An appeasing pheromone ameliorates fear responses in the brown rat (Rattus norvegicus).

The brown rat (Rattus norvegicus) is one of the major animals both in the laboratory and in urban centers. Brown rats communicate various types of information using pheromones, the chemicals that mediate intra-species communication in minute amounts. Therefore, analyses of pheromones would further our understanding of the mode of life of rats. We show that a minute amount of 2-methylbutyric acid (2-MB) released from the neck region can ameliorate fear responses both in laboratory rats and in wild brown rats. Based on these findings, we conclude that 2-MB is an appeasing pheromone in the brown rat. A better understanding of rats themselves would allow us to perform more effective ecologically based research on social skills and pest management campaigns with low animal welfare impacts, which might contribute to furthering the advancement of science and improving public health.

Pharmacist-led sedative-hypnotic deprescribing in team-based primary care practice.

BACKGROUND: Sedative-hypnotic (SH) medications are often used to treat chronic insomnia, with potentially serious long-term side effects. The objective of this study is to evaluate an interprofessional SH deprescribing program within a community team-based, primary care practice, with or without cognitive behavioural therapy for insomnia (CBT-I). METHODS: Retrospective chart review for patients referred to the team pharmacist for SH deprescribing from February 2016 to June 2019. RESULTS: A total of 121 patients were referred for SH deprescribing, with 111 (92%) patients who attempted deprescribing (average age 69, range 29-97 years) and 22 patients who also received CBT-I. Overall, 36 patients (32%) achieved complete abstinence, and another 36 patients (32%) reduced their dosage by ≥50%. For the 36 patients who achieved complete abstinence, 26 (72%) patients remained abstinent at 6 months (9 patients resumed using SH and 1 patient was lost to follow-up). The proportion of patients achieving complete abstinence or reduced dosage of ≥50% (successful tapering) was higher with CBT-I than without CBT-I but did not reach statistical significance (77% vs 62%, p = 0.22). There were also no statistically significant differences detected in the success between those who took a benzodiazepine and those who took a Z-drug (67% vs 61%, p = 0.55) or for those who took SH daily and those who took them intermittently (67% vs 44%, p = 0.09). CONCLUSION: Almost two-thirds of patients participating in our pharmacist-led program were able to stop or taper their SH medications by ≥50%. The role of CBT-I in SH deprescribing remains to be further elucidated. Can Pharm J (Ott) 2021;154:xx-xx.

Dissociative Effects of Neuropeptide S Receptor Deficiency and Nasal Neuropeptide S Administration on T-Maze Discrimination and Reversal Learning.

Cognitive flexibility refers to the ability to modify learned behavior in response to changes in the environment. In laboratory rodents, cognitive flexibility can be assessed in reversal learning, i.e., the change of contingencies, for example in T-maze discrimination learning. The present study investigated the role of the neuropeptide S (NPS) system in cognitive flexibility. In the first experiment, mice deficient of NPS receptors (NPSR) were tested in T-maze discrimination and reversal learning. In the second experiment, C57BL/6J mice were tested in the T-maze after nasal administration of NPS. Finally, the effect of nasal NPS on locomotor activity was evaluated. NPSR deficiency positively affected the acquisition of T-maze discrimination but had no effects on reversal learning. Nasal NPS administration facilitated reversal learning and supported an allocentric learning strategy without affecting acquisition of the task or locomotor activity. Taken together, the present data show that the NPS system is able to modulate both acquisition of T-maze discrimination and its reversal learning. However, NPSR deficiency only improved discrimination learning, while nasal NPS administration only improved reversal learning, i.e., cognitive flexibility. These effects, which at first glance appear to be contradictory, could be due to the different roles of the NPS system in the brain regions that are important for learning and cognitive flexibility.

Regulation of CREB Phosphorylation in Nucleus Accumbens after Relief Conditioning.

Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKIIα, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning.

Neuropeptide-S-receptor deficiency affects sex-specific modulation of safety learning by pre-exposure to electric stimuli.

Neuropeptide S (NPS) is a neuropeptide involved in the regulation of fear. Because safety learning is impaired in patients suffering from anxiety-related psychiatric disorders, and polymorphisms of the human neuropeptide S receptor (NPSR) gene have also been associated with anxiety disorders, we wanted to investigate whether NPSR-deficiency interferes with safety learning, and how prior stress would affect this type of learning. We first investigated the effect of pre-exposure to two different types of stressors (electric stimuli or immobilization) on safety learning in female and male C57Bl/6 mice, and found that while stress induced by electric stimuli enhanced safety learning in males, there were no differences in safety learning following immobilization stress. To further investigate the role of the NPS system in stress-induced modulation of safety learning, we exposed NPSR-deficient mice to stress induced by electric stimuli 10 days before safety learning. In nonstressed male mice, NPSR-deficiency enhanced safety learning. As in male C57Bl/6 mice, pre-exposure to electric stimuli increased safety learning in male NPSR +/+ mice. This pre-exposure effect was blocked in NPSR-deficient male mice showing impaired, but still intact, safety learning in comparison to their NPSR +/+ and NPSR +/- littermates. There was neither a pre-exposure nor a genotype effect in female mice. Our findings provide evidence that pre-exposure to stress induced by electric stimuli enhances safety learning in male mice, and that NPSR-deficiency prevents the beneficial effect of stress exposure on safety learning. We propose an inverted U-shape relationship between stress and safety learning.

