The periaqueductal gray (PAG) acts as a relay station by processing afferent input from the lower urinary tract and is involved in top-down control by facilitating communication between higher (sub)cortical areas and the pontine micturition center (PMC). The hypothalamus plays a complementary role, contributing to both sensory processing of the bladder and descending modulation of motor output. Specifically, the anterior, caudal, and preoptic hypothalamus have direct projections to the PAG and PMC, whereas the paraventricular nucleus projects to all autonomic spinal cord motor neurons . Dysfunction of this network has been implicated in urgency symptoms (1), and is central to improving personalized therapy selection.
This is the first study to examine functional connectivity between the hypothalamus, PAG, and PMC in relation to bladder filling using 7 Tesla resting-state fMRI (rs-fMRI).

Rs-fMRI data of 15 healthy females was acquired during controlled bladder filling on a 7T MRI scanner, while they reported bladder sensations on a 4-point desire-to-void-scale. The regions of interest were the hypothalamus, and PAG and PMC as seed regions. Firstly, functional connectivity between 13 hypothalamic subregions(2), three subdivisions of the PAG (3), and the PMC were evaluated using Fisher Z-transformed correlation coefficients. The average rs-fMRI signal in hypothalamic subregions was correlated with seed regions in empty bladder and strong desire to void states.  Secondly, voxelwise correlation maps between (sub)regions in bladder states, and a difference map of the two states were generated. Hereby, the rs-fMRI signal of every voxel in the hypothalamus was correlated with the seed regions, resulting in a spatial correlation map. Ultimately, spatial connectivity patterns were assessed using Moran’s I applied to the voxelwise correlation group maps for the empty bladder state, strong desire to void state, and the difference map. Moran’s I quantifies the similarity of neighboring voxels, providing a measure of spatial clustering within the hypothalamus. Multiple comparisons were controlled using the false discovery rate (FDR).

The correlation between the spatial mean rs-fMRI signal in the hypothalamic subregions and the seed regions showed no significant difference between bladder states after FDR correction (all p>0.05).  Spatial autocorrelation analyses using Moran’s I showed significant spatial clustering in empty bladder (I=0.27043-0.81265, p<0.05 for 12/13 subregions) and strong desire to void state (I=0.16045-0.70533, p<0.05 for 12/13 subregions) (see Figure 1 and Table 1). Difference maps showed significant spatial autocorrelation (I=0.20198-0.77023, p<0.05 for 12/13 subregions), indicating spatially organized connectivity changes across sensory states. Clustering strength did not differ between states (Delta I, p>0.999 in all regions). Visual inspection revealed positive and negative clusters co-occurring within individual hypothalamic nuclei.

Averaging across entire hypothalamic nuclei masked the within-nucleus heterogeneity in both bladder states. The clustering reflects that functional organization surpasses anatomical borders. Maintained clustering strength across states suggests spatial organization in both states, though this does not specify whether the spatial pattern is preserved or reorganized. Spatial coherence in the difference map provides evidence for spatially structured rather than random connectivity changes in the hypothalamus. These functional microregions likely reflect that bladder filling affects specific circuits and might reveal different functional roles in afferent/efferent processing.

The current findings suggest that bladder-filling-related connectivity is organized by functional microregions in the hypothalamus rather than by more macroscopic subregions. This offers a new framework for examination of small brain regions involved in micturition. The findings create an opportunity to further investigate functional connectivity patterns at high resolution in patients with urgency symptoms, thereby broadening our understanding of a heterogenous patient population with a need for tailored therapeutic interventions.

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