Serotonin Signaling in Hippocampus during Initial Cocaine Abstinence Drives Persistent Drug Seeking.

The initiation of abstinence after chronic drug self-administration is stressful. Cocaine-seeking behavior on the first day of the absence of the expected drug (Extinction Day 1, ED1) is reduced by blocking 5-HT signaling in dorsal hippocampal cornu ammonis 1 (CA1) in both male and female rats. We hypothesized that the experience of ED1 can substantially influence later relapse behavior and that dorsal raphe (DR) serotonin (5-HT) input to CA1 may be involved. We inhibited 5-HT1A/1B receptors (WAY-100635 plus GR-127935), or DR input (chemogenetics), in CA1 on ED1 to test the role of this pathway on cocaine-seeking persistence 2 weeks later. We also inhibited 5-HT1A or 5-HT1B receptors in CA1 during conditioned place preference (CPP) for cocaine, to examine mechanisms involved in the persistent effects of ED1 manipulations. Inhibition of DR inputs, or 5-HT1A/1B signaling, in CA1 decreased drug seeking on ED1 and decreased cocaine seeking 2 weeks later revealing that 5-HT signaling in CA1 during ED1 contributes to persistent drug seeking during abstinence. In addition, 5-HT1B antagonism alone transiently decreased drug-associated memory performance when given prior to a CPP test, whereas similar antagonism of 5-HT1A alone had no such effect but blocked CPP retrieval on a test 24 h later. These CPP findings are consistent with prior work showing that DR inputs to CA1 augment recall of the drug-associated context and drug seeking via 5-HT1B receptors and prevent consolidation of the updated nondrug context via 5-HT1A receptors. Thus, treatments that modulate 5-HT-dependent memory mechanisms in CA1 during initial abstinence may facilitate later maintenance of abstinence.

It is all About the Chase: Neurosteroidogenesis in Male Rats is Driven by Control of Mating Pace.

BACKGROUND: Masculine sexual behaviors are dependent on androstane-derived steroids; however, the modulatory effects of mating, and of mating control, on androstane neurosteroidogenesis remain largely unknown. OBJECTIVE: Herein, we investigated the effects of mating control, prior sexual experience, and age on brain region specific neurosteroidogenic responses in male rats. METHODS: Effects of acute sexual experience were tested in naïve male rats that either remained sexually- naïve, were exposed to a standard mating chamber, or were either given control of the mating pace in a standard mating chamber (male control) or mated wherein the female stimulus rat controlled the mating pace in a paced-mating chamber (female control). Aged (10-12 months) sexually responsive male rats were similarly euthanized from the homecage or engaged in male controlled or female controlled mating. All rats were euthanized immediately following exposure conditions for radioimmunoassay of steroids in midbrain, hypothalamus, hippocampus and cortex. RESULTS: Consummatory sexual behavior in male vs. female-controlled mating paradigms was altered by age and prior sexual experience. Male-controlled mating increased androstane neurosteroid metabolism, such that complementary increases in the testosterone (T) metabolite 5α-androstane-3α-17ß- diol (3α-diol) in the midbrain and hypothalamus of male rats corresponded to decreases in the prohormone, T. 3α-diol were increased in the hippocampus in response to the context alone, and to a lesser degree in response to mating. Mating diminished neurosteroidogenesis in the cortex. Neurosteroidogenesis was overall reduced in aged male rats compared to naïve controls, however, these effects were more prominent in sexually non-responsive aged male rats. CONCLUSION: Extending previous findings, these results indicate differential production of androstane neurosteroids in a mating exposure, age and brain region dependent manner.

Hormonal milieu drives economic demand for cocaine in female rats.

