Priming Injection Research Articles

O-076 Novel method to enhance preantral follicle growth by granulocyte colony-stimulating factor administration improves embryos and pregnancy rate in poor ovarian reserve: a randomized controlled trial

Abstract Study question Whether priming with granulocyte colony-stimulating factor (G-CSF) before ART improves embryo and pregnancy rate while increasing anti-Müllerian hormone (AMH) in patients with poor ovarian reserve. Summary answer G-CSF priming improved embryonic development and pregnancy rate in ART with poor ovarian reserve, simultaneously increasing serum AMH. Enhanced preantral follicle growth likely was responsible. What is known already Eleven years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering G-CSF with embryo transfers. No implantation succeeded directly, but serendipitously 3 of the 10 spontaneously became pregnant 2 months later. One of these pregnancies involved twins in a 45-year-old woman without ovulation induction. We hypothesized that G-CSF administration might have stimulated preantral follicle growth, with improved ovulation after 2 months. In animal models with diminished ovarian reserve, G-CSF administration consistently increased ovarian preantral follicles and AMH. Therefore, we designed this clinical trial. Study design, size, duration Patients were prospectively randomized to priming or control groups. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of IVF/ICSI and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Participants/materials, setting, methods Hundred ART patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. Main results and the role of chance Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by G-CSF priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts (53% with priming vs. 24% in controls). The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. The efficacy of G-CSF priming was not associated with genotypes for killer-cell immunoglobulin-like receptors. No adverse effects of priming were observed. Limitations, reasons for caution Enhancement of preantral follicle growth by G-CSF administration needs to be confirmed histologically as this clinical trial could not allow ovarian biopsies for its purpose of infertility treatments. Wider implications of the findings This study proposes a novel, simple, and safe treatment for poor ovarian reserve. In addition, enhancement of preantral follicle growth by G-CSF priming may also improve ovulatory dysfunctions in polycystic ovary syndrome with elevated AMH, reflecting pooling of retarded/arrested growing preantral follicles. Trial registration number UMIN000013956

Abstract 21: Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency <4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p <0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p <0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 21.

Nalmefene attenuates reinstatement of methamphetamine-seeking behavior in rats through group II metabotropic glutamate receptors (mGluR2/3)

Nalmefene, an analog to naltrexone, is an antagonist at the μ opioid receptor and a partial agonist at the κ opioid receptor. Both agents are approved for the treatment of alcohol use disorder and opioid addiction. Here, we evaluated the potential of nalmefene for treating psychostimulant dependence using a methamphetamine (METH) self-administration rat model. Rats were trained to press a lever for 0.02-mg intravenous METH infusions paired with drug-associated cues (light and tone) under a fixed ratio 1 schedule. After a 10-day METH self-administration acquisition phase, rats underwent extinction training. A reinstatement test was conducted after fulfilment of the extinction criterion under saline infusions. Re-exposure to METH-associated cues or a priming injection of METH (1.0 mg/kg, i.p.) significantly reinstated METH-seeking behaviors. Pretreatment with nalmefene (10 mg/kg, i.p.) immediately before reinstatement tests significantly attenuated the METH-seeking behaviors induced by both cues and METH priming injection. To investigate the mechanism of effect of nalmefene, we also tested the ability of a group II metabotropic glutamate receptors (mGluR2/3) antagonist, LY341495, to the ameliorating effects of nalmefene. Pretreatment with LY341495 (1.0 mg/kg, i.p.) before nalmefene administration antagonized the effect of nalmefene on reinstatement. LY341495 alone did not affect the reinstatement of lever pressing. We found that nalmefene attenuates METH-seeking behaviors during withdrawal, and this attenuation of reinstatement is mediated by the activation of mGluR2/3. The present findings suggest that nalmefene could decrease incentive motivation for drug use in psychostimulant dependence.

