Hippocampal and amygdalar increased BDNF expression in the extinction of opioid-induced place preference.

The opioid crisis was exacerbated during the COVID-19 pandemic in the United States with alarming statistics about overdose-related deaths. Current treatment options, such as medication assisted treatments, have been unable to prevent relapse in many patients, whereas cue-based exposure therapy have had mixed results in human trials. To improve patient outcomes, it is imperative to develop animal models of addiction to understand molecular mechanisms and identify potential therapeutic targets. We previously found increased brain derived neurotrophic factor (bdnf) transcript in the ventral striatum/nucleus accumbens (VS/NAc) of rats that extinguished morphine-induced place preference. Here, we expand our study to determine whether BDNF protein expression was modulated in mesolimbic brain regions of the reward system in animals exposed to extinction training. Drug conditioning and extinction sessions were followed by Western blots for BDNF in the hippocampus (HPC), amygdala (AMY) and VS/NAc. Rears, as a measure of withdrawal-induced anxiety were also measured to determine their impact on extinction. Results showed that animals who received extinction training and successfully extinguished morphine CPP significantly increased BDNF in the HPC when compared to animals deprived of extinction training (sham-extinction). This increase was not significant in animals who failed to extinguish (extinction-resistant). In AMY, all extinction-trained animals showed increased BDNF, regardless of behavior phenotype. No BDNF modulation was observed in the VS/NAc. Finally, extinction-trained animals showed no difference in rears regardless of extinction outcome, suggesting that anxiety elicited by drug withdrawal did not significantly impact extinction of morphine CPP. Our results suggest that BDNF expression in brain regions of the mesolimbic reward system could play a key role in extinction of opioid-induced maladaptive behaviors and represents a potential therapeutic target for future combined pharmacological and extinction-based therapies.

Neuroplasticity transcript profile of the ventral striatum in the extinction of opioid-induced conditioned place preference.

Persistent drug-seeking behavior has been associated with deficits in neural circuits that regulate the extinction of addictive behaviors. Although there is extensive data that associates addiction phases with neuroplasticity changes in the reward circuit, little is known about the underlying mechanisms of extinction learning of opioid associated cues. Here, we combined morphine-conditioned place preference (CPP) with real-time polymerase chain reaction (RT-PCR) to identify the effects of extinction training on the expression of genes (mRNAs) associated with synaptic plasticity and opioid receptors in the ventral striatum/nucleus accumbens (VS/NAc). Following morphine extinction training, we identified two animal subgroups showing either extinction (low CPP) or extinction-resistance (high CPP). A third group were conditioned to morphine but did not receive extinction training (sham-extinction; high CPP). RT-PCR results showed that brain derived neurotrophic factor (Bdnf) was upregulated in rats showing successful extinction. Conversely, the lack of extinction training (sham-extinction) upregulated genes associated with kinases (Camk2g), neurotrophins (Ngfr), synaptic connectivity factors (Ephb2), glutamate neurotransmission (Grm8) and opioid receptors (µ1, Δ1). To further identify genes modulated by morphine itself, comparisons with their saline-counterparts were performed. Results revealed that Bdnf was consistently upregulated in the extinction group. Alternatively, widespread gene modulation was observed in the group with lack of extinction training (i.e. Drd2, Cnr1, Creb, µ1, Δ1) and the group showing extinction resistance (i.e. Crem, Rheb, Tnfa). Together, our study builds on the identification of putative genetic markers for the extinction learning of drug-associated cues.

Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids.

The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 µM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

Bidirectional Modulation of Extinction of Drug Seeking by Deep Brain Stimulation of the Ventral Striatum.

BACKGROUND: Recent research in humans and rodents has explored the use of deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VS) as a possible treatment for drug addiction. However, the optimum electrode placement and optimum DBS parameters have not been thoroughly studied. Here we varied stimulation sites and frequencies to determine whether DBS of the VS could facilitate the extinction of morphine-induced conditioned place preference in rats. METHODS: Rats were implanted with DBS electrodes in the dorsal or ventral subregions of the VS and trained to the morphine conditioned place preference. Subsequently, rats received extinction sessions over 9 days, combined with 60 min of either high- (130 Hz) or low- (20 Hz) frequency DBS. To study circuit-wide activations after DBS of the VS, c-fos immunohistochemistry was performed in regions involved in the extinction of drug-seeking behaviors. RESULTS: High-frequency DBS of the dorsal-VS impaired both extinction training and extinction memory, whereas high-frequency DBS of the ventral-VS had no effect. In contrast, low-frequency DBS of the dorsal-VS strengthened extinction memory when tested 2 or 9 days after the cessation of stimulation. Both DBS frequencies increased c-fos expression in the infralimbic prefrontal cortex, but only low-frequency DBS increased c-fos expression in the basal amygdala and the medial portion of the central amygdala. CONCLUSIONS: Our results suggest that low-frequency (rather than high-frequency) DBS of the dorsal-VS strengthens extinction memory and may be a potential adjunct for extinction-based therapies for treatment-refractory opioid addiction.