There are substantial sex differences in drug abuse, and a key feature of cocaine addiction is pathologically high motivation for drug. We investigated the role of ovarian hormones on cocaine demand in female rats using a within-session threshold behavioral economics (BE) procedure, which allows us to compare motivation for drug across hormonal states and sex while controlling for differences in dose and intake. This approach quantifies demand elasticity (α) and free consumption (Q0, consumption at null effort) to determine motivation for cocaine. Overall, female rats showed greater motivation for cocaine compared to males. However, this difference was cycle phase-dependent - motivation for cocaine when females were in proestrus was lower compared to the same animals across cycle phases, and overall similar to that of males. Hormonal cycle phase accounted for 70% of the within-subject variance in demand elasticity, obscuring other individual differences in female demand. High serum progesterone (P4; e.g., in proestrus) predicted decreased cocaine motivation (high demand elasticity), whereas serum estradiol (E2) correlated to greater intake at null effort (Q0). However, individual differences were revealed across OVX females, who displayed a range of demand elasticity, as seen in males. E2 replacement in OVX females increased motivation for cocaine, whereas P4 replacement decreased motivation. We also found that as few as 4 weeks of cocaine self-administration accelerated estropause in female rats as young as 12 weeks old. By 13 weeks of self-administration, proestrus epochs were no longer observed, and cocaine demand was potentiated by persistent estrus in all females. Thus, P4 signaling is a key modulator of cocaine demand in females that may underlie previously observed sex differences in addiction phenotypes.

Effects of non-contingent cocaine on 3alpha-androstanediol. I. Disruption of male sexual behavior.

One of the hallmarks of drug abuse is a reduction in the salience of, and motivation for, natural rewards, such as mating. The effects of psychostimulants on male sexual interest and performance are conflicting; use of psychostimulants can produce increases in risky sexual behaviors but have detrimental effects on sexual ability. We hypothesize that these conflicting effects on sexual behavior are due to interactions between cocaine and androgens, such as testosterone and its neuroactive metabolite, 3α-androstanediol (3α-diol). Male rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug-administration, and then sexual responding was assessed for 15min. Levels of androgens (testosterone, 3ɑ-diol, and testosterone's aromatized metabolite, estradiol) were measured in circulation and brain regions (frontal cortex, hippocampus, hypothalamus/striatum (hypo/str), and midbrain). Cocaine had no effect on measures of sexual interest (i.e. anogenital investigation). However, cocaine had substantial effects on consummatory sexual behaviors, such as the latency to mount/intromit and the number of sexual contacts. Frontal cortex and hypo/str 3α-diol levels were strongly correlated with consummatory behaviors in saline administered rats; however, this relationship was disrupted by cocaine at all dosages, concomitant with impaired sexual behaviors. Additionally, there was a shift in metabolism at low dosages of cocaine to push testosterone metabolism in the midbrain towards 3α-diol. On the contrary, moderate and high dosages of cocaine shifted testosterone metabolism towards estradiol. These data demonstrate that the association between cortical and hypo/str 3α-diol levels and sexual behavior of male rats is disrupted by non-contingent cocaine and that there may be dose-dependent effects of acute cocaine on androgen metabolism.

Effects of non-contingent cocaine on 3 alpha-androstanediol. II. Disruption of lordosis of proestrous rats.

Drug use influences sexual behavior, performance, and can be associated with increased sexual risk-taking. Our prior results using an animal model indicate that progestogens contribute to hormonally-mediated changes in sexual behavior of female rodents during acute cocaine exposure. Androgens, such as testosterone, and its metabolite 3ɑ-androstanediol (3α-diol), and estradiol, are known to influence male sexual behavior, but can also alter the expression of sexual behavior of female rodents. As such, we investigated the influence of endogenous androgen and estradiol fluctuations on cocaine-mediated changes in motor behavior and sexual receptivity of rats during diestrous or proestrous phases of the estrous cycle. Female rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug administration, and then sexual responding was assessed for 15min. Cocaine decreased aggressive behavior in response to attempted mounts by a male among non-receptive (diestrous) rats and inhibited sexual behavior among sexually receptive (proestrous) rats. Cocaine dose-dependently altered concentrations of testosterone metabolites (estradiol and 3α-diol), but not testosterone, which correlated to motor and sexual behaviors of diestrous and proestrous rats, respectively. These data suggest that actions of 3α-diol may be involved in female sexual and motor behavior in response to cocaine, in a cycle-dependent manner.

Attenuated cocaine-seeking after oxytocin administration in male and female rats.

