice daily by oral gavage for 28days. On day 29, the withdrawal rats received water instead of alcohol, and all groups were reallocated to receive 100mg/kg taurine or vehicle intraperitoneally, once a day for 5days. On day 33, the rats were exposed to OFT; 18h later, they were euthanized, and the frontal cortex was dissected for GABAAR α2 subunit detection and BDNF mRNA expression determination by real-time quantitative PCR. Taurine administration restored rearing behavior to the control levels in the withdrawal rats. Taurine also showed anxiolytic-like effects in control rats and did not change the behaviors in the chronic alcohol group. Chronic alcohol treatment or withdrawal did not change the GABAAR α2 subunit or BDNF mRNA expression in the frontal cortex, but taurine decreased the α2 subunit level in control rats and to the BDNF levels in the alcohol rat group. We conclude that taurine restored exploratory behavior after alcohol withdrawal but that this effect was not related to the GABAAR α2 subunit or BDNF mRNA expression in the frontal cortex of the rats.

Antidepressant dose of taurine increases mRNA expression of GABAA receptor α2 subunit and BDNF in the hippocampus of diabetic rats.

Diabetes mellitus is a metabolic disorder associated with higher risk for depression. Diabetic rats present depressive-like behaviors and taurine, one of the most abundant free amino acids in the brain, reverses this depressive behaviors. Because taurine is a GABAA agonist modulator, we hypothesize that its antidepressant effect results from the interaction on this system by changing α2 GABAA receptor subunit expression, beside changes on BDNF mRNA, and memory in diabetic rats. Streptozotocin-diabetic and non-diabetic Wistar rats were daily injected with 100mg/kg of taurine or saline, intraperitoneally, for 30 days. At the end of the experiment, rats were exposed to the novel object recognition memory. Later they were euthanized, the brains were weighed, and the hippocampus was dissected for α2 GABAA subunit and BDNF mRNA expression. Real-time quantitative PCR (qPCR) showed that diabetic rats presented lower α2 GABAA subunit and BDNF mRNA expression than non-diabetic rats and taurine increased both parameters in these sick rats. Taurine also reversed the lower brain weight and improved the short-term memory in diabetic rats. Thus, the taurine antidepressant effect may be explained by interference with the GABA system, in line to its neuroprotective effect showed here by preventing brain weight loss and improving memory in diabetic rats.