Barrington's nucleus: Neuroanatomic landscape of the mouse "pontine micturition center"

Abstract

Barrington's nucleus (Bar) is thought to contain neurons that trigger voiding and thereby function as the "pontine micturition center." Lacking detailed information on this region in mice, we examined gene and protein markers to characterize Bar and the neurons surrounding it. Like rats and cats, mice have an ovoid core of medium-sized Bar neurons located medial to the locus coeruleus (LC). Bar neurons express a GFP reporter for Vglut2, develop from a Math1/Atoh1 lineage, and exhibit immunoreactivity for NeuN. Many neurons in and around this core cluster express a reporter for corticotrophin-releasing hormone (Bar^CRH ). Axons from Bar^CRH neurons project to the lumbosacral spinal cord and ramify extensively in two regions: the dorsal gray commissural and intermediolateral nuclei. Bar^CRH neurons have unexpectedly long dendrites, which may receive synaptic input from the cerebral cortex and other brain regions beyond the core afferents identified previously. Finally, at least five populations of neurons surround Bar: rostral-dorsomedial cholinergic neurons in the laterodorsal tegmental nucleus; lateral noradrenergic neurons in the LC; medial GABAergic neurons in the pontine central gray; ventromedial, small GABAergic neurons that express FoxP2; and dorsolateral glutamatergic neurons that express FoxP2 in the pLC and form a wedge dividing Bar from the dorsal LC. We discuss the implications of this new information for interpreting existing data and future experiments targeting Bar^CRH neurons and their synaptic afferents to study micturition and other pelvic functions.

Keywords: FoxP2; bladder; continence; dorsal gray commissure nucleus; locus coeruleus; math1/atoh1; micturition; neuroanatomy; neurourology; pelvic autonomic; pontine central gray; pontospinal; pre-locus coeruleus; sacral intermediolateral nucleus; urethral sphincter; voiding.

FIGURE 1

Nissl cytoarchitecture in the mouse brainstem region that contains Barrington’s nucleus (Bar). Panels (a–d): successive rostral-to-caudal sections through the pons of a Crh reporter mouse. Every third section (35 μm-thick) is shown across a total span of ~0.5 mm. Bar neurons, using Nissl cytoarchitecture alone, are evident as an ovoid cluster of medium-large neurons located medial to the ventral locus coeruleus (dashed line in panel c). Rostral and caudal to this core, we cannot distinguish Bar neurons from surrounding populations in a Nissl preparation. Panels (a1–d1) show a GFP reporter for Crh in the same tissue sections as (a–d), at the same magnification, in fluorescence images taken prior to Nissl counterstaining. The location of Bar^CRH neurons in each section is indicated by a green dashed line in panels (a–d). Panels (a2–d2) show the distribution of Bar^CRH neurons throughout each section by blending images of GFP and thionin counterstaining from the same sections. Scale bars are all 500 μm (d–d2), and apply to the column above. Approximate level in mm caudal to bregma is shown in the lower left corner of panels (a2–d2) and applies to all panels in each row. 4V = fourth ventricle; Bar-CRH = corticotropin releasing hormone neurons in Barrington’s nucleus; LC = locus coeruleus; Me5 = mesencephalic nucleus and tract of the trigeminal nerve; Mo5 = motor nucleus of the trigeminal nerve. PCG = pontine central gray matter; RtTg = reticular tegmental nucleus; scp, superior cerebellar peduncle

FIGURE 2

The crescent-shaped locus coeruleus (LC) cradles the lateral surface of mouse Bar^CRH neurons. Panels (a–f) show the close relationship between LC (immunofluorescence labeling for tyrosine hydroxylase; TH, red) and Bar^CRH (GFP reporter for Crh, green). Bar^CRH neurons are found at middle-to-rostral levels of the LC (panels B–E, corresponding to the same approximate bregma levels as Figure 1, panels a–d). No neurons are double-labeled. Rostral to the LC, there are no more Bar^CRH neurons, though sparse, large neurons immunoreactive for TH extend rostrally into the periaqueductal gray. At caudal levels (panels e, f), there are few or no Bar^CRH neurons. At these caudal levels, LC dendrites project medially into a zone immediately behind the core of Bar. At central levels containing the core of Bar (panel d), its dorsolateral surface diverges from the dorsal LC; the resulting gap between Bar^CRH and the dorsal LC (dashed outline in panel d) is filled by a wedge of FoxP2/glutamatergic neurons, which are shown in Figure 6. Lines in panels (d–f) mark the ependymal surface of the fourth ventricle. Scale bar is 100 μm and applies to all panels