RATIONALE: Initial drug abstinence (modeled here as Extinction Day 1, ED1) is a critical time point in the progression of addiction that is strongly influenced by stress and sex. ED1 induces corticosterone release in both sexes, and cocaine-seeking during ED1 can be mitigated by corticotrophin-releasing factor (CRF) antagonists more effectively in female rats. Oxytocin (OXT) is a neuropeptide that has several biological functions, including regulation of stress pathways. METHODS: To investigate a relationship between OXT, sex, and cocaine-seeking, we examined Fos on ED1 in OXT neurons of paraventricular (PVN) and supraoptic nuclei (SON) compared to homecage (cocaine experienced) or naïve male and female rats. We also administered OXT 30 min prior to ED1 testing or cued reinstatement testing. RESULTS: OXT neurons had decreased activity (as reflected by Fos protein) in PVN and SON on withdrawal day 1 (homecage) compared to naïve rats. Fos in OXT neurons was further decreased on ED1, compared to homecage controls, in both males and females even though in SON, cocaine exposure increased the number of OXT-expressing neurons. In addition, systemically administered OXT reduced cocaine-seeking during ED1 and cue-induced reinstatement of cocaine-seeking but delayed extinction, similarly among male and female rats. CONCLUSIONS: These data indicate that OXT neurons in PVN and SON may be involved in cocaine-seeking during ED1 and support OXT as a possible therapeutic to decrease cocaine-seeking during initial abstinence and in response to cocaine-associated cues.

Insulin-Like Growth Factor II Targets the mTOR Pathway to Reverse Autism-Like Phenotypes in Mice.

Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social interaction and repetitive behavior, and is also associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASD symptomatology; thus, identifying novel therapies is urgently needed. We used male BTBR T+Itpr3tf /J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, to test the effects of systemic administration of insulin-like growth factor II (IGF-II), a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-II treatments reverse the typical defects in social interaction, cognitive/executive functions, and repetitive behaviors reflective of ASD-like phenotypes. In BTBR mice, IGF-II, via IGF-II receptor, but not via IGF-I receptor, reverses the abnormal levels of the AMPK-mTOR-S6K pathway and of active translation at synapses. Thus, IGF-II may represent a novel potential therapy for ASD.SIGNIFICANCE STATEMENT Currently, there is no effective treatment for autism spectrum disorder (ASD), a developmental disability affecting a high number of children. Using a mouse model that expresses most of the key core as well as associated behavioral deficits of ASD, that are, social, cognitive, and repetitive behaviors, we report that a systemic administration of the polypeptide insulin-like growth factor II (IGF-II) reverses all these deficits. The effects of IGF-II occur via IGF-II receptors, and not IGF-I receptors, and target both basal and learning-dependent molecular abnormalities found in several ASD mice models, including those of identified genetic mutations. We suggest that IGF-II represents a potential novel therapeutic target for ASD.

Cocaine Seeking During Initial Abstinence Is Driven by Noradrenergic and Serotonergic Signaling in Hippocampus in a Sex-Dependent Manner.

There is evidence for sex differences in cocaine addiction from both clinical and preclinical studies. In particular, preclinical studies indicate that females may be more sensitive than males to stress-induced drug seeking. The dorsal hippocampus (DH) is prominently involved in the stress response, as are the locus coeruleus norepinephrine (LC-NE) and dorsal raphe serotonin (DR 5-HT) systems. Moreover, DH receives strong inputs from LC-NE and DR 5-HT neurons. We hypothesized that the stress associated with non-reinforced drug seeking during early abstinence (on extinction day 1 (ED1)) may contribute to drug seeking via ß-adrenergic and 5-HT neurotransmission in DH. We observed decreased drug-seeking behavior on ED1 following 10 mg/kg S-propranolol (ß-adrenergic and 5-HT1A/1B receptor antagonist), R-propranolol (5-HT1A/1B receptor antagonist), or racemic propranolol in both male and female rats. ED1 increased Fos expression in DH, LC, and DR, and DH Fos was decreased by systemic S-propranolol. Based on these results, we investigated the effects of blocking 5-HT and ß-adrenoceptor transmission in DH on drug seeking during ED1 by infusing a cocktail of WAY100635 plus GR127935 (5-HT1A/1B receptor antagonists), betaxolol plus ICI-118 551 (ß1 and ß2 antagonists), or S-propranolol alone. In males, WAY100635/GR127935 was most effective in reducing drug-seeking on ED1, whereas betaxolol/ICI-118 551 was ineffective. In contrast, S-propranolol was most effective in females in reducing drug seeking on ED1, and WAY100635/GR127935 and betaxolol/ICI-118 551 were each partially effective. Our results indicate that drug seeking during initial abstinence involves 5-HT and ß-adrenergic signaling in female DH, but only 5-HT signaling in male DH.

