L-NAC and L-NAC methyl ester prevent and overcome physical dependence to fentanyl in male rats.

N-acetyl-L-cysteine (L-NAC) is a proposed therapeutic for opioid use disorder. This study determined whether co-injections of L-NAC (500 µmol/kg, IV) or its highly cell-penetrant analogue, L-NAC methyl ester (L-NACme, 500 µmol/kg, IV), prevent acquisition of acute physical dependence induced by twice-daily injections of fentanyl (125 µg/kg, IV), and overcome acquired dependence to these injections in freely-moving male Sprague Dawley rats. The injection of the opioid receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IV), elicited a series of withdrawal phenomena (i.e. behavioral and cardiorespiratory responses, hypothermia and body weight loss) in rats that received 5 or 10 injections of fentanyl and similar numbers of vehicle co-injections. With respect to the development of dependence, the NLX-precipitated withdrawal phenomena were reduced in rats that received had co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme. In regard to overcoming established dependence, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) were reduced in rats that had received co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme beginning with injection 6 of fentanyl. This study provides compelling evidence that co-injections of L-NAC and L-NACme prevent the acquisition of physical dependence and overcome acquired dependence to fentanyl in male rats. The higher efficacy of L-NACme is likely due to its greater cell penetrability in brain regions mediating dependence to fentanyl and interaction with intracellular signaling cascades, including redox-dependent processes, responsible for the acquisition of physical dependence to fentanyl.

L-cysteine ethyl ester prevents and reverses acquired physical dependence on morphine in male Sprague Dawley rats.

The molecular mechanisms underlying the acquisition of addiction/dependence on morphine may result from the ability of the opioid to diminish the transport of L-cysteine into neurons via inhibition of excitatory amino acid transporter 3 (EAA3). The objective of this study was to determine whether the co-administration of the cell-penetrant L-thiol ester, L-cysteine ethyl ester (L-CYSee), would reduce physical dependence on morphine in male Sprague Dawley rats. Injection of the opioid-receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IP), elicited pronounced withdrawal phenomena in rats which received a subcutaneous depot of morphine (150 mg/kg) for 36 h and were receiving a continuous infusion of saline (20 µL/h, IV) via osmotic minipumps for the same 36 h period. The withdrawal phenomena included wet-dog shakes, jumping, rearing, fore-paw licking, 360° circling, writhing, apneas, cardiovascular (pressor and tachycardia) responses, hypothermia, and body weight loss. NLX elicited substantially reduced withdrawal syndrome in rats that received an infusion of L-CYSee (20.8 µmol/kg/h, IV) for 36 h. NLX precipitated a marked withdrawal syndrome in rats that had received subcutaneous depots of morphine (150 mg/kg) for 48 h) and a co-infusion of vehicle. However, the NLX-precipitated withdrawal signs were markedly reduced in morphine (150 mg/kg for 48 h)-treated rats that began receiving an infusion of L-CYSee (20.8 µmol/kg/h, IV) at 36 h. In similar studies to those described previously, neither L-cysteine nor L-serine ethyl ester (both at 20.8 µmol/kg/h, IV) mimicked the effects of L-CYSee. This study demonstrates that 1) L-CYSee attenuates the development of physical dependence on morphine in male rats and 2) prior administration of L-CYSee reverses morphine dependence, most likely by intracellular actions within the brain. The lack of the effect of L-serine ethyl ester (oxygen atom instead of sulfur atom) strongly implicates thiol biochemistry in the efficacy of L-CYSee. Accordingly, L-CYSee and analogs may be a novel class of therapeutics that ameliorate the development of physical dependence on opioids in humans.

Fentanyl-induced reward seeking is sex and dose dependent and is prevented by D-cysteine ethylester.