The effect of the anabolic steroid, nandrolone, in conditioned place preference and D1 dopamine receptor expression in adolescent and adult mice.

Adolescents and adults engage in anabolic-androgenic steroid (AAS) misuse seeking their anabolic effects, even though later on, many could develop neuropsychological dependence. Previously, we have shown that nandrolone induces conditioned place preference (CPP) in adult male mice. However, whether nandrolone induces CPP during adolescence remains unknown. In this study, the CPP test was used to determine the rewarding properties of nandrolone (7.5 mg/kg) in adolescent mice. In addition, since D1 dopamine receptors (D1DR) are critical for reward-related processes, the effect of nandrolone on the expression of D1DR in the nucleus accumbens (NAc) was investigated by Western blot analysis. Similar to our previous results, nandrolone induced CPP in adults. However, in adolescents, nandrolone failed to produce place preference. At the molecular level, nandrolone decreased D1DR expression in the NAc only in adult mice. Our data suggest that nandrolone may not be rewarding in adolescents at least during short-term use. The lack of nandrolone rewarding effects in adolescents may be due, in part to differences in D1DR expression during development.

Sex-specific effect of the anabolic steroid, 17α-methyltestosterone, on inhibitory avoidance learning in periadolescent rats.

The illicit use of anabolic androgenic steroids (AAS) has gained popularity among adolescents in the last decade. However, although it is known that exposure to AAS impairs cognition in adult animal models, the cognitive effects during adolescence remain undetermined. An inhibitory avoidance task (IAT) was used to assess the effect of AAS (17α-methyltestosterone; 17α-meT--7.5 mg/kg) in male and female periadolescent rats. A single injection of 17α-meT immediately before the footshock produced significant impairment of inhibitory avoidance learning in males but not females. Generalized anxiety, locomotion, and risk assessment behaviors (RAB) were not affected. Our results show that exposure to a single pharmacological dose of 17α-meT during periadolescence exerts sex-specific cognitive effects without affecting anxiety. Thus, disruption of the hormonal milieu during this early developmental period might have negative impact on learning and memory.

The testosterone metabolite 3α-diol enhances female rat sexual motivation when infused in the nucleus accumbens shell.

AIM: The purpose of this study was to provide a quantitative assessment of female rat sexual behaviors after acute exposure to the A-ring reduced testosterone metabolite, androstanediol (3α-Diol), through the nucleus accumbens (NA) shell. MAIN OUTCOME MEASURES: Quantitative analyses of female rat sexual behaviors and assessment of protein levels for the enzyme glutamic acid decarboxylase isoform 67 (GAD67) and gephyrin, a protein that participates in the clustering of GABA-A receptors in postsynaptic cells, were accomplished. METHODS: Female rats were ovariectomized and primed with estrogen and progesterone to induce sexual behaviors. Females received a 3α-Diol infusion via guided cannula that aimed to the NA shell five minutes prior to a sexual encounter with a stud male. The following parameters were videotaped and measured in a frame by frame analysis: lordosis quotient (LQ), Lordosis rating (LR), frequency and duration of proceptive behaviors (hopping/darting and ear wiggling). Levels of GAD67 and gephyrin were obtained by Western blot analysis two or twenty-four hours after the sexual encounter. RESULTS: Acute exposure to 3α-Diol in the NA shell enhanced LR, ear wiggling, and hopping/darting but not LQ. Some of these behavioral effects were counteracted by co-infusion of 3α-Diol plus the GABAA-receptor antagonist GABAzine. A transient reduction of GAD67 levels in the NA shell was detected. CONCLUSIONS: The testosterone metabolite 3α-Diol enhances sexual proceptivity, but not receptivity, when infused into the NA shell directly. The GABAergic system may participate in the androgen-mediated enhancement of female rat sexual motivation.

The anabolic steroids testosterone propionate and nandrolone, but not 17alpha-methyltestosterone, induce conditioned place preference in adult mice.