FIGURE 3

Bar neurons have prominent NeuN nuclear immunoreactivity. (a) NeuN immunofluorescence (green) is prominent in many neurons in the pontine tegmentum, including the PB and pontine central gray (PCG) medial to Bar. However, NeuN is not ubiquitous, and most LC neurons lack it. It is also largely absent in the region bordering Bar on its ventromedial surface (arrowhead). (b) TH-immunofluorescence (red) is shown for reference, showing LC neurons and their dendrites projecting medially above and below Bar. (c) Shows NeuN and TH in combination with DAPI nuclear staining for all cells. Scale bar is 200 μm

FIGURE 4

Bar contains predominantly glutamatergic neurons, as shown by the presence of a GFP reporter for the type 2 vesicular glutamate transporter (Vglut2), and absence of a reporter for the vesicular GABA/glycine transporter (Vgat). Panels (a) and (b) compare the distributions of Vglut2 (glutamatergic) and Vgat (GABAergic/glycinergic) neurons, respectively, in the pontine tegmentum at levels containing the core of Bar. TH immunofluorescence (magenta) is labeled as a landmark. The core of Bar contains a prominent cluster of glutamatergic neurons, and is virtually devoid of GABAergic neurons. Conversely, a region of the pontine central gray (PCG) just medial and ventral to Bar contains many Vgat/GABAergic neurons and relatively few Vglut2/glutamatergic neurons. Scale bar in (b) is 200 μm and applies to both panels. Additional abbreviations: Cb = cerebellum; DR = dorsal raphe nucleus; DTN = dorsal tegmental nucleus; Me5 = mesencephalic trigeminal nucleus; Su5 = supratrigeminal nucleus

FIGURE 5

Glutamatergic neurons in Bar are surrounded by mutually exclusive populations of cholinergic and catecholaminergic neurons, as well as other glutamatergic populations. Panels (a–f) show, in successive rostral to caudal levels, the relative positions of neurons in each population. Laterodorsal tegmental nucleus (LDT) neurons, labeled by immunofluorescence for choline acetyltransferase (ChAT, magenta), are located rostral and medial-dorsal to Bar. Vglut2 GFP reporter is found in many LC catecholaminergic (red +green = yellow) and LDT cholinergic neurons (magenta +green = white), as well the large Me5 neurons dorsolateral to the LC, and most neurons in the PB. The dashes in panels (c–e) outline the approximate location of Bar, estimated from the distribution of Bar^CRH neurons shown above. Scale bars are all 200 μm (panel c applies to a–c; panel f applies to d–f). Approximate levels in mm caudal to bregma are given at the bottom left or right of each panel

FIGURE 6

Bar is surrounded by two populations of neurons that express FoxP2. (a–c) At its dorsolateral surface, Bar borders a cluster of small neurons with FoxP2-immunoreactive nuclei (red) and GFP reporter for Vglut2 (but not for Vgat, see panel i). This population of FoxP2/Vglut2 neurons is referred to as the pre-LC (pLC), and forms a wedge between Bar and dorsal LC, corresponding to the dashed outline in Figure 2d; pLC neurons also distribute laterally through the LC and Me5 and into the medial PB. Panels (d–f) the double-labeling for FoxP2 and GFP (reporter for Vglut2) in a subset of these neurons. (g–i) Across from the pLC, Bar borders a separate cluster of FoxP2-expressing neurons. These neurons fill a region lacking NeuN (highlighted in Figure 3) and are a subset of the larger population of GABAer-gic neurons in the pontine central gray (PCG). They express GFP reporter for Vgat (but not for Vglut2, see panel c). Panels (j–l) show double-labeling for FoxP2 and GFP (reporter for Vgat) in a subset of these neurons. Scale bars are 200 μm in panels (c and i) and 50 μm in panels (f and l)