Effects of adolescent methylphenidate administration on methamphetamine conditioned place preference in an animal model of attention-deficit/hyperactivity disorder: Examination of potential sex differences

BackgroundIndividuals with attention-deficit/hyperactivity disorder (ADHD) are more likely to be diagnosed with a substance use disorder; however, the effects of long-term psychostimulant treatment on addiction are mixed. Preclinical studies are useful for further elucidating the relationship between ADHD and addiction-like behaviors, but these studies have focused on male subjects only. The goal of the current study was to determine if early-life administration of methylphenidate (MPH) augments methamphetamine (METH) conditioned place preference (CPP) and/or potentiates reinstatement of CPP in both male and female rats. MethodsMale and female spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) received either MPH (1.5mg/kg; p.o.) or vehicle (1.0ml/kg) during adolescence (postnatal day [PND] ~29–57). Two weeks after cessation of MPH treatment, rats were tested for METH CPP (1.0mg/kg or 2.0mg/kg; s.c.). Rats were then given extinction sessions. Once rats met extinction criteria, they were tested for reinstatement of CPP following a priming injection of METH (0.25mg/kg; s.c.). ResultsAll groups developed METH CPP, except vehicle-treated SHR males and vehicle-treated WKY females conditioned with the higher dose of METH (2.0mg/kg). Female SHRs treated with MPH showed greater reinstatement of METH CPP compared to female SHRs treated with vehicle. Adolescent MPH treatment did not augment the locomotor-stimulant effects of METH in adulthood. ConclusionsThese results demonstrate the importance of considering biological sex when prescribing psychostimulant medications for ADHD as long-term MPH administration may increase the risk of continued drug use in females with ADHD following a period of abstinence.

Liraglutide attenuates nicotine self-administration as well as nicotine seeking and hyperphagia during withdrawal in male and female rats.

Nicotine cessation is associated with increased consumption of highly palatable foods and body weight gain in most smokers. Concerns about body weight gain are a major barrier to maintaining long-term smoking abstinence, and current treatments for nicotine use disorder (NUD) delay, but do not prevent, body weight gain during abstinence. Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce food intake and are FDA-approved for treating obesity. However, the effects of GLP-1R agonist monotherapy on nicotine seeking and withdrawal-induced hyperphagia are unknown. We screened the efficacy of the long-lasting GLP-1R agonist liraglutide to reduce nicotine-mediated behaviors including voluntary nicotine taking, as well as nicotine seeking and hyperphagia during withdrawal. Male and female rats self-administered intravenous nicotine (0.03 mg/kg/inf) for ~21 days. Daily liraglutide administration (25 μg/kg, i.p.) started on the last self-administration day and continued throughout the extinction and reinstatement phases of the experiment. Once nicotine taking was extinguished, the reinstatement of nicotine-seeking behavior was assessed after an acute priming injection of nicotine (0.2 mg/kg, s.c.) and re-exposure to conditioned light cues. Using a novel model of nicotine withdrawal-induced hyperphagia, intake of a high fat diet (HFD) was measured during home cage abstinence in male and female rats with a history of nicotine self-administration. Liraglutide attenuated nicotine self-administration and reinstatement in male and female rats. Repeated liraglutide attenuated withdrawal-induced hyperphagia and body weight gain in male and female rats at a dose that was not associated with malaise-like effects. These findings support further studies investigating the translational potential of GLP-1R agonists to treat NUD.

The Effect of [(D-Ala6, Pro9NEt)mGnRH-a + Metoclopramide] (Ovopel) on Propagation Effectiveness of Two Breeding Lines of Common Carp (Cyprinus carpio L.) and on Luteinizing Hormone and 17α,20β-Dihydroxyprogesterone Levels in Females during Ovulation Induction.

The study evaluates the impact of Ovopel on the reproductive effectiveness of carp from Polish line 6 and Lithuanian line B and the release of luteinizing hormone (LH) and 17α,20β-dihydroxyprogesterone (17α,20β-DHP) in females from these lines during ovulation induction. The levels of both hormones were determined in blood plasma samples taken just before the priming injection of Ovopel (0 h), at the time of administering the resolving dose of Ovopel (12 h), and after the next 12 h (24 h). Following Ovopel treatment, the mean egg weight obtained for line 6 was higher, but not statistically different, than that observed for line B. Egg quality, on the other hand, was significantly higher in line B. Female provenance did not significantly affect the number of eggs and living embryos after 70 h incubation. However, the total egg number for line 6 was higher. The mean number of living embryos (70 h) was similar for both lines. LH concentrations at 0, 12, and 24 h were not statistically different between the lines. A comparison of LH concentrations between ovulated and non-ovulated females at different sampling times revealed no significant differences either within or between the lines. Statistically significant differences in LH levels were found for both ovulated and non-ovulated females from a given line between the sampling times. The results for 17α,20β-DHP were similar, with only one difference: 24 h after the priming dose of Ovopel, 17α,20β-DHP levels in ovulated fish were significantly higher compared with non-ovulated females, but only in line 6.

