Computational protocol: Oxytocin-receptor-expressing neurons in the parabrachial nucleus regulate fluid intake

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Protocol publication

[…] Mice were prepared for calcium imaging as described. Briefly, 3 weeks after AAV1-DIO-GCaMP6m and AAV1-DIO-hM3Dq:mCherry viral injection, mice were anesthetized (as described above) and implanted with a microendoscope lens (6.1 mm length, 0.5 mm diameter; Inscopix, catalog #100-000588) with assistance of a ProView implant kit (Inscopix, catalog #100-000754) that allowed visualization of the fluorescent activity during the lens implantation. Because basal fluorescence from Oxtr neurons was low, we treated the mice with CNO before lens implantation to facilitate visualization and lens placement. The lens was targeted to be ~200–300 µm above the neurons using the following coordinates: –4.80 mm posterior to bregma, –1.40 mm lateral from midline, and –3.00 mm ventral to skull surface. One week after lens implantation, mice were anesthetized and a baseplate (Inscopix, catalog #100-000279) was implanted above the lens. The baseplate provides an interface for attaching the miniature microscope during calcium imaging experiments, but at other times a baseplate cover (Inscopix, catalog #100-000241) was attached to prevent damage to the microendoscope lens. Calcium fluorescence was recorded at five frames per second, 200-ms exposure time, and 50% LED power using a miniature microscope from Inscopix (nVista). The recording parameters were based on pilot studies that demonstrated the least amount of photobleaching while allowing sufficient detection of fluorescent activity. We used Ethovision (XT 10, Noldus Technology) to trigger and synchronize calcium recordings with behavioral video recordings. [...] Data analysis and generation of histograms were performed using GraphPad Prism Version 6.01 for Windows (GraphPad Software). Results are expressed as mean ± s.e.m. Statistical significance was determined by different tests appropriate for each dataset: for comparing the means of two groups, unpaired two-tailed Student’s t test; for comparing the means of three or more groups, a one-way ANOVA with Tukey’s post hoc tests; for correlation studies, a Pearson product-moment correlation; for comparing food or fluid intake over time, a repeated measures two-way ANOVA with Sidak’s post hoc tests, unless otherwise noted. Three-way mixed design ANOVAs were performed using IBM SPSS Statistics for Windows v.20 (IBM). If Mauchly’s test of sphericity was violated, a Greenhouse-Geisser correction was applied; if significant interaction was determined, a simple two-way interaction was performed with statistical significance accepted at a Bonferroni-adjusted α level of 0.025. We also tested for equality of variance (Brown-Forsythe test for one-way ANOVAs; or Levene’s test of equality of variances). Data distribution was assumed to be normal, but this was not formally tested. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Calcium fluorescence data were analyzed using OriginPro v.2016 (OriginLab).A power analysis was performed for an effective sample size using http://powerandsamplesize.com. Based on a pilot study for fluid deprivation, we used a mean of 1.8 and s.d. of 0.4. Assuming a significance level of 0.05 and power of 0.8, we calculated a sample size of 7 per group if means were 1.5-fold different with a two-tailed Student’s t test. Data and figures were exported into Adobe Illustrator CS6 (Adobe Systems) to prepare figures. Images of sagittal mouse brain were taken from the Motifolio toolkit.Supplementary statistical analyses and methods checklist are provided in Supplementary Table  and the Life Sciences Reporting Summary. […]

Pipeline specifications

Software tools EthoVision, SPSS, Adobe Illustrator
Applications Miscellaneous, Macroscope & basic digital camera imaging
Organisms Mus musculus
Chemicals Oxytocin