Deficiency of the immunoproteasome subunit ß5i/LMP7 supports the anxiogenic effects of mild stress and facilitates cued fear memory in mice.

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.

Role of the mesolimbic dopamine system in relief learning.

The relief from an aversive event is rewarding. Since organisms are able to learn which environmental cues can cease an aversive event, relief learning helps to better cope with future aversive events. Literature data suggest that relief learning is affected in various psychopathological conditions, such as anxiety disorders. Here, we investigated the role of the mesolimbic dopamine system in relief learning. Using a relief learning procedure in Sprague Dawley rats, we applied a combination of behavioral experiments with anatomical tracing, c-Fos immunohistochemistry, and local chemogenetic and pharmacological interventions to broadly characterize the role of the mesolimbic dopamine system. The present study shows that a specific part of the mesolimbic dopamine system, the projection from the posterior medial ventral tegmental area (pmVTA) to the nucleus accumbens shell (AcbSh), is activated by aversive electric stimuli. 6-OHDA lesions of the pmVTA blocked relief learning but fear learning and safety learning were not affected. Chemogenetic silencing of the pmVTA-AcbSh projection using the DREADD approach, as well as intra-AcbSh injections of the dopamine D2/3 receptor antagonist raclopride inhibited relief learning. Taken together, the present data demonstrate that the dopaminergic pmVTA-AcbSh projection is critical for relief learning but not for similar learning phenomena. This novel finding may have clinical implications since the processing of signals predicting relief and safety is often impaired in patients suffering from anxiety disorders. Furthermore, it may help to better understand psychological conditions like non-suicidal self-injury, which are associated with pain offset relief.

Nucleolar stress in Drosophila melanogaster: RNAi-mediated depletion of Nopp140.

Nucleolar stress results when ribosome biogenesis is disrupted. An excellent example is the human Treacher Collins syndrome in which the loss of the nucleolar chaperone, Treacle, leads to p53-dependent apoptosis in embryonic neural crest cells and ultimately to craniofacial birth defects. Here, we show that depletion of the related nucleolar phosphoprotein, Nopp140, in Drosophila melanogaster led to nucleolar stress and eventual lethality when multiple tissues were depleted of Nopp140. We used TEM, immuno-blot analysis and metabolic protein labeling to show the loss of ribosomes. Targeted loss of Nopp140 in larval wing discs caused Caspase-dependent apoptosis which eventually led to defects in the adult wings. These defects were not rescued by a p53 gene deletion, as the craniofacial defects were in the murine model of TCS, thus suggesting that apoptosis caused by nucleolar stress in Drosophila is induced by a p53-independent mechanism. Loss of Nopp140 in larval polyploid midgut cells induced premature autophagy as marked by the accumulation of mCherry-ATG8a into autophagic vesicles. We also found elevated phenoloxidase A3 levels in whole larval lysates and within the hemolymph of Nopp140-depleted larvae vs. hemolymph from parental genotype larvae. Phenoloxidase A3 enrichment was coincident with the appearance of melanotic tumors in the Nopp140-depleted larvae. The occurrence of apoptosis, autophagy and phenoloxidase A3 release to the hemolymph upon nucleolar stress correlated well with the demonstrated activation of Jun N-terminal kinase (JNK) in Nopp140-depleted larvae. We propose that JNK is a central stress response effector that is activated by nucleolar stress in Drosophila larvae.

Osteogenesis and expression of the bone marrow niche in endothelial cell-depleted HipOPs.

The identification and purification of murine multipotent mesenchymal stem cells (MSCs) have been difficult due to their low frequency, the presence of contaminating cell types and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOP) population that was also enriched for other mesenchymal precursors, including MSCs [Itoh and Aubin, 2009]. We now report that HipOPs are also highly enriched in vascular endothelial cells (VECs), which we hypothesized were an accessory cell type regulating osteogenesis. However, when VECs were immunodepleted from HipOPs with anti-CD31 antibodies, the resulting CD31(-) HipOP population had equal osteogenic capacity to the HipOPs in vitro and in vivo. Analysis of gene expression of Ncad, Pth1r, Ang1, Cxcl12, Jag1, Pdgfr-ß, α-sma, Desmin, and Ng2 suggested that both HipOPs and CD31(-) HipOPs are hemopoietic stem cell (HSC) niche populations. However, the data support the view that osteoblast differentiation and depletion of VECs modulate the HSC niche.

Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375.

Morphine is a poor inducer of micro-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy-terminal residue 375 (Ser(375)). Ser(375) phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser(375)-phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser(375).