Granulocyte colony-stimulating factor priming improves embryos and pregnancy rate in patients with poor ovarian reserve: a randomized controlled trial

BackgroundGranulocyte colony-stimulating factor (G-CSF) administration increased ovarian preantral follicles and anti-Müllerian hormone (AMH) in animal models with diminished ovarian reserve. We investigated whether G-CSF priming before treatment with assisted reproductive technology (ART) improved embryo development and pregnancy rate while increasing serum AMH in patients with poor ovarian reserve.MethodsIn this prospective randomized open-label controlled trial, 100 patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of in vitro fertilization/intracytoplasmic sperm injection and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming.ResultsFertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts. The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04–7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. No adverse effects of priming were observed.ConclusionG-CSF priming improved embryonic development and pregnancy rate during ART treatment and increased AMH in patients with poor ovarian reserve. Enhanced preantral follicle growth likely was responsible.Trial registrationUMIN registration in Japan (UMIN000013956) on May 14, 2014. https://www.umin.ac.jp/ctr/index.htm.

Development of a polyclonal antibody Edwardsiella ictaluri serum for diagnosis of Enteric Septicemia of Catfish (ESC) in Patin Catfish (Pangasianodon hypopthalmus)

Enteric Septicemia of Catfish (ESC), caused by Edwarsiella ictaluri, has been considered a major bacterial disease in catfish, including Patin (Pangasionodon hypophthalmus), causing economic losses. Monitoring activities and identification of bacterial disease are performed using a series of biochemical tests, including Gram stain, Ryu, motility, and glucose; the test is at a certain temperature, requires specific conditions and facilities, and takes a long time. We developed a method based on antigen-antibody reactions to diagnose the disease, which provides more specific/accurate results in a shorter time to be used as a more flexible and rapid "diagnosis tool" as an anticipatory step in disease control. The development of antibody-serum is based on the specific character (specificity) of bacteria for antigen and performed according to standard methods using rabbits as test animals. The process starts with a mass culture of E. ictaluri, antigen in-activation, priming and booster injection, bleeding, and deriving polyclonal antibody serum. This diagnostic tool is expected to identify the target bacteria within 24 h, indicated by agglutination in the antigen-antibody reaction called the serum antibodies method. In this research, we produced serum, and ready to use, a 0.05-0.1 mL of serum-containing antibody is reacted with the bacterial isolate suspension.

Changes in Ovulation-Related Gene Expression during Induced Ovulation in the Amur Sturgeon (Acipenser schrenckii) Ovarian Follicles.

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17α,20β-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons.

Absence of active systemic anaphylaxis in guinea pigs upon intramuscular injection of inactivated SARS-CoV-2 vaccine (Vero cells)

Background: The safety of novel vaccines against COVID-19 is currently a major focus of preclinical research. As a part of the safety evaluation testing package, 24 healthy guinea pigs were used to determine whether repeated administration of inactivated SARS-CoV-2 vaccine could induce active systemic anaphylaxis (ASA), and to evaluate its degree of severity. Method: According to sex and body weight, the animals were randomly divided into three experimental groups (eight animals per group). The negative control group received 0.9% sodium chloride (priming dose: 0.5 mL/animal; challenge dose: 1 mL/animal); the positive control group received 10% ovalbumin (priming dose: 0.5 mL/animal; challenge dose: 1 mL/animal); and the inactivated SARS-CoV-2 vaccine group received inactivated SARS-CoV-2 vaccines (priming dose: 100 U in 0.5 mL/animal; challenge dose: 200 U in 1 mL/animal). Priming dose administration was conducted by multi-point injection into the muscles of the hind limbs, three times, once every other day. On days 14 and 21 after the final priming injection, a challenge test was conducted. Half of the animals in each group were injected intravenously with twice the dose and volume of the tested substance used for immunization. During the experimental course, the injection site, general clinical symptoms, body weight, and systemic allergic reaction symptoms were monitored. Result: After intramuscular injection of inactivated SARS-CoV-2 vaccine, there were no abnormal reactions at the injection site, clinical symptoms, or deaths. There was no difference in body weight between the groups, and there were no allergic reactions. Conclusion: Thus, inactivated SARS-CoV-2 vaccine injected intramuscularly in guinea pigs did not produce ASA and had a good safety profile, which can provide actual data on vaccine risks and important reference data for clinical research on this vaccine.

