How do Barrington's nucleus Penk neurons control sex-biased micturition?

Penk marks a distinct excitatory Bar subtype

Barrington's nucleus is a brainstem hub for lower urinary tract function, but its neuronal subtypes were poorly defined at the molecular level. The authors built a single-nucleus transcriptional atlas of the mouse Bar region, profiling 60,135 nuclei and resolving 39 clusters grouped into nine major cell types; sub-clustering of glutamatergic neurons covered 13,076 nuclei and produced 13 clusters, one of which was defined by Penk expression.

Penk neurons are not simply a relabeled version of the better-studied Crh population. RNAscope and immunohistochemistry showed that 95% of Bar-Penk neurons expressed Vglut2 mRNA, confirming their excitatory nature, while only 5% co-expressed Crh mRNA. Penk+ neurons made up about one-fifth of the total Bar population, Crh+ neurons about 40%, and cells negative for both about 38%; only 1% of the Bar pool expressed both Penk and Crh. Just over half of Bar-Penk neurons were Esr1-positive (57%, four males and three females, with no significant sex difference), which matters because Esr1 is broadly expressed in Bar rather than marking a single subtype.

Penk and Crh axons divide the spinal cord differently

Both populations project to the lumbosacral spinal cord, but their innervation patterns diverge. At L6, where the intermediolateral cell column (IML) begins to emerge, the distribution of synapses between the dorsal gray commissure (DGC) and IML was similar for the two populations. At S1 and S2 the patterns differed significantly: Bar-Penk boutons were enriched in the DGC, while Bar-Crh boutons were more enriched in the IML. Sparse dorsal horn labeling also differed, with Crh boutons preferentially in superficial laminae and Penk boutons spread more broadly.

This anatomical split is the first reason to expect different jobs. The IML contains parasympathetic preganglionic neurons that drive detrusor contraction, while the DGC region is associated with the sphincter-related output the paper examines. The authors use this comparison to argue that Penk neurons are not a redundant duplicate of Crh neurons but a separate channel with its own spinal weighting.

Bar-Penk activity tracks voiding, not pressure rise

To test when these neurons fire in behaving animals, the authors combined fiber photometry with cystometry in awake, freely moving mice. Bar-Penk neurons consistently activated around void onset and stayed active for the duration of the void. Crucially, the increase in neural activity did not precede the rise in intravesical pressure. Small transient GCaMP changes accompanied pressure changes during non-voiding contractions, but these were attenuated relative to voids.

That timing is the paper's central mechanistic clue. If Penk neurons were driving detrusor contraction, their activity should lead the pressure rise; instead it coincides with the void, when the external urethral sphincter relaxes. The authors therefore interpret Bar-Penk activity as coordinating sphincter relaxation rather than initiating bladder contraction. This is an interpretation built on correlation plus the optogenetic result below, not a direct demonstration of causation from the photometry alone.

Optogenetic activation suppresses sphincter activity in time

Optogenetic activation of Bar-Penk neurons elicited time-locked suppression of external urethral sphincter (EUS) activity, measured by EMG electrodes implanted in the sphincter. This is the most direct functional evidence in the paper: stimulating the defined population produces an immediate, temporally locked drop in the sphincter signal that the authors use to link Penk neurons to outlet control.

The result also sharpens the division of labor within Bar. The authors note that Bar-Crh activity closely tracks bladder pressure during voiding contractions and likely augments detrusor contraction, whereas Bar-Penk activity aligns with sphincter relaxation. Read together, the two subtypes appear to handle the two sides of voiding — bladder contraction and outlet opening — rather than duplicating one function.

Chemogenetic activation splits by sex

The sex-specific claim comes from chemogenetic activation with hM3Dq, not from the optogenetic experiments. In males (n = 6), activation significantly changed urine volume per void (p = 0.027) and the number of leaks (p = 0.017), while voiding frequency did not change significantly (p = 0.33). In females (n = 7), none of the three measures changed significantly (frequency p = 0.17, volume per void p = 0.67, leaks p > 0.99). Control animals lacking viral expression showed no significant off-target effects (four males, all p > 0.99; six females, p = 0.97, p > 0.99, p = 0.44).

The behavioral readout was micturition video thermography, which counts void spots and leaks on filter paper. Activation produced an aberrant pattern — more void spots and smaller voids scattered through the cage rather than a typical void in the corner — and the quantified male-specific changes were in volume per void and leaks, not in frequency. When the authors restricted activation to spinally projecting Bar-Penk neurons (n = 7), voiding frequency again did not change significantly (p = 0.51), but volume per void (p = 0.035) and leaks (p = 0.0012) did. The sex difference is therefore a difference in which voiding parameters shift under activation, and it should not be read as males voiding more often.

Ablation removes cue-driven marking behavior

Targeted ablation of Bar-Penk neurons impaired voluntary marking behavior in response to female cues. This is a different assay from the cage-floor void counts: it tests a socially motivated urinary behavior rather than reflexive emptying, and it is the evidence behind the paper's claim that these neurons are needed for voluntary, context-dependent micturition.

The distinction matters for interpreting the whole study. Reflexive voiding and scent-marking are not the same output, and the paper treats Bar-Penk neurons as contributing to both. The authors frame this as support for Bar-Penk neurons integrating internal state and environmental context, a role reinforced by the input mapping described next.

Visceromotor and behavioral regions feed Bar-Penk

Retrograde rabies tracing from Bar-Penk neurons identified inputs from the subcoeruleus nuclei, the ventrolateral, lateral, and dorsomedial periaqueductal gray, the lateral hypothalamic area, the bed nucleus of the stria terminalis (concentrated in the laterodorsal division), and the central amygdala. The authors also traced inputs to the spinally projecting subset of Penk neurons.

These are regions associated with visceromotor regulation and behavioral state control, which is why the authors describe Bar-Penk neurons as integrators of internal state and environmental context. The periaqueductal gray is highlighted as a likely gate: the paper's model is that upstream inhibition is transiently lifted at void onset so that sphincter relaxation can be synchronized with bladder contraction. That model is the authors' interpretation of the tracing and timing data, not a directly manipulated circuit in this study.

Where the sex-specific claim stops

The strongest, most specific finding is that Bar-Penk neurons are a molecularly distinct excitatory subtype, active at void onset, whose optogenetic activation suppresses EUS activity in a time-locked way. The sex-specific behavioral phenotype is narrower: it appears under chemogenetic activation, differs in which parameters move in males versus females, and was not explained by a hormonal manipulation in this study. The authors themselves call for functional studies under varying physiological and hormonal conditions to capture more nuanced male-female differences.

Two further boundaries belong with the methods that produced them. The atlas used DroNc-seq, which the authors note has relatively low sensitivity and may have missed low-abundance transcripts, rare populations, or sex-specific expression differences; the tracing focused primarily on spinal projections, leaving local brainstem circuits and brain-wide afferents to each subtype for future work. The practical priority for readers is therefore not that Penk neurons control micturition in general, but that they form a sphincter-oriented channel separable from Crh neurons — and that any sex difference reported here is tied to a specific activation assay and a specific set of voiding measures.

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