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The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains. [ABSTRACT FROM AUTHOR]

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools. A Cre recombinase dependent on red fluorescent protein (RFP) is generated, which expands the repertory of nanobody-based genetic tools by enabling selective targeting of RFP-dependent gene expression in the mouse and rat brain. [ABSTRACT FROM AUTHOR]

Oxytocin is involved in pain transmission, although the detailed mechanism is not fully understood. Here, we generate a transgenic rat line that expresses human muscarinic acetylcholine receptors (hM3Dq) and mCherry in oxytocin neurons. We report that clozapine-N-oxide (CNO) treatment of our oxytocin-hM3Dq-mCherry rats exclusively activates oxytocin neurons within the supraoptic and paraventricular nuclei, leading to activation of neurons in the locus coeruleus (LC) and dorsal raphe nucleus (DR), and differential gene expression in GABA-ergic neurons in the L5 spinal dorsal horn. Hyperalgesia, which is robustly exacerbated in experimental pain models, is significantly attenuated after CNO injection. The analgesic effects of CNO are ablated by co-treatment with oxytocin receptor antagonist. Endogenous oxytocin also exerts anti-inflammatory effects via activation of the hypothalamus-pituitary-adrenal axis. Moreover, inhibition of mast cell degranulation is found to be involved in the response. Taken together, our results suggest that oxytocin may exert anti-nociceptive and anti-inflammatory effects via both neuronal and humoral pathways. The analgesic effect of oxytocin appears to be mediated via an oxytocin receptor and endogenous oxytocin contributes to the anti-nociception and anti-inflammatory response. [ABSTRACT FROM AUTHOR]

Arginine vasopressin (AVP) is a hypothalamic neurosecretory hormone well known as an antidiuretic, and recently reported to be involved in pain modulation. The expression kinetics of AVP and its potential involvement in the descending pain modulation system (DPMS) in neuropathic pain (NP) remains unclear. We investigated AVP expression and its effects on mechanical and thermal nociceptive thresholds using a unilateral spinal nerve ligation (SNL) model. All rats with SNL developed NP. Intensities of enhanced green fluorescent protein (eGFP) in the supraoptic and paraventricular nuclei, median eminence, and posterior pituitary were significantly increased at 7 and 14 days post-SNL in AVP-eGFP rats. In situ hybridisation histochemistry revealed significantly increased AVP mRNA expression at 14 days post-SNL compared with the sham control group. The chemogenetic activation of AVP neurones significantly attenuated mechanical and thermal hyperalgesia with elevated plasma AVP concentration. These analgesic effects were suppressed by pre-administration with V1a receptor antagonist. AVP neurones increased the neuronal activity of serotonergic dorsal raphe, noradrenergic locus coeruleus, and inhibitory interneurones in the spinal dorsal horn. These results suggest that the hypothalamo-neurohypophysial system of AVP is upregulated in NP and activated endogenous AVP exerts analgesic effects via the V1a receptors. AVP neurones may activate the DPMS. [ABSTRACT FROM AUTHOR]

Gentle touch contributes to affiliative interactions. We investigated the effects of gentle stroking in female rats on the development of affiliative behaviors toward humans and we exploratively examined brain regions in which activity was influenced by stroking. Rats that had received stroking stimuli repeatedly after weaning emitted 50-kHz calls, an index of positive emotion, and showed affiliative behaviors toward the experimenter. Hypothalamic paraventricular oxytocin neurons were activated in the rats after stroking. The septohypothalamic nucleus (SHy) in the post-weaningly stroked rats showed decreased activity in response to stroking stimuli compared with that in the non-stroked control group. There were negative correlations of neural activity in hypothalamic regions including the SHy with the number of 50-kHz calls. These findings revealed that post-weaning stroking induces an affiliative relationship between female rats and humans, possibly via activation of oxytocin neurons and suppression of the activity of hypothalamic neurons. [ABSTRACT FROM AUTHOR]

Multiple sequential actions, performed during parental behaviors, are essential elements of reproduction in mammalian species. We showed that neurons expressing melanin concentrating hormone (MCH) in the lateral hypothalamic area (LHA) are more active in rodents of both sexes when exhibiting parental nursing behavior. Genetic ablation of the LHA-MCH neurons impaired maternal nursing. The post-birth survival rate was lower in pups born to female mice with congenitally ablated MCH neurons under control of tet-off system, exhibiting reduced crouching behavior. Virgin female and male mice with ablated MCH neurons were less interested in pups and maternal care. Chemogenetic and optogenetic stimulation of LHA-MCH neurons induced parental nursing in virgin female and male mice. LHA-MCH GABAergic neurons project fibres to the paraventricular hypothalamic nucleus (PVN) neurons. Optogenetic stimulation of PVN induces nursing crouching behavior along with increasing plasma oxytocin levels. The hypothalamic MCH neural relays play important functional roles in parental nursing behavior in female and male mice. [ABSTRACT FROM AUTHOR]