Novelty preference does not predict trait cocaine behaviors in male rats

Heightened novelty seeking is a risk factor for the initiation of drug use and development of substance use disorders. In rats, novelty seeking can be examined by assessing preference for a novel environment. Some evidence indicates that high novelty preferring (HNP) rats have higher drug intake compared to low novelty preferring (LNP) rats, although these data are mixed. Moreover, the extent to which the HNP phenotype can predict other initial drug behaviors, including economic demand for cocaine, has not been tested. Here, we screened a cohort (n=60) of male rats for novelty preference and several subsequent cocaine behaviors, including locomotor reactivity to a cocaine priming injection, acquisition of cocaine self-administration, as well as cocaine demand using a within-session behavioral economics procedure. Novelty preference did not correlate with cocaine behaviors, nor were there any differences between HNP and LNP rats identified using a median split strategy. Moreover, regression analyses indicated that novelty preference did not have predictive utility for any of the cocaine behaviors tested. Thus, the extent to which the novelty preference trait can predict initial cocaine-related behaviors in male rats may be limited. This is in contrast to the novel locomotor reactivity phenotype, which is strongly linked with initial cocaine intake, indicating that these traits are distinct and differentially predict cocaine behaviors in rats.

Modeling Stimulant and Opioid Co-use in Rats Provided Concurrent Access to Methamphetamine and Fentanyl.

Concurrent use of stimulants (e.g., methamphetamine) and opioids (e.g., fentanyl) has become increasingly common in recent years and continues to pose an enormous health burden, worldwide. Despite the prevalence, relatively little is known about interactions between the reinforcing effects of stimulants and opioids in this pattern of polysubstance use. The goals of the current study were to evaluate the relative reinforcing and relapse-related effects of methamphetamine and fentanyl using a concurrent access, drug-vs.-drug choice procedure. Male Sprague-Dawley rats were first allowed to acquire self-administration for either 0.1 mg/kg/infusion methamphetamine or 0.0032 mg/kg/infusion fentanyl, independently, after which concurrent access to both drugs was provided. When training doses of methamphetamine and fentanyl were concurrently available, a subset of rats self-administered both drugs, either within a session or alternating across sessions, whereas the remaining rats responded exclusively for one drug. When the cost of the preferred drug was increased (i.e., unit dose reduced), or the cost of the non-preferred drug was decreased (i.e., unit dose increased), choice was largely allocated toward the cheaper alternative. Following extinction of responding, methamphetamine- and fentanyl-paired cues reinstated responding on both levers. Responding reinstated by a priming injection of methamphetamine or fentanyl allocated more responding to the lever previously reinforced by the priming drug. The current studies suggest that choice of methamphetamine and fentanyl is largely allocated to the cheaper alternative, although more co-use was observed than would be expected for economic substitutes. Moreover, they lay the groundwork for more fully evaluating interactions between commonly co-abused drugs (e.g., stimulants and opioids) in order to better understand the determinants of polysubstance use and develop effective treatment strategies for individuals suffering from a polysubstance use disorder.