Anabolic androgenic steroids (AAS) are often misused by adolescents and athletes. Their effects vary according to chemical structure and metabolism, route of administration, and AAS regimen. In this study, adult C57Bl/6 male mice were systemically exposed to testosterone propionate (TP), nandrolone or 17alpha-methyltestosterone (17alpha-meT), type I, type II and type III AAS, respectively, in order to determine the hedonic or aversive properties of each drug. For this purpose, the conditioned place preference (CPP) test was employed at three different AAS doses (0.075, 0.75 and 7.5 mg/kg). Other behavioral domains monitored were light-dark transitions (side changes) and general activity. TP shifted place preference at all doses tested, and nandrolone shifted place preference at 0.75 and 7.5 mg/kg, but not at 0.075 mg/kg, the lower dose tested. Conversely, mice receiving 17alpha-meT did not show alteration in the preference score. The lower dose of nandrolone did modify exploratory-based anxiety showing a decrease in light-dark transitions if compared to vehicle-treated animals, while mice treated with TP or 17alpha-meT were not affected. Our data suggest that when studying hedonic and rewarding properties of synthetic androgens, distinction has to be made based on type of AAS and metabolism.

Exposure to an anabolic steroid changes female mice's sexual responses according to sex partner.

INTRODUCTION: Anabolic androgenic steroids (AAS) affect human female sexual behaviors. Animal models have been useful in uncovering the neural mechanisms governing changes in female sexual response upon AAS exposure. AIM: We quantify the sexual response of AAS-exposed gonadally intact female mice when paired with gonadally intact female or male pairs. METHODS: C57Bl/6 female mice were systemically exposed to the AAS 17alpha-methyltestosterone (7.5 mg/kg) for 17 days via a subcutaneous osmotic implant. On days 15-17, these females were allowed to mate with males or proestrus female partners in familiar and unfamiliar testing arenas for 10 minutes. MAIN OUTCOME MEASURES: The following behavioral responses were registered: parameters related to mounting behavior such as the frequency of mounts, attempts to mount, and the latency to the first mount, anogenital investigation, fights and escapes, rejections, pelvic thrusts, and lordotic responses. RESULTS: We found that males displayed a significant decrease in the frequency of mounts to AAS-exposed females, when compared with mating encounters with control females. We found no difference in the lordosis strength when control females were mounted by either a male or AAS-exposed females. However, females under androgen exposure attempted to mount control females, but not males, and their behavior was accompanied by significant increases in the number of fights, escapes, and rejections to the male. There were no differences between AAS-exposed females and males when the frequency of mounts and pelvic thrusts toward control females were compared. The lordotic quotient of control females was similar for either partner. CONCLUSIONS: Aside from showing a male-like pattern, AAS-exposed females displayed a higher frequency of anogenital investigations toward control females than males, and their latency to the first mount was as fast as that of males. Taken together, we conclude that the sex partner greatly influences the sexual response of AAS-exposed female mice.

Modulation of affect after chronic exposure to the anabolic steroid 17alpha-methyltestosterone in adult mice.

A battery of behavioral tasks in C57BL/6J mice was used to assess changes in affective components of behavior after systemic exposure to the anabolic-androgenic steroid (AAS) 17alpha-methyltestosterone (7.5 mg/kg). Gonadal weight in both sexes was reduced after 16 days of AAS exposure. Changes in discrete components of social behaviors were observed. No changes were recorded in the elevated plus-maze, the light-dark transition, and defensive behavior tests on exposure to 17alpha-methyltestosterone. When compared with controls, AAS-exposed females received a greater number of shocks, and AAS-exposed males displayed a shorter recovery time to consume water after a negative reinforcer in the modified Vogel conflict test. Results show that systemic exposure to a single AAS modified social behaviors, whereas minimal effects on anxiety-related behaviors were observed according to sex.

The morphological and biochemical response of avian embryonic sympathoadrenal cells to nerve growth factor is developmentally regulated.

Cellular differentiation is a stepwise process where environmental factors are essential key components to direct cells to their final phenotype. The sympathoadrenal (SA) system is one of the principal models used to study the role of environmental factors in the development of the peripheral nervous system. Two major cell types originate from the SA progenitor: the principal neurons of the sympathetic ganglia and the chromaffin cells of the adrenal medulla. These cells are directed to their final phenotype by a series of environmental factors, of which nerve growth factor (NGF) and glucocorticoids, are the best studied. Previously, we have shown that 11-day embryonic chick sympathetic cell cultures increased their neuropeptide Y (NPY) protein and mRNA levels in the presence of NGF. In contrast, NGF had no such effect in chromaffin cell cultures from the same developmental stage. These results were unexpected since both cells types respond morphologically to NGF. To determine if these cells can gain or lose their capacity to respond to NGF, morphological and biochemical studies were done at earlier stages using immunocytochemical, radioimmunoassay and polymerase chain reaction (PCR) techniques. Interestingly we found that in E-7 chromaffin cells there is a biochemical and morphological response to NGF, while E-7 sympathetic cells lack this response. Our observations show a developmental point of regulation of morphological and biochemical properties by NGF and reveal an age dependent capacity of SA cells to acquire or lose competence to an environmental factor.