Corticotropin-releasing factor (CRF) expression in postnatal and adult rat sacral parasympathetic nucleus (SPN)

Abstract

The neural control of micturition undergoes marked changes during the early postnatal development. During the first few postnatal weeks, the spinal micturition reflex is gradually replaced by a spinobulbospinal reflex pathway that is responsible for micturition in adult animals. Upregulation of brainstem regulation of spinal micturition pathways may contribute to development of mature voiding patterns. We examined the expression of corticotropin-releasing factor (CRF), present in descending projections from Barrington's nucleus to the sacral parasympathetic nucleus (SPN), in postnatal (P0-P36) and adult Wistar rats (P60-90). CRF-immunoreactivity (IR) was present predominantly in the SPN region, although some staining was also observed in the dorsal horn and dorsal commissure in L5-S1 spinal segments. CRF-IR in spinal cord regions was age dependent (R2=0.87-0.98). The majority of the CRF-IR in the lumbosacral spinal cord was eliminated by complete spinalization (2-3 weeks). Double-label immunohistochemistry was combined with quantitative confocal laser scanning microscopy to quantify the number and percentage of colocalization between CRF-immunoreactive varicosities and preganglionic somas or proximal neurites in the SPN in postnatal and adult rats. Results demonstrate an age-dependent upregulation of CRF-IR in the SPN region and specifically in association with preganglionic parasympathetic neurons identified with neuronal nitric oxide synthase (nNOS)-IR. CRF-immunoreactive varicosities on or within a 1 microm perimeter of nNOS-immunoreactive somas or proximal neurites also increased with postnatal age. The upregulation of CRF-IR in bulbospinal projections to the SPN may contribute to mature voiding reflexes.

Figure 1

Fluorescence photographs of corticotropin releasing factor (CRF)-immunoreactivity (IR) in lumbosacral (L6-S1) spinal cord of postnatal (P) (A-F) and adult (G) rats. Expression of CRF-IR at birth (P0)(A) and at P3(B) was not observed in the spinal cord. Dotted line in A, B outlines the edge of the grey matter that is difficult to discern in young, postnatal ages. CRF-IR was present at P3(C) primarily in the region of the sacral parasympathetic nucleus (SPN) although faint CRF-IR was present in the superficial dorsal horn (DH). With increasing postnatal age (P14, P21, P36, adult), increased CRF-IR was observed in the SPN region (E-G, I-K) and in the superficial DH, dorsal commissure (DCM; D,G) and in the lateral collateral pathway of Lissauer (LCP)(J, K; arrows). Higher power photographs of CRF-IR (E-G; regions in white box) in the SPN at P21 (I), P36 (J) and in the adult rat (K) are shown. CRF-IR was observed to make pericellular baskets around cells in the SPN region. Complete spinalization at T8-T10, eliminated the majority of CRF-IR in the SPN region (H, L), some faint CRF-IR fibers were occasionally observed projecting from the SPN to the region of the DCM (L). Faint CRF-IR was still observed in the superficial DH after complete spinalization (H). CC, central canal. Calibration bar represents 80 μm in A-E, G, H, 100 μm in F, 40 μm in I, K, L and 50 μm in J.

Figure 2

Histogram summarizing changes in CRF staining density (O.D., optical density) in specific regions of the L6-S1 spinal cord with increasing postnatal (P) age (P0-Adult). The spinal cord inset depicts the areas analyzed: lateral dorsal horn (LDH), lateral collateral pathway of Lissauer (LCP), sacral parasympathetic nucleus (SPN), dorsal commissure (DCM). The density of CRF-IR within each region analyzed was correlated with increasing postnatal age (R² = 0.87–0.98; p ≤ 0.001). At each age examined, CRF-IR in the SPN was significantly greater than all other regions. *, p ≤ 0.001.