Ibogaine Blocks Cue- and Drug-Induced Reinstatement of Conditioned Place Preference to Ethanol in Male Mice

Ibogaine is a psychedelic extracted from the plant Tabernanthe iboga Baill. (Apocynaceae), natural from Africa, and has been proposed as a potential treatment for substance use disorders. In animal models, ibogaine reduces ethanol self-administration. However, no study to date has investigated the effects of ibogaine on ethanol-induced conditioned place preference (CPP). The present study aimed to investigate the effects of repeated treatment with ibogaine on the reinstatement of CPP to ethanol in male mice. The rewarding effects of ethanol (1.8 g/kg, i. p.) or ibogaine (10 or 30 mg/kg, p. o.) were investigated using the CPP model. Furthermore, we evaluated the effects of repeated treatment with ibogaine (10 or 30 mg/kg, p. o.) on the reinstatement of ethanol-induced CPP. Reinstatement was evaluated under two conditions: 1) during a priming injection re-exposure test in which animals received a priming injection of ethanol and had free access to the CPP apparatus; 2) during a drug-free test conducted 24 h after a context-paired re-exposure, in which subjects received an injection of ethanol and were confined to the compartment previously conditioned to ethanol. Our results show that ethanol, but not ibogaine, induced CPP in mice. Treatment with ibogaine after conditioning with ethanol blocked the reinstatement of ethanol-induced CPP, both during a drug priming reinstatement test and during a drug-free test conducted after re-exposure to ethanol in the ethanol-paired compartment. Our findings add to the literature suggesting that psychedelics, in particular ibogaine, may have therapeutic properties for the treatment of alcohol use disorder at doses that do not have rewarding effects per se.

The interaction between sexual reward/ deprivation and the acquisition, extinction and reinstatement of morphine-seeking behavior

Natural rewards and abused drugs affect the function of the common brain's reward system. Interaction between social and drug rewards can change the vulnerability to development of drug addiction. Here, we investigate the effects of sexual experience and sex deprivation on the acquisition, maintenance, and drug prime-induced reinstatement of morphine-seeking behavior in male mice using conditioned place preference (CPP). CPP induced with morphine (3, 5, 7 mg/kg, s.c. for 3 days) lasted for 10 days after cessation of morphine treatment and priming dose of morphine (2 mg/kg, s.c.) reinstated the extinguished CPP. In the post-test phase, sexually experienced animals showed a lower preference for morphine compared to sex-deprived males. In the extinction phase, sex deprivation shortened maintenance time compared to control animals. The preference for morphine in sexually experienced animals did not diminish by the seventeenth extinction day. In both groups, the priming injection of morphine after the extinction period could reinstate the extinguished morphine-induced CPP. Together, these data showed the interaction between sex and drug reward and that sexual behavior -a natural rewarding stimulus- can prolong, whereas sex deprivation can block the maintenance of morphine-seeking behaviors. Sexual experience may induce functional and morphological alterations in brain reward areas particularly the mesolimbic system similar to repeated exposure to abused drugs which can affect morphine-seeking behaviors.

Memantine Prevents the WIN 55,212-2 Evoked Cross-Priming of Ethanol-Induced Conditioned Place Preference (CPP).

The activation of the endocannabinoid system controls the release of many neurotransmitters involved in the brain reward pathways, including glutamate. Both endocannabinoid and glutamate systems are crucial for alcohol relapse. In the present study, we hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors regulate the ability of a priming dose of WIN 55,212-2 to cross-reinstate ethanol-induced conditioned place preference (CPP). To test this hypothesis, ethanol-induced (1.0 g/kg, 10% w/v, i.p.) CPP (unbiased method) was established using male adult Wistar rats. After CPP extinction, one group of animals received WIN 55,212-2 (1.0 and 2.0 mg/kg, i.p.), the cannabinoid receptor 1 (CB1) agonist, or ethanol, and the other group received memantine (3.0 or 10 mg/kg, i.p.), the NMDA antagonist and WIN 55,212-2 on the reinstatement day. Our results showed that a priming injection of WIN 55,212-2 (2.0 mg/kg, i.p.) reinstated (cross-reinstated) ethanol-induced CPP with similar efficacy to ethanol. Memantine (3.0 or 10 mg/kg, i.p.) pretreatment blocked this WIN 55,212-2 effect. Furthermore, our experiments indicated that ethanol withdrawal (7 days withdrawal after 10 days ethanol administration) down-regulated the CNR1 (encoding CB1), GRIN1/2A (encoding GluN1 and GluN2A subunit of the NMDA receptor) genes expression in the prefrontal cortex and dorsal striatum, but up-regulated these in the hippocampus, confirming the involvement of these receptors in ethanol rewarding effects. Thus, our results show that the endocannabinoid system is involved in the motivational properties of ethanol, and glutamate may control cannabinoid induced relapse into ethanol seeking behavior.