Gentle tactile stimuli have been shown to play an important role in the establishment and maintenance of affiliative social interactions. Oxytocin has also been shown to have similar actions. We investigated the effects of gentle stroking on affiliative relationships between humans and rats and the effects of gentle stroking on activation of oxytocin neurons. Male rats received 5-min stroking stimuli from an experimenter every other day for 4 weeks between 3 and 6 weeks of age (S3–6 group), for 4 weeks between 7 and 10 weeks of age (S7–10 group), or for 8 weeks between 3 and 10 weeks of age (S3–10 group). Control rats did not receive stroking stimuli. Rats in the S7–10 and S3–10 groups emitted 50-kHz calls, an index of positive emotion, more frequently during stroking stimuli. Rats in the S3–6, S7–10, and S3–10 groups showed affiliative behaviors toward the experimenter. Oxytocin neurons in the hypothalamic paraventricular nucleus of rats in the S3–6, S7–10, and S3–10 groups were activated following stroking stimuli. These findings revealed that post-weaning repeated stroking stimuli induce an affiliative relationship between rats and humans and activation of oxytocin neurons. [ABSTRACT FROM AUTHOR]

Circulating levels of fibroblast growth factor-21 (FGF21) start increasing in patients with chronic kidney disease (CKD) since early stages during the cause of disease progression. FGF21 is a liver-derived hormone that induces responses to stress through acting on hypothalamus to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal endocrine axis. However, roles that FGF21 plays in pathophysiology of CKD remains elusive. Here we show in mice that FGF21 is required to survive CKD but responsible for blood pressure dysregulation. When introduced with CKD, Fgf21−/− mice died earlier than wild-type mice. Paradoxically, these Fgf21−/− CKD mice escaped several complications observed in wild-type mice, including augmentation of blood pressure elevating response and activation of the sympathetic nervous system during physical activity and increase in serum noradrenalin and corticosterone levels. Supplementation of FGF21 by administration of an FGF21-expressing adeno-associated virus vector recapitulated these complications in wild-type mice and restored the survival period in Fgf21−/− CKD mice. In CKD patients, high serum FGF21 levels are independently associated with decreased baroreceptor sensitivity. Thus, increased FGF21 in CKD can be viewed as a survival response at the sacrifice of blood pressure homeostasis. [ABSTRACT FROM AUTHOR]

Many peptides, when released as chemical messengers within the brain, have powerful influences on complex behaviours. Most strikingly, vasopressin and oxytocin, once thought of as circulating hormones whose actions were confined to peripheral organs, are now known to be released in the brain, where they have fundamentally important roles in social behaviours. In humans, disruptions of these peptide systems have been linked to several neurobehavioural disorders, including Prader–Willi syndrome, affective disorders and obsessive–compulsive disorder, and polymorphisms of V1a vasopressin receptor have been linked to autism. Here we report that the rat olfactory bulb contains a large population of interneurons which express vasopressin, that blocking the actions of vasopressin in the olfactory bulb impairs the social recognition abilities of rats and that vasopressin agonists and antagonists can modulate the processing of information by olfactory bulb neurons. The findings indicate that social information is processed in part by a vasopressin system intrinsic to the olfactory system. [ABSTRACT FROM AUTHOR]

Diet affects health through ingested calories and macronutrients, and macronutrient balance affects health span. The mechanisms regulating macronutrient-based diet choices are poorly understood. Previous studies had shown that NAD-dependent deacetylase sirtuin-1 (SIRT1) in part influences the health-promoting effects of caloric restriction by boosting fat use in peripheral tissues. Here, we show that neuronal SIRT1 shifts diet choice from sucrose to fat in mice, matching the peripheral metabolic shift. SIRT1-mediated suppression of simple sugar preference requires oxytocin signalling, and SIRT1 in oxytocin neurons drives this effect. The hepatokine FGF21 acts as an endocrine signal to oxytocin neurons, promoting neuronal activation and Oxt transcription and suppressing the simple sugar preference. SIRT1 promotes FGF21 signalling in oxytocin neurons and stimulates Oxt transcription through NRF2. Thus, neuronal SIRT1 contributes to the homeostatic regulation of macronutrient-based diet selection in mice. SIRT1 is a NAD+-dependent deacetylase whose functions have been linked to organismal longevity, aging and metabolism. Here, Matsui and colleagues show that neuronal SIRT1 can affect nutrient-related dietary choice in mice, and this effect is mediated by FGF21 signalling and oxytocin. [ABSTRACT FROM AUTHOR]