Individual difference in addiction-like behaviors and choice between cocaine versus food in Heterogeneous Stock rats

Rationale and objectives: Recent studies reported that when given a mutually exclusive choice between cocaine and palatable food, most rats prefer the non-drug reward over cocaine. However, these studies used rat strains with limited genetic and behavioral diversity. Here, we used a unique outbred strain of rats (Heterogeneous Stock, HS) that mimic the genetic variability of humans. Methods: We first identified individual differences in addiction-like behaviors (low and high). Next, we tested choice between cocaine and palatable food using a discrete choice procedure. We characterized the individual differences using an Addiction score that incorporates key features of addiction: escalated intake, highly motivated responding (progressive ratio), and responding despite adverse consequences (footshock punishment). We assessed food vs. cocaine choice at different drug-free days (without pre-trial cocaine self administration) during acquisition of cocaine self-administration or after escalation of cocaine self-administration. We also assessed drug vs. food choice immediately after 1-, 2-, or 6-h cocaine self-administration. Results: Independent of the addiction score, without pre-trial coccaine (1 or more abstinence days) HS rats strongly preferred the palatable food over cocaine, even if the food reward was delayed or its size was reduced. However, rats with high but not low addiction score modestly increased cocaine choice immediately after 1-, 2- or 6-h cocaine self-administration. Conclusions: Like other strains, HS rats strongly prefer palatable food over cocaine. Individual differences in addiction score were associated with increased drug choice in the presence but not absence (abstinence) of cocaine. The HS strain may be useful in studies on mechanisms of addiction vulnerability.

PACAP induces light aversion in mice by an inheritable mechanism independent of CGRP

The neuropeptides CGRP and PACAP have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about a third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex-linked, although there was generally a trend for greater responses among male mice. RNA-seq analysis of trigeminal ganglia yielded hieriechial clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of pituitary hormones and receptors in a subset of responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice.SignificanceThe relationship between the neuropeptides CGRP and PACAP in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

Development of experimental silicosis in inbred and outbred mice depends on instillation volume

There is considerable variation in methods to induce experimental silicosis with the effects of dose and route of exposure being well documented. However, to what extent the volume of silica suspension alters the dispersion and severity of silicosis has not been adequately investigated. In this study, the optimal volume of a crystalline silica suspension required to obtain uniform distribution and greatest incidence and severity of silicosis was determined in inbred and outbred mice. Silica dispersal, detected by co-inspiration with India ink and polarized light microscopy, was highly dependent upon volume. Furthermore, although peribronchitis, perivasculitis, and increases in bronchoalveolar lavage fluid cell numbers were detected a lower doses and volumes, significant alveolitis required exposure to 5 mg of silica in 50 μl. This dose and volume of transoral instillation led to a greater penetrance of silicosis in the genetically heterogeneous Diversity Outbred strain as well as greater alveolar inflammation typical of the silicosis in human disease. These findings underscore the critical importance of instillation volume on the induction, severity, and type of inflammatory pathology in experimental silicosis.

Puffing activity in Drosophila melanogaster Meig. political chromosomes after exposure to microwave radiation

Aim. The aim of the work was to study the effect of microwave radiation of varying intensity on the polytene chromosomes puffing activity in larvae salivary glands of Drosophila melanogaster. Methods. The wild type outbred strain Oregon-R was used as the material. Microwave radiation with a frequency of 36.64 GHz and a power density of 0.1 and 1 W / m2 was used. Exposure to microwaves was applied in early embryogenesis after 3-hour oviposition. Exposure time was 30 sec. The puff sizes were studied on the squashed preparations of larvae salivary glands stained with acetoorcein. Dimensions of four puffs were investigated^ 2B5-6 (X chromosome); 62E, 71CE and 72CD (chromosome 3L). The measurements were carried out using an ocular-micrometer. Results. There were no significant changes in the size of the puffs in any of the four loci studied, regardless of the applied power density. Conclusions. Microwave radiation in early embryogenesis at a frequency of 36.64 GHz, a power density of 0.1 and 1 W/m2, and an exposure of 30 sec does not have a significant effect on the puff sizes in the Drosophila polytene chromosomes. Keywords: Drosophila melanogaster Meig., giant chromosomes, puff sizes, non-ionizing radiation.