Escalation and reinstatement of fentanyl self-administration in male and female rats.

Escalation of drug intake and craving are two DSM-5 hallmark symptoms of opioid use disorder (OUD). This study determined if escalation of intake as modeled by long access (LgA) self-administration (SA) and craving measured by reinstatement are related. Adult male and female Sprague-Dawley rats were trained to self-administer fentanyl across 7 daily 1-h short access (ShA) sessions, followed by 21 SA sessions of either 1- or 6-h duration (ShA or LgA). Following 14 1-h extinction sessions, Experiment 1 assessed reinstatement induced by either fentanyl (10 or 30µg/kg) or yohimbine (1 or 2mg/kg), and Experiment 2 assessed reinstatement induced by a drug-associated cue light. Females acquired fentanyl SA faster than males. When shifted to LgA sessions, LgA rats escalated fentanyl intake, but ShA rats did not; no reliable sex difference in the rate of escalation was observed. In extinction, compared to ShA rats, LgA rats initially responded less and showed less decay of responding across sessions. A priming injection of fentanyl induced reinstatement, with LgA rats reinstating more than ShA rats at the 30µg/kg dose. Yohimbine (1mg/kg) also induced reinstatement, but there was no effect of access group or sex. With cue-induced reinstatement, LgA females reinstated less than LgA males and ShA females. Among the different reinstatement tests assessed, escalation of fentanyl SA increased only drug-primed reinstatement, suggesting a limited relationship between escalation of drug intake and craving (reinstatement) for OUD.

Linalool attenuates acquisition and reinstatement and accelerates the extinction of nicotine-induced conditioned place preference in male mice

ABSTRACT Background: Nicotine is the addictive agent in tobacco products. The monoterpene linalool is the main ingredient in the essential oils of various aromatic plants. It has previously been demonstrated that linalool has beneficial effects on some mechanisms that are important in drug addiction. Objectives: The goal of the current study was to investigate the effect of linalool on nicotine-induced conditioned place preference (CPP) in male mice. Methods: CPP was induced by administering intraperitoneal (i.p.) injection of nicotine (0.5 mg/kg) during the conditioning phase. The effects of nicotinic acetylcholine receptor partial agonist varenicline and linalool on the rewarding characteristics of nicotine were tested in mice with administration of linalool (12.5, 25, and 50 mg/kg, i.p.), varenicline (2 mg/kg, i.p.) or saline 30 minutes before nicotine injection. CPP was extinguished by repeated testing, during which conditioned mice were administered varenicline and linalool every day. One day after the last extinction trial, mice that received linalool, varenicline or saline 30 minutes before a priming injection of nicotine (0.1 mg/kg, i.p.) were immediately tested for reinstatement of CPP. Results: Linalool attenuated nicotine acquisition (50 mg/kg, p < .01) and reinstatement (25 and 50 mg/kg, respectively p < .05, p < .01) and accelerated the extinction of nicotine-induced CPP (50 mg/kg, p < .05). Linalool exhibited similar effects on the reference drug varenicline in the CPP phases. Conclusion: These results suggest that linalool may be helpful as an adjuvant for the treatment of nicotine use disorder.

Deep Brain Stimulation of the Lateral Hypothalamus Facilitates Extinction and Prevents Reinstatement of Morphine Place Preference in Rats

ObjectivesWe have previously shown that high-frequency (HF) deep brain stimulation (DBS) of the lateral hypothalamus (LH) during the acquisition phase of morphine-induced conditioned place preference (CPP) abolished the development of morphine reward. In the present study, we investigated the effect of DBS in the LH during the extinction phase of morphine CPP. Materials and MethodsRats were implanted with electrodes in the LH and went through conditioning trials for morphine CPP (40 min each, for three days), followed by extinction trials (20 min, for nine days). DBS-like stimulation (square pulses at 13 or 130 Hz, 200 μA, 100 μsec) was applied during the extinction trials. ResultsRats that received HF-DBS (130 Hz) accomplished extinction of morphine place preference by day 5 of the phase, whereas those in sham-stimulation or low-frequency-DBS (LF-DBS, 13 Hz) groups reached the criterion for extinction at day 8. One day later, rats received a priming injection of morphine (2 mg/kg) to reinstate the extinguished preference. While rats in the sham-DBS and LF-DBS relapsed into the state of preferring morphine-associated context, those in the HF-DBS group did not show such preference. Rats were then proceeded into an additional phase of extinction training (20 min, once daily, three to five days) with DBS, followed by restraint stress-induced reinstatement test. Again, sham-DBS and LF-DBS had no effect on relapse to the morphine place preferring state, but HF-DBS completely prevented the relapse. ConclusionHF-DBS facilitated extinction of morphine place preference and disrupted drug priming- and stress-induced renewal of morphine place preference.