Figure 3

Pictorial representation of the methods used to quantify CRF-immunoreactive varicosities (red, A, B; yellow, C, D) on neuronal nitric oxide synthase (nNOS)-immunoreactive cells (green, A, B) (presumptive preganglionic neurons) in the region of the sacral parasympathetic nucleus. CRF-immunoreactive varicosities on or within a 1 μm perimeter (A-D, blue dashed lines) of a selected nNOS-immunoreactive cell (A, C) or a nNOS-immunoreactive proximal neurite (B, D) were thresholded (yellow, C, D) and quantified (number, area occupied and colocalization) as detailed in the methods section. Calibration bar represents 5 μm.

Figure 4

Fluorescence images of CRF-IR surrounding nNOS-IR (presumptive preganglionic parasympathetic neurons) in the sacral parasympathetic nucleus (SPN) in postnatal (P)3 (A, D, G, J), P7 (B, E, H, K) and P14 (C, F, I, L) rat pups. CRF-IR in the SPN region (A-F; CRF-IR, red; nNOS-IR, green) increased with increasing postnatal age and CRF-IR in close apposition to nNOS-immunoreactive cells also increased with increasing postnatal age (thresholded images of CRF-immunoreactive varicosities, G-I, yellow; thresholded images of nNOS-immunoreactive cells, J-L, yellow). Higher power images of the SPN region (D-F) showing increasing CRF-IR (red) in the SPN and in association with nNOS-immunoreactive cells (green). CRF-IR and nNOS-IR was thresholded separately as described in the methods section and only CRF- or nNOS-IR that exceeded threshold (G-L, yellow) was quantified. Only nNOS-immunoreactive preganglionic parasympathetic cells that exceeded threshold (J-L) were used to determine CRF-immunoreactive varicosity measurements (number, density, colocalization) in association with nNOS-immunoreactive cells (J-L, yellow). Calibration bar represents 40 μm in A-C and 20 μm in D-L.

Figure 5

Fluorescence images of CRF-IR surrounding nNOS-IR (presumptive preganglionic parasympathetic neurons) in the sacral parasympathetic nucleus (SPN) in postnatal (P)21 (A, D, G, J), P28 (B, E, H, K) rat pups and Adult (C, F, I, L) rat. CRF-IR in the SPN region (A-F; CRF-IR, red; nNOS-IR, green) increased with increasing postnatal age and CRF-IR in close apposition to nNOS-IR cells also increased with increasing postnatal age (thresholded images of CRF-immunoreactive varicosities, G-I, yellow; thresholded images of nNOS-IR cells, J-L, yellow). Higher power images of the SPN region (D-F) showing increasing CRF-IR (red) in the SPN and in association with nNOS-immunoreactive cells (green). CRF-IR and nNOS-IR was thresholded separately as described in the methods section and only CRF- or nNOS-IR that exceeded threshold (G-L, yellow) was quantified. Only nNOS-immunoreactive preganglionic parasympathetic cells that exceeded threshold (J-L) were used to determine CRF-immunoreactive varicosity measurements (number, density, colocalization) in association with nNOS-immunoreactive cells (J-L, yellow). Calibration bar represents 40 μm in A-C and 20 μm in D-L.

Figure 6

Summary histogram of the average number of CRF-immunoreactive varicosities on or within a 1 μm distance from nNOS-immunoreactive cell bodies or proximal neurites in the sacral parasympathetic nucleus (presumptive preganglionic parasympathetic neurons). There was an age dependent increase in the numbers of CRF-immunreactive varicosities closely apposed to nNOS-immunoreactive cells (R² = 0.80; p ≤ 0.05) or proximal neurites (R² = 0.91; p ≤ 0.01). The number of CRF-immunoreactive varicosities in close apposition to nNOS-immunoreactive cell bodies at P27 and in the adult rat was significantly (p ≤ 0.01) greater than other ages examined (P3-P21). The number of CRF-immunoreactive varicosities in close apposition to nNOS-immunoreactive proximal neurites in the adult rat was significantly (p ≤ 0.001) greater than all other ages examined (P3-P27). #, p ≤ 0.01; *, p ≤ 0.001.