Ventrolateral Periaqueductal Gray Neurons Are Active During Urination

Abstract

The ventrolateral periaqueductal gray (VLPAG) is thought to be the main PAG column for bladder control. PAG neurons (especially VLPAG neurons) and neurons in the pontine micturition center (PMC) innervating the bladder detrusor have anatomical and functional synaptic connections. The prevailing viewpoint on neural control of the bladder is that PAG neurons receive information on the decision to void made by upstream brain regions, and consequently activate the PMC through their direct projections to initiate urination reflex. However, the exact location of the PMC-projecting VLPAG neurons, their activity in response to urination, and their whole-brain inputs remain unclear. Here, we identified the distribution of VLPAG neurons that may participate in control of the bladder or project to the PMC through retrograde neural tracing. Population Ca2⁺ signals of PMC-projecting VLPAG neurons highly correlated with bladder contractions and urination as shown by in vivo recording in freely moving animals. Using a RV-based retrograde trans-synaptic tracing strategy, morphological results showed that urination-related PMC-projecting VLPAG neurons received dense inputs from multiple urination-related higher brain areas, such as the medial preoptic area, medial prefrontal cortex, and lateral hypothalamus. Thus, our findings reveal a novel insight into the VLPAG for control of bladder function and provide a potential therapeutic midbrain node for neurogenic bladder dysfunction.

Keywords: cystometry; fiber photometry; pontine micturition center; rabies virus; urination; ventrolateral periaqueductal gray (vlPAG).

Figure 1

Identification of ventrolateral periaqueductal gray (VLPAG) neurons after PRV531 injection into the bladder wall. (A) Schematic for PRV531 injection into the bladder detrusor of adult mouse. (B) Sagittal brain section showing PRV531-infected VLPAG neurons from one mouse (red dotted box, enlarged on the right). (C,D) Serial coronal brain sections showing PRV531-infected neurons in the VLPAG of one mouse. (E). Drawings of brain anatomy overlay showing major PRV531-labeled areas in the VLPAG/LPAG of adult mice (n = 6 mice, each mouse was indicated with different colors). (F) Quantification of cell numbers of VLPAG PRV531-labeled neurons on contralateral/ipsilateral sides (n = 6 mice). Wilcoxon signed-rank test, P > 0.05. (G) Quantification of cell numbers of LPAG PRV531-labeled neurons on contralateral/ipsilateral sides (n = 6 mice). Wilcoxon signed-rank test, P > 0.05. (H) Anatomical scheme of neural pathways innervating the bladder detrusor. SPN, sacral parasympathetic nuclei; MPG, major pelvic ganglion.

Figure 2

Identification of VLPAG neurons that project to PMC^CRH neurons. (A) Schematic of the experimental timeline and injection procedure for helper AAVs and RV into the pontine micturition center (PMC) of corticotropin releasing hormone (CRH)-Cre mice. (B) Strategy for retrograde trans-synaptic tracing of monosynaptic inputs from the VLPAG in PMC^CRH neurons using cell-type-specific RV system. (C) A representative image containing the PMC immunostained with tyrosine-hydroxylase (TH) (left). The starter neurons expressing helper AAVs and RV (yellow dotted box, enlarged on the right) were restricted to the unilateral PMC of one CRH-Cre mouse. (D,E) Serial coronal brain sections showing RV-labeled neurons in the VLPAG of one mouse. (F) Distributions of RV-labeled neurons in the whole ipsilateral VLPAG (Bregma: −4.12 to −5.20 mm, n = 6 mice, each mouse was indicated with different colors). The black line represents the average number of RV-labeled VLPAG neurons in each section. Data are presented as mean ± s.e.m. (G) Quantification of cell numbers of RV-labeled neurons in the ipsilateral LPAG (Bregma: −4.12 to −5.20 mm, n = 6 mice, each mouse was indicated with different colors). The black line represents the average number of RV-labeled LPAG neurons in each section. Data are presented as mean ± s.e.m.

Figure 3

In vivo fiber photometry recording of the activity of PMC-projecting VLPAG neurons during urination in freely moving mice. (A) Schematic of AAVRetro-hSyn-Cre injection into the PMC and AAV-Dio-GCaMP6f injection into the VLPAG. (B) The representative picture shows that PMC-projecting VLPAG neurons are labeled with GCaMP6f (yellow dotted box, enlarged image on the right). (C) Confocal images from the same mouse in (B) of CTB555 (red) and the axon terminals of GCaMP6f-labeled VLPAG neurons (green) in the PMC. The locus coeruleus (LC) is stained with TH (magenta). (D) Schematic of fiber photometry recording experiment during urination in freely moving mouse. (E) An example trace for GCaMP6f fluorescence change of PMC-projecting VLPAG neurons during urination events. Red arrows and dotted bars indicate initiation of three urination events.

Figure 4

The activities of VLPAG neurons projecting to the PMC correlate with urination in freely moving mice. (A) Example individual recording traces from GCaMP6f-labeled (left) and EGFP-labeled (right) PMC-projecting VLPAG neurons. Dashed red lines and red arrows indicate urination onsets. Dashed black lines indicate urination offsets. (B) Top, overlay of all trials in the GCaMP6f-labeled group (199 urination events, n = 8 mice) and the EGFP-labeled control group (174 urination events, n = 7 mice). Bottom, heatmaps of individual recording traces aligned to urination event onsets. The bold black lines indicate urination offsets in the left heatmap. (C) Quantification of the percentage of Ca²⁺ transients correlated with urination in the GCaMP6f-labeled group and EGFP-labeled control group. (D) Quantification of amplitudes of all trials in GCaMP6f-labeled group and EGFP-labeled control group. Wilcoxon rank-sum test, ^***P < 0.001. (E) Averaged Ca²⁺ signal in GCaMP6f-labeled PMC-projecting VLPAG neurons aligned to the urination onset or to the shuffled urination onset.

Figure 5

Population Ca²⁺ transients of VLPAG neurons projecting to the PMC correlate with increased bladder pressure in freely moving mice. (A) Schematic of fiber photometry recording experiment during cytometry in freely moving mouse. (B,C) Representative time-locked GCaMP6f or EGFP fluorescence (black) and bladder pressure (orange) traces from GCaMP6f-labeled (B) and EGFP-labeled (C) PMC-projecting VLPAG neurons. Dashed red lines indicate bladder contraction onsets. (D) Averaged bladder pressure (orange) and time-locked fluorescence (black) trace from GCaMP6f-labeled (left) group (n = 63 events from seven mice) and EGFP-labeled (right) control group (n = 59 events from seven mice). Averaged fluorescence trace aligned to the onset of bladder contractions. (E) A representative example of cross-correlation between Ca²⁺ signals of VLPAG neurons projecting to the PMC (green) and bladder pressure from one mouse compared to the shuffled data (black). (F) Quantification of cross-correlation coefficients. n = 7 mice. Wilcoxon signed-rank test, ^*P < 0.05.

Figure 6

PMC-projecting VLPAG neurons receive monosynaptic inputs from upstream brain areas. (A) Schematic of the experimental timeline and injection procedure. (B) Representative images showing starter neurons expressing helper AAVs and RV (yellow, enlarged in the right) restricted to the unilateral VLPAG. (C) Representative coronal sections of RV-labeled neurons distributed in many ipsilateral upstream brain regions of PMC-projecting VLPAG neurons. (D) Quantification of the total number of RV-labeled neurons in each ipsilateral upstream brain region of PMC-projecting VLPAG neurons.