Cross-reinstatement of mitragynine and morphine place preference in rats

Kratom is a medicinal plant that exhibits promising results as an opiate substitute. However, there is little information regarding the abuse profile of its main psychoactive constituent, mitragynine (MG), particularly in relapse to drug abuse. Using the place conditioning procedure as a model of relapse, this study aims to evaluate the ability of MG to induce conditioned place preference (CPP) reinstatement in rats. To evaluate the cross-reinstatement effects, MG and morphine were injected to rats that previously extinguished a morphine- or MG-induced CPP. Following a CPP acquisition induced by either MG (10 and 30 mg/kg, i.p.) or morphine (10 mg/kg, i.p.), rats were subjected to repeated CPP extinction sessions. A low dose priming injection of MG or morphine produced a reinstatement of the previously extinguished CPP. In the second experiment of this study, a priming injection of morphine (1, 3 and 10 mg/kg, i.p.) dose-dependently reinstated an MG-induced CPP. Likewise, a priming injection of MG (3, 10 and 30 mg/kg, i.p.) was able to dose-dependently reinstate a morphine-induced CPP. The present study demonstrates a cross-reinstatement effect between MG and morphine, thereby suggesting a similar interaction in their rewarding motivational properties. The findings from this study also suggesting that a priming exposure to kratom and an opioid may cause relapse for a previously abused drug.

Multidose Priming and Delayed Boosting Improve Plasmodium falciparum Sporozoite Vaccine Efficacy Against Heterologous P. falciparum Controlled Human Malaria Infection.

A live-attenuated Plasmodium falciparum sporozoite (SPZ) vaccine (PfSPZ Vaccine) has shown up to 100% protection against controlled human malaria infection (CHMI) using homologous parasites (same P. falciparum strain as in the vaccine). Using a more stringent CHMI, with heterologous parasites (different P. falciparum strain), we assessed the impact of higher PfSPZ doses, a novel multi-dose prime regimen, and a delayed vaccine boost upon vaccine efficacy (VE). We immunized 4 groups that each contained 15 healthy, malaria-naive adults. Group 1 received 5 doses of 4.5 x 105 PfSPZ (Days 1, 3, 5, and 7; Week 16). Groups 2, 3, and 4 received 3 doses (Weeks 0, 8, and 16), with Group 2 receiving 9.0 × 105/doses; Group 3 receiving 18.0 × 105/doses; and Group 4 receiving 27.0 × 105 for dose 1 and 9.0 × 105 for doses 2 and 3. VE was assessed by heterologous CHMI after 12 or 24 weeks. Volunteers not protected at 12 weeks were boosted prior to repeat CHMI at 24 weeks. At 12-week CHMI, 6/15 (40%) participants in Group 1 (P = .04) and 3/15 (20%) participants in Group 2 remained aparasitemic, as compared to 0/8 controls. At 24-week CHMI, 3/13 (23%) participants in Group 3 and 3/14 (21%) participants in Group 4 remained aparasitemic, versus 0/8 controls (Groups 2-4, VE not significant). Postboost, 9/14 (64%) participants versus 0/8 controls remained aparasitemic (3/6 in Group 1, P = .025; 6/8 in Group 2, P = .002). Administering 4 stacked priming injections (multi-dose priming) resulted in 40% VE against heterologous CHMI, while dose escalation of PfSPZ using single-dose priming was not significantly protective. Boosting unprotected subjects improved VE at 24 weeks, to 64%. NCT02601716.