Introduction: Despite their inclination to induce tolerance, addictive states, and respiratory depression, synthetic opioids are among the most effective clinically administered drugs to treat severe acute/chronic pain and induce surgical anesthesia. Current medical interventions for opioid-induced respiratory depression (OIRD), wooden chest syndrome, and opioid use disorder (OUD) show limited efficacy and are marked by low success in the face of highly potent synthetic opioids such as fentanyl. D-Cysteine ethylester (D-CYSee) prevents OIRD and post-treatment withdrawal in male/female rats and mice with minimal effect on analgesic status. However, the potential aversive or rewarding effects of D-CYSee have yet to be fully characterized and its efficacy could be compromised by interactions with opioid-reward pathology. Methods: Using a model of fentanyl-induced conditioned place preference (CPP), this study evaluated 1) the dose and sex dependent effects of fentanyl to induce rewarding states, and 2) the extent to which D-CYSee alters affective state and the acquisition of fentanyl-induced seeking behaviors. Results: Fentanyl reward-related effects were found to be dose and sex dependent. Male rats exhibited a range-bound dose response centered at 5 µg/kg. Female rats exhibited a CPP only at 50 µg/kg. This dose was effective in 25% of females with the remaining 75% showing no significant CPP at any dose. Pretreatment with 100 mg/kg, but not 10 mg/kg, D-CYSee prevented acquisition of fentanyl seeking in males while both doses were effective at preventing acquisition in females. Discussion: These findings suggest that D-CYSee is an effective co-treatment with prescribed opioids to reduce the development of OUD.

Lipophilic analogues of D-cysteine prevent and reverse physical dependence to fentanyl in male rats.

We examined whether co-injections of the cell-permeant D-cysteine analogues, D-cysteine ethyl ester (D-CYSee) and D-cysteine ethyl amide (D-CYSea), prevent acquisition of physical dependence induced by twice-daily injections of fentanyl, and reverse acquired dependence to these injections in freely-moving male Sprague Dawley rats. Injection of the opioid receptor antagonist, naloxone HCl (NLX, 1.5 mg/kg, IV), elicited a series of withdrawal phenomena that included cardiorespiratory and behavioral responses, and falls in body weight and body temperature, in rats that received 5 or 10 injections of fentanyl (125 µg/kg, IV), and the same number of vehicle co-injections. Regarding the development of physical dependence, the NLX-precipitated withdrawal phenomena were markedly reduced in fentanyl-injected rats that had received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV). Regarding reversal of established dependence to fentanyl, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) was markedly reduced in rats that received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV), starting with injection 6 of fentanyl. This study provides evidence that co-injections of D-CYSee and D-CYSea prevent the acquisition of physical dependence, and reverse acquired dependence to fentanyl in male rats. The lack of effect of D-cysteine suggests that the enhanced cell-penetrability of D-CYSee and D-CYSea into cells, particularly within the brain, is key to their ability to interact with intracellular signaling events involved in acquisition to physical dependence to fentanyl.

Infralimbic Estradiol Enhances Neuronal Excitability and Facilitates Extinction of Cocaine Seeking in Female Rats via a BDNF/TrkB Mechanism.

Women are more susceptible to developing cocaine dependence than men, but paradoxically, are more responsive to treatment. The potent estrogen, 17ß-estradiol (E2), mediates these effects by augmenting cocaine seeking but also promoting extinction of cocaine seeking through E2's memory-enhancing functions. Although we have previously shown that E2 facilitates extinction, the neuroanatomical locus of action and underlying mechanisms are unknown. Here we demonstrate that E2 infused directly into the infralimbic-medial prefrontal cortex (IL-mPFC), a region critical for extinction consolidation, enhances extinction of cocaine seeking in ovariectomized (OVX) female rats. Using patch-clamp electrophysiology, we show that E2 may facilitate extinction by potentiating intrinsic excitability of IL-mPFC neurons. Because the mnemonic effects of E2 are known to be regulated by brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), we examined whether BDNF/TrkB signaling was necessary for E2-induced enhancement of excitability and extinction. We found that E2-mediated increases in excitability of IL-mPFC neurons were abolished by Trk receptor blockade. Moreover, blockade of TrkB signaling impaired E2-facilitated extinction of cocaine seeking in OVX female rats. Thus, E2 enhances IL-mPFC neuronal excitability in a TrkB-dependent manner to support extinction of cocaine seeking. Our findings suggest that pharmacological enhancement of E2 or BDNF/TrkB signaling during extinction-based therapies would improve therapeutic outcome in cocaine-addicted women.

17ß-estradiol is necessary for extinction of cocaine seeking in female rats.