FIGURE 7

In this Cre-reporter mouse (Atoh1-Cre; R26-lsl-YFP), YFP expression (ice-blue) reveals cells derived from embryonic Math1/Atoh1-expressing precursors in the rhombic lip neurothelium in a P0 mouse. Among the Math1/Atoh1-derived neurons in the pontine tegmentum are a dense cluster with the expected location, size, and shape of Bar. These images also contain FoxP2 immunolabeling (red) in cells surrounding Bar. LC neurons, which are derived from separate precursors, do not express YFP. Cerebellar cells (granule cell precursors) and other neurons express YFP, including FoxP2 (pLC) neurons bordering the dorsolateral surface of Bar and winding through the LC. In contrast, FoxP2 neurons in the PCG, including those bordering the ventromedial surface of Bar (FoxP2/Vgat), do not express YFP. Other populations highlighted in this image include a group of non-Math1/Atoh1-derived, FoxP2+ neurons in the caudal PB/Kolliker-Fuse nucleus (PB/KF), which are GABAergic and are separate from the larger population of Math1-derived (glutamatergic) FoxP2 expressing neurons through much of the PB found at levels rostral to this. Scale bars in panels (b) and (c) are 200 μm

FIGURE 8

The dendritic arbors of Bar^CRH neurons are extensive, and may receive synaptic inputs up to 500 μm outside the cytoarchitectural borders of Bar. Panels (a–h) show successive rostral-to-caudal levels of the caudal midbrain (a–c) and pontine tegmentum (d–h) of mice expressing a membrane-targeted fluorescent protein in Bar^CRH neurons (Cre-dependent hM3Dq-mCherry in a Crh-IRES-Cre mouse). Cell bodies expressing mCherry (white dots) were concentrated in or near Bar and were few in number, yet extend thick, brightly labeled dendritic processes primarily in three directions: (1) Rostral- medial, extending as far as to 500 μm along the ventral aspect of cholinergic neurons in the LDT and pedunculopontine tegmental nucleus (PPN); this dendritic bundle is indicated by white arrows in panels (b–f). (2) Dorsal, extending up to and fanning out along the fourth ventricle (4V) ependyma (highlighted by arrowheads in panels d–h). And (3) caudal, producing an extensive dendritic arbor immediately behind Bar^CRH cell bodies and overlapping some dendrites that extend medially from neurons in the caudal LC. Smaller numbers of Bar^CRH dendrites project ventrally or laterally around and through the LC (f–g). The scale bar (panel h) is 200 μm and applies to all panels. TH was labeled in this case with a mouse monoclonal antibody that also cross-reacts with an antigen in the pia and ependyma, producing green immunofluorescence lining the 4V (d–h) and brainstem surface dorsolateral to the PB (d–g). Approximate level in mm caudal to bregma is given in the lower left corner of each panel

FIGURE 9

ChR2-mCherry expressed selectively in Bar^CRH neurons after injecting AAV into a Crh-IRES-Cre; Chat-GFP mouse, which also expresses GFP in cholinergic neurons. (a) In addition to cell bodies (marked with dots) and dendrites, ChR2-mCherry robustly labels axons projecting to the spinal cord (b, c). Upon reaching low lumbar and upper sacral levels, Bar^CRH axons ramify extensively in a horizontal stripe that envelops two major targets: (1) cholinergic neurons (green) in the intermediolateral nucleus (IML) and (2) a large region of the central gray matter known as the dorsal gray commissure (DGC). All scale bars are 200 μm. Spinal cord sections in (b) were scaled proportionally and assigned approximate lumbar (L), sacral (S), and coccygeal (Cx) levels

FIGURE 10

Injecting CTb into the lumbosacral spinal cord retrogradely labels neurons in Bar and LC. (a) In this CRH reporter mouse (Crh-IRES-Cre; L10-GFP), a slight majority of retrogradely labeled neurons in Bar express GFP. These CTb-labeled, Bar^CRH neurons appear yellow-orange due to labeling for both CTb (red) and GFP (green). Outside Bar, CTb-labeled neurons were located in the ventral LC and subcoeruleus, and all of them express TH. CTb-labeled LC neurons appear pink due to labeling for both CTb (red) and TH (magenta). The diagram in panel (b) shows the CTb injection located in the lumbosacral spinal cord. Panel (c) shows a level of the sacral spinal cord in this mouse at the center of the injection site resulting in the ipsilateral, retrograde labeling shown in (a); the contralateral sacral gray matter contains several CTb retrogradely labeled neurons. Scale bars are 200 μm

FIGURE 11

Summary drawings of the mouse pontine tegmentum show (a) the relative distributions of neurons in Bar and five surrounding populations and (b) the unexpectedly large territory covered by dendritic processes emerging from Bar^CRH neurons (as in Figure 8). These long dendrites may receive a broader range of synaptic (input) connections than those identified in previous tracing studies targeting the somatic core. The neuron populations abbreviated in the legend (a) are detailed in the text and figures above