Impact of gender on corticotropin-releasing factor and noradrenergic sensitivity in cocaine use disorder.

Responses to stress may be important in understanding gender differences in substance use disorders and may also be a target for development of treatment interventions. A growing body of both preclinical and clinical research supports important underlying gender differences in the corticotropin-releasing factor (CRF) and noradrenergic systems, which may contribute to drug use. Preclinical models have demonstrated increased sensitivity of females to CRF and noradrenergic-induced drug reinstatement compared with males, and, consistent with these findings, human laboratory studies have demonstrated greater sensitivity to corticotropin-releasing hormone (CRH) and noradrenergic stimulation in cocaine-dependent women compared with men. Furthermore, neuroimaging studies have demonstrated increased neural response to stressful stimuli in cocaine-dependent women compared with men as well as showing significant sex differences in the sensitivity of brain regions responsible for regulating the response to CRH. Development of interventions targeting the noradrenergic system and stress response in drug-dependent individuals could have important clinical implications for both women and men. © 2016 Wiley Periodicals, Inc.

Enhancement of memories by systemic administration of insulin-like growth factor II.

To treat cognitive disorders in humans, new effective therapies that can be easily delivered systemically are needed. Previous studies showed that a bilateral injection of insulin-like growth factor II (IGF-II) into the dorsal hippocampus of rats or mice enhances fear memories and facilitates fear extinction. Here, we report that, in mice, systemic treatments with IGF-II given before training significantly enhance the retention and persistence of several types of working, short-term and long-term memories, including fear conditioning, object recognition, object placement, social recognition, and spatial reference memory. IGF-II-mediated memory enhancement does not alter memory flexibility or the ability for new learning and also occurs when IGF-II treatment is given in concert with memory retrieval. Thus IGF-II may represent a potentially important and effective treatment for enhancing human cognitive and executive functions.

Progesterone-facilitated lordosis of estradiol-primed mice is attenuated by knocking down expression of membrane progestin receptors in the midbrain.

Evidence is emerging of the role of membrane progestin receptors (referred to as mPRs herein: members of Progestin and AdipoQ Receptor (Paqr) family) as a novel brain target in mammals, such as rats. In the present study, the role of mPRs in mice was assessed to further elucidate the conservation of this mechanism across species. The brain target investigated was the midbrain ventral tegmental area (VTA) given its described role for rapid actions of progestins for reproduction. Studies tested the hypothesis that if mPRs are required for progestin-facilitated lordosis through actions in the VTA, then knockdown of mPRs in the VTA will attenuate lordosis. Ovariectomized (OVX) mice were subcutaneously injected with estradiol (E2) and progesterone (P4), and infused with antisense oligodeoxynucleotides (AS-ODNs) to mPRα (Paqr7) and/or mPRß (Paqr8) or vehicle to the lateral ventricle or VTA. Mice were assessed for reproductive behavior (lordosis and aggression/rejection quotients) in a standard mating task. Results supported our hypothesis. E2+P4-facilitated lordosis was significantly reduced, and aggression/rejection increased, with infusions of mPRα, mPRß, or mPRαß AS-ODNs to the lateral ventricle, compared to vehicle. E2+P4-facilitated lordosis was significantly decreased, and aggression/rejection increased, with mPRß or mPRαß AS-ODNs to the VTA of C57/BL6 mice. Both mPRɑ and mPRß AS-ODNs reduced lordosis, and increased aggression/rejection, of wildtype (C57/BL6x129) mice, but not nuclear PR knockout mice. Thus, mPRs may be a novel target of progestins for reproductive behavior of mice.

Membrane progestin receptors in the midbrain ventral tegmental area are required for progesterone-facilitated lordosis of rats.

