Figure 5. Selective ablation of glutamatergic or glutamatergic-Crh+ Bar neurons causes retention or delays voiding contractions.

A) Schematic of Cre-dependent diphtheria toxin subunit A (DTA) construct and viral targeting bilaterally to Bar. mCherry is located upstream of the DIO-DTA sequence, thus labeling non-Cre expressing transduced neurons.

Lower panel: Upper image, right side: DIO-DTA expression in transduced Bar^Vglut2 neurons and uninjected Bar^Vglut2 (GFP+) neurons in the control side (up left), TH-i.r. labeled neurons shown in magenta. Unilateral selective neuronal ablation of GFP+ Bar^Crh/Vglut2 neurons (lower image, right side) and remaining Bar^Crh/Vglut2 (GFP+) neurons in the uninjected side (down left). See also Figure S5A–C.

B) Representative thermal camera screenshots of the voiding pattern after 2 hours awake thermography recording, from a Vglut2-IRES-Cre mouse injected with DIO-DTA (upper panel), and a Crh-IRES-Cre mouse injected with DIO-DTA (lower panel) at POD 15. Next to the screenshots are images with thresholding applied to the void spot(s) for void size calculation.

C) Frequency of voiding (upper graph) and volume per void (lower graph) during a 2-hour video thermography awake behavior recording on POD 5, 10, 15 or 20. Data is represented as population mean ± s.e.m. for C57Bl6/J (Bar^Control, n = 9 mice), Crh-IRES-Cre (Bar^Crh/Vglut2, n = 7 mice) and Vglut2-IRES-Cre (Bar^Vglut2, n = 4 mice) injected bilaterally with DIO-DTA. RM one-way ANOVA; **, p = 0.0013 for frequency, with Dunnett’s multiple comparisons test; ns control vs Bar^Crh/Vglut2, **, p = 0.0017 control vs. Bar^Vglut2. RM one-way ANOVA; **, p = 0.0018 for voided volume, with Dunnett’s multiple comparisons test; *, p = 0.0169 and *, p = 0.0139 for control vs Bar^Crh/Vglut2 and Bar^Vglut2 respectively. See also Figure S5E,F. Some Vglut2-IRES-Cre mice became moribund before POD 20.

D) An example of a CMG bladder pressure trace in an anesthetized Vglut2-IRES-Cre (upper panel) and a representative CMG bladder trace in an anesthetized Crh-IRES-Cre mouse (lower panel), injected with DIO-DTA in Bar. See also Figure S5G.

Scale bar is 200 μm. Droplet icon indicates urine drops or voiding contraction leading to a void. Abbreviations: 4V, 4^th ventricle; Bar, Barrington’s Nucleus; CMG, cystometrogram; DTA, diphtheria toxin A; i.r., immunoreactive; MVT, Micturition Video Thermography; LC, locus coeruleus; ns, not significant; PMC, pontine micturition center; POD, post-operative day; TH, tyrosine hydroxylase. See also Figure S5.