Sex differences in 50 kHz call subtypes emitted during tickling-induced playful behaviour in rats.

‘Tickling’ induces positive affective states in laboratory rats as evidenced by the production of 50-kHz ultrasonic vocalisations (USVs), although this has mostly been investigated in males. Juvenile rats emit distinctive 50-kHz USV subtypes. Frequency-modulated (FM) 50-kHz USVs are thought to be associated with positive affect and flat 50-kHz USVs with social communication. FM and flat USVs are produced by both sexes during tickling, but it is unclear whether these calls are produced in relation to particular play-related behaviours, and whether USV subtypes are used in a sexually dimorphic manner during tickling. We tested the hypotheses that FM USVs are associated with tickle-induced play behaviours in a sex-specific way, and that flat USVs are associated with non-play activities. Rats were allocated to one of two treatment groups: tickling (tickled, n = 16/sex) or no hand contact (control, n = 16/sex). Play behaviours (hopping, darting and hand approaches) and FM and flat USVs emitted during the testing session were quantified for each rat, with the frequency of FM and flat USVs made in anticipation of, and during, each behaviour analysed. In females, play behaviours were associated with more flat USVs than in males (before and during; p < 0.001), irrespective of treatment. FM USVs were paired with hopping and darting (before and during; p < 0.001), and in anticipation of hand approaches (p < 0.001) in both tickled females and males compared to controls (both sexes) suggesting that FM USVs are linked with play behaviour. The higher call rate of flat USVs paired with play behaviour in females suggests that there may be sex differences in the role of flat USVs during play. This result is evidence of sex differences in tickle-induced behaviours and has implications for our understanding of the function of different USVs in juvenile female and male rats

An exploratory interview study of researchers’ and technicians’ perceptions of rat tickling

This paper highlights the main themes which emerged from a study carried out with Animal Technicians and researchers to better understand:• perceptions of rat tickling• potential drivers and barriers to the uptake of tickling in a laboratory environmentThe interviewees indicated they had positive attitudes towards rats and the idea of rat tickling with positive comments about rats’ social behaviour, their intelligenceand their capacity to interact with Animal Technicians andresearchers.The participants indicated that barriers to wider uptake of rat tickling including time constraints, a lack of training in the specifics of rat tickling and how to interpret rat responses to tickling.In addition, there was mention of concerns over tickling affecting experimental integrity and the need to maintain professional detachment from rats as experimental animals

Odour conditioning of positive affective states: Rats can learn to associate an odour with being tickled.

Most associative learning tests in rodents use negative stimuli, such as electric shocks. We investigated if young rats can learn to associate the presence of an odour with the experience of being tickled (i.e. using an experimenter’s hand to mimic rough-and-tumble play), shown to elicit 50 kHz ultrasonic vocalisations (USVs), which are indicative of positive affect. Male, pair-housed Wistar rats (N = 24) were all exposed to two neutral odours (A and B) presented in a perforated container on alternate days in a test arena. Following 60s of exposure, the rats were either tickled on days when odour A (n = 8) or odour B (n = 8) was present, or never tickled (n = 8). When tickled, rats produced significantly more 50 kHz USVs compared to the days when not being tickled, and compared to control rats. The level of anticipatory 50 kHz USVs in the 60s prior to tickling did not differ significantly between the tickled and control rats. As a retrieval test following the odour conditioning, rats were exposed successively in the same arena to three odours: an unknown neutral odour, extract of fox faeces, and either odours A or B. Compared to controls, 50 kHz USVs of tickled rats increased when exposed to the odour they had previously experienced when tickled, indicating that these rats had learned to associate the odour with the positive experience of being tickled. In a test with free access for 5 min to both arms of a T-maze, each containing one of the odours, rats tickled with odour A spent more time in the arm with this odour. This work is the first to test in a fully balanced design whether rats can learn to associate an odour with tickling, and indicates that positive odour conditioning has potential to be used as an alternative to negative conditioning tests

Refinement of tickling protocols to improve positive animal welfare in laboratory rats.:Stage 1 Registered Report

Rat tickling is a heterospecific interaction for experimenters to mimic the interactions of rat play, where they produce 50 kHz ultrasonic vocalisations (USV), symptoms of positive affect; tickling can improve laboratory rat welfare. The standard rat tickling protocol involves gently pinning the rat in a supine position. However, individual response to this protocol varies. This suggests there is a risk that some rats may perceive tickling as only a neutral experience, while others as a positive one, depending on how tickling is performed. Based on our research experiences of the standard tickling protocol we have developed a playful handling (PH) protocol, with reduced emphasis on pinning, intended to mimic more closely the dynamic nature of play. We will test whether our PH protocol gives rise to more uniform increases in positive affect across individuals relative to protocols involving pinning. We will compare the response of juvenile male and female Wistar rats as: Control (hand remains still against the side of the test arena), P0 (PH with no pinning), P1 (PH with one pin), P4 (PH with four pins). P1 and P4 consist of a background of PH, with treatments involving administration of an increasing dosage of pinning per PH session. We hypothesise that rats exposed to handling protocols that maximise playful interactions (where pinning number per session decreases) will show an overall increase in total 50 kHz USV as an indicator of positive affect, with less variability. We will explore whether behavioural and physiological changes associated with alterations in PH experience are less variable. We propose that maximising the numbers of rats experiencing tickling as a positive experience will reduce the variation in response variables affected by tickling and increase the repeatability of research where tickling is applied either as a social enrichment or as a treatment