Progesterone (P4) and its metabolites, rapidly facilitate lordosis of rats partly through actions in the ventral tegmental area (VTA). The study of membrane progestin receptors (mPRs), of the Progestin and AdipoQ Receptor (PAQR) superfamily, has been limited to expression and regulation, instead of function. We hypothesized that if mPRs are required for progestin-facilitated lordosis in the VTA, then mPRs will be expressed in this region and knockdown will attenuate lordosis. First, expression of mPR was examined by reverse-transcriptase polymerase chain reaction (RT-PCR) in brain and peripheral tissues of proestrous Long-Evans rats. Expression of mPRα (paqr7) was observed in peripheral tissues and brain areas, including hypothalamus and midbrain. Expression of mPRß (paqr8) was observed in brain tissues and was abundant in the midbrain and hypothalamus. Second, ovariectomized rats were estrogen (E2; 0.09 mg/kg, SC), and P4 (4 mg/kg, SC) or vehicle-primed, and infused with antisense oligodeoxynucleotides (AS-ODNs) targeted against mPRα and/or mPRß intracerebroventricularly or to the VTA. Rats were assessed for motor (open field), anxiety (elevated plus maze), social (social interaction), and sexual (lordosis) behavior. P4-facilitated lordosis was significantly reduced with administration of AS-ODNs for mPRα, mPRß, or co-administration of mPRα and mPRß to the lateral ventricle, compared to vehicle. P4-facilitated lordosis was reduced, compared to vehicle, by administration of mPRß AS-ODNs, or co-administration of mPRα and mPRß AS-ODNs, but not mPRα AS-ODNs alone, to the VTA. No differences were observed for motor, anxiety, or social behaviors. Thus, mPRs in the VTA are targets of progestin-facilitated lordosis of rats.

Dissociating behavioral, autonomic, and neuroendocrine effects of androgen steroids in animal models.

Developments in behavioral assessment, autonomic and/or baseline reactivity, psychopharmacology, and genetics, have contributed significantly to the assessment of performance-enhancing drugs in animal models. Particular classes of steroid hormones: androgenic steroids are of interest. Anecdotally, the performance enhancing effects of androgens are attributed to anabolic events. However, there is a discrepancy between anecdotal evidence and investigative data. While some androgen steroids may promote muscle growth (myogenesis), effects of androgens on performance enhancement are not always seen. Indeed, some effects of androgens on performance may be attributable to their psychological and cardiovascular effects. As such, we consider androgen effects in terms of their behavioral, autonomic, and neuroendocrine components. Techniques are discussed in this chapter, some of which are well established, while others have been more recently developed to study androgen action. Androgens may be considered for their positive impact, negative consequence, or psychotropic properties. Thus, this review aims to elucidate some of the effects and/or mechanisms of androgens on behavioral, autonomic, and/or neuroendocrine assessment that may underlie their controversial performance enhancing effects.

Low doses of cocaine decrease, and high doses increase, anxiety-like behavior and brain progestogen levels among intact rats.

There are sex and hormonal differences in response to cocaine that have been demonstrated in people and animal models. Cocaine can alter secretion of progestogens, such as progesterone (P), and its neuroactive metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP). However, little research has been done on the neuroendocrine effects in the initiation phase of cocaine use. We hypothesize that some sex/hormonal differences in initiation phase responses to cocaine may be related to formation of progestogens. To investigate the role of progestogens in sex differences in response to acute cocaine, male and female rats in the high (proestrous) or low (diestrous) progestogen phase of the estrous cycle were administered cocaine (0, 5, 10, or 20mg/kg, IP). We examined cocaine's acute neuroendocrine effects on P and 3alpha,5alpha-THP levels, as well as its effects on acute psychomotor stimulation, anxiety, and sexual behaviors. Among rats that had P and/or 3alpha,5alpha-THP levels increased in response to cocaine, enhanced acute psychomotor stimulation was observed. Results suggest that cocaine produces U-shaped curves for progestogens, and anxiety-like behaviors. Male rats were less susceptible to these effects of cocaine than were proestrous or diestrous female rats. However, cocaine's disruption of sexual behaviors was similar among males and proestrous females. These data suggest a complex interaction between hormonal milieu and the neuroendocrine and behavioral effects of cocaine.