Human and preclinical models of addiction demonstrate that gonadal hormones modulate acquisition of drug seeking. Little is known, however, about the effects of these hormones on extinction of drug-seeking behavior. Here, we investigated how 17ß-estradiol (E2) affects expression and extinction of cocaine seeking in female rats. Using a conditioned place preference (CPP) paradigm, ovariectomized rats were maintained throughout conditioning with 2 d of E2 treatment followed by 2 d of vehicle treatment, or were injected with E2 daily. Hormone injections were paired or explicitly unpaired with place conditioning sessions. Expression of a cocaine CPP was of equal magnitude regardless of conditioning protocol, suggesting that E2 levels during conditioning did not affect subsequent CPP expression. During extinction, daily E2 administration initially enhanced expression of the cocaine CPP, but resulted in significantly faster extinction compared to controls. Whereas E2-treated rats were extinguished within 8 d, vehicle-treated rats maintained CPP expression for more than a month, indicative of perseveration. To determine whether E2 could rescue extinction in these rats, half were given daily E2 treatment and half were given vehicle. E2-treated rats showed rapid extinction, whereas vehicle-treated rats continued to perseverate. These data demonstrate for the first time that E2 is necessary for extinction of cocaine seeking in female rats, and that it promotes rapid extinction when administered daily. Clinically, these findings suggest that monitoring and maintaining optimal E2 levels during exposure therapy would facilitate therapeutic interventions for female cocaine addicts.

Amphetamine pretreatment facilitates appetitive sexual behaviors in the female rat.

RATIONALE: Intermittent treatment of rats with psychomotor stimulants induces behavioral sensitization to their motor-stimulating effects. This sensitization involves an increase in mesolimbic and nigrostriatal dopamine release, and in male rats, facilitates sexual behavior. OBJECTIVES: The aim of this study is to investigate the effect of repeated injections of D-amphetamine on appetitive and consummatory sexual behaviors in female rats. MATERIALS AND METHODS: Sexually experienced or naïve females were injected with either D-amphetamine (1 mg/kg, i.p.) or saline every other day for three injections each. After each amphetamine injection, females were placed either in a bilevel testing chamber or in their home cages. After saline injections, females were placed in bilevel chambers. Following a 3-week washout period, females were tested for sexual behavior in bilevel chambers in a drug-free state. RESULTS: Amphetamine pre-exposure facilitated the display of solicitations, hops and darts, and female-male mounting (FMM), regardless of whether the drug was paired with the testing environment. CONCLUSION: Intermittent amphetamine pretreatment that induces behavioral sensitization facilitates appetitive sexual behaviors in female rats, as has been shown previously in male rats. This suggests that the physiological substrates that modulate sensitized responses to psychomotor stimulants also mediate sensitized appetitive responses to sexual cues, including solicitation, hops and darts, and FMM. As in male rats, this facilitation was a direct consequence of amphetamine sensitization and not due to conditioned associations between drug and test environment.

Noradrenergic signaling in infralimbic cortex increases cell excitability and strengthens memory for fear extinction.

Emotional arousal strengthens memory. This is most apparent in aversive conditioning, in which the stress-related neurotransmitter norepinephrine (NE) enhances associations between sensory stimuli and fear-inducing events. In contrast to conditioning, extinction decreases fear responses, and is thought to form a new memory. It is not known, however, whether NE is necessary for extinction learning. Previous work has shown that the infralimbic prefrontal cortex (IL) is a site of extinction consolidation. Here, we show that blocking noradrenergic beta-receptors in IL before extinction training impaired retrieval of extinction the following day, consistent with a weakened extinction memory. We further found that the sequelae of beta-receptor activation, including protein kinase A (PKA), gene transcription and translation in IL, are necessary for extinction. To determine whether activation of this cascade modulates IL excitability, we measured the response of IL pyramidal neurons to injected current. NE increased the excitability of IL neurons in a beta-receptor- and PKA-dependent manner. We suggest that NE released in IL during fear extinction activates a PKA-mediated molecular cascade that strengthens extinction memory. Thus, emotional arousal evoked by conditioned fear paradoxically promotes the subsequent extinction of that fear, thereby ensuring behavioral flexibility.