Crying with laughter: adapting the tickling protocol to address individual differences among rats in their response to playful handling

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Estrogen receptor related beta is expressed in human endometrium throughout the normal menstrual cycle

BACKGROUND: Estrogen receptor related beta (ERRβ, ESRRB/NR3B2) is an orphan receptor that shares significant sequence homology with estrogen receptors ERα and ERβ. ERR family members are reported to exhibit constitutive transcriptional activity; however, little is known about the biological function of ERRβ. In an attempt to delineate its role, we examined expression of ERRβ in normal human endometrium, a tissue that undergoes cyclic remodelling under the influence of estrogen and progesterone. METHODS: Well-characterized endometrial tissue (n = 31), including full-thickness biopsies, was obtained from women with regular menstrual cycles. RT–PCR was used to measure mRNA encoding ERRβ, the peroxisome proliferator activated receptor gamma coactivators (PGC)-1α and β and to determine whether ERRβ splice variant mRNAs were expressed. ERRβ was immunolocalized using both single and double antibody immunohistochemistry. RESULTS: Total ERRβ mRNA appeared higher in proliferative phase samples but results did not reach significance. Transcripts corresponding to the long- and short-splice variants of ERRβ as well as PGC1α and β were detected but ERRβΔ10 was absent. ERRβ protein was localized to cell nuclei within multiple endometrial cell types including the glands, stroma, endothelium and immune cells, including uterine natural killer (uNK) cells and macrophages. Fluorescent immunohistochemistry revealed that some cells co-expressed ERRβ and ERα or ERβ, for example, endothelial and uNK cells were ERRβ+/ERβ+. CONCLUSIONS: ERRβ mRNA and protein are expressed in healthy human endometrium. Further studies are warranted to characterize the functional impact of ERRβ on endometrial biology

Expression of oestrogen receptors, ERα, ERβ, and ERβ variants, in endometrial cancers and evidence that prostaglandin F may play a role in regulating expression of ERα

<p>Abstract</p> <p>Background</p> <p>Endometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERα and ERβ) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERβ lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors.</p> <p>Methods</p> <p>We compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n ≥ 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2α on expression of ERs and PR.</p> <p>Results</p> <p>Full length ERβ (ERβ1) and two ERβ variants (ERβ2, ERβ5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERα^neg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERα. Treatment of adenocarcinoma cells with PGF2α reduced expression of ERα but had no impact on ERβ1. Cells incubated with PGF2α were unable to increase expression of PR mRNA when they were incubated with E2.</p> <p>Conclusion</p> <p>We have demonstrated that ERβ5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERβ variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERβ^pos/ERα^neg. We found evidence of a link between COX-2, its product PGF2α, and expression of ERα and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.</p

Fetal brain 11β-hydroxysteroid dehydrogenase type 2 selectively determines programming of adult depressive-like behaviors and cognitive function, but not anxiety behaviors in male mice

Stress or elevated glucocorticoids during sensitive windows of fetal development increase the risk of neuropsychiatric disorders in adult rodents and humans, a phenomenon known as glucocorticoid programming. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyses rapid inactivation of glucocorticoids in the placenta, controls access of maternal glucocorticoids to the fetal compartment, placing it in a key position to modulate glucocorticoid programming of behavior. However, the importance of the high expression of 11β-HSD2 within the midgestational fetal brain is unknown. To examine this, a brain-specific knockout of 11β-HSD2 (HSD2BKO) was generated and compared to wild-type littermates. HSD2BKO have markedly diminished fetal brain 11β-HSD2, but intact fetal body and placental 11β-HSD2 and normal fetal and placental growth. Despite normal fetal plasma corticosterone, HSD2BKO exhibit elevated fetal brain corticosterone levels at midgestation. As adults, HSD2BKO show depressive-like behavior and have cognitive impairments. However, unlike complete feto-placental deficiency, HSD2BKO show no anxiety-like behavioral deficits. The clear mechanistic separation of the programmed components of depression and cognition from anxiety implies distinct mechanisms of pathogenesis, affording potential opportunities for stratified interventions