Regulatory Lymphocytes Are Key Factors in MHC-Independent Resistance to EAE

Background and Objectives. Resistant and susceptible mouse strains to experimental autoimmune encephalomyelitis (EAE), an inducible demyelinating experimental disease serving as animal model for multiple sclerosis, have been described. We aimed to explore MHC-independent mechanisms inducing resistance to EAE.Methods. For EAE induction, female C57BL/6 (susceptible strain) and CD1 (resistant outbred strain showing heterogeneous MHC antigens) mice were immunized with the 35–55 peptide of myelin oligodendrocyte glycoprotein (MOG35−55). We studied T cell proliferation, regulatory and effector cell subpopulations, intracellular and serum cytokine patterns, and titers of anti-MOG serum antibodies.Results. Upon immunization with MOG35−55, T lymphocytes from susceptible mice but not that of resistant strain were capable of proliferating when stimulated with MOG35−55. Accordingly, resistant mice experienced a rise in regulatory B cells (P=0.001) and, to a lower extent, in regulatory T cells (P=0.02) compared with C57BL/6 susceptible mice. As a consequence, MOG35−55-immunized C57BL/6 mice showed higher percentages of CD4+ T cells producing both IFN-gamma (P=0.02) and IL-17 (P=0.009) and higher serum levels of IL-17 (P=0.04) than resistant mice.Conclusions. Expansion of regulatory B and T cells contributes to the induction of resistance to EAE by an MHC-independent mechanism.

Loss of Roquin induces early death and immune deregulation but not autoimmunity

The substitution of one amino acid in the Roquin protein by the sanroque mutation induces a dramatic autoimmune syndrome in mice. This is believed to occur through ectopic expression of inducible T cell co-stimulator (ICOS) and unrestrained differentiation of follicular T helper cells, which induce spontaneous germinal center reactions to self-antigens. In this study, we demonstrate that tissue-specific ablation of Roquin in T or B cells, in the entire hematopoietic system, or in epithelial cells of transplanted thymi did not cause autoimmunity. Loss of Roquin induced elevated expression of ICOS through T cell–intrinsic and –extrinsic mechanisms, which itself was not sufficient to break self-tolerance. Instead, ablation of Roquin in the hematopoietic system caused defined changes in immune homeostasis, including the expansion of macrophages, eosinophils, and T cell subsets, most dramatically CD8 effector–like T cells, through cell-autonomous and nonautonomous mechanisms. Germline Roquin deficiency led to perinatal lethality, which was partially rescued on the genetic background of an outbred strain. However, not even complete absence of Roquin resulted in overt self-reactivity, suggesting that the sanroque mutation induces autoimmunity through an as yet unknown mechanism.

Multiple Antigen Peptide Vaccines against Plasmodium falciparum Malaria

ABSTRACT The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-142, and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-142) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.

Clonidine and Dexmedetomidine Produce Antinociceptive Synergy in Mouse Spinal Cord

Background Synergy between drugs manifests with increased potency and/or efficacy of the combination relative to either agonist given alone. Synergy is typically observed between drugs of different classes, as is the case with the alpha-adrenergic-opioid receptor synergy often observed in preclinical studies. However, rare studies report synergy between agonists of the same class. The current study examined the analgesic interaction between two intrathecally injected alpha2-adrenergic receptor (AR) agonists previously thought to act at the same receptor subtype when given spinally. Methods Mice were given clonidine, dexmedetomidine, or the combination spinally to evaluate the interaction between these two agonists. The ED50 values were calculated, and the interactions were tested by isobolographic analysis. The rotarod test was performed in the same mice after the completion of analgesic assessment to assess motor or sedative effects. These experiments were performed in outbred mice as well as in mice with mutant alpha2A ARs, alpha2C AR knockout mice, or wild-type controls. Finally, analgesic cross-tolerance between clonidine and dexmedetomidine was evaluated. Results Clonidine and dexmedetomidine interacted synergistically in all lines except the alpha2C AR knockout line, implicating alpha2C ARs in the interaction. In addition, clonidine and dexmedetomidine did not show analgesic cross-tolerance in the outbred strain, suggesting that the two drugs have distinct mechanisms of action. Conclusions The current study introduces a new synergistic agonist pair, clonidine-dexmedetomidine. These two drugs seem to require the alpha2A AR for spinal analgesia when given separately; when delivered as a combination, the resultant synergistic interaction requires the alpha2C AR as well.