Estrogen receptor beta is essential for sprouting of nociceptive primary afferents and for morphogenesis and maintenance of the dorsal horn interneurons

Abstract

Estrogen is known to influence pain, but the specific roles of the two estrogen receptors (ERs) in the spinal cord are unknown. In the present study, we have examined the expression of ERalpha and ERbeta in the spinal cord and have looked for defects in pain pathways in ERbeta knockout (ERbeta(-/-)) mice. In the spinal cords of 10-month-old WT mice, ERbeta-positive cells were localized in lamina II, whereas ERalpha-positive cells were mainly localized in lamina I. In ERbeta(-/-) mice, there were higher levels of calcitonin gene-regulated peptide and substance P in spinal cord dorsal horn and isolectin B4 in the dorsal root ganglion. In the superficial layers of the spinal cord, there was a decrease in the number of calretinin (CR)-positive neurons, and in the outer layer II, there was a loss of calbindin-positive interneurons. During embryogenesis, ERbeta was first detectable in the spinal cord at embryonic day 13.5 (E13.5), and ERalpha was first detectable at E15.5. During middle and later embryonic stages, ERbeta was abundantly expressed in the superficial layers of the dorsal horn. ERalpha was also expressed in the dorsal horn but was limited to fewer neurons. Double staining for ERbeta and CR showed that, in the superficial dorsal horn of WT neonates [postnatal day 0 (P0)], most CR neurons also expressed ERbeta. At this stage, few CR-positive cells were detected in the dorsal horn of ERbeta(-/-) mice. Taken together, these findings suggest that, early in embryogenesis, ERbeta is involved in dorsal horn morphogenesis and in sensory afferent fiber projections to the dorsal horn and that ERbeta is essential for survival of dorsal horn interneurons throughout life.

Fig. 1.

ERβ and ERα expression in the spinal cord during embryogenesis. (A) At E13.5, ERβ is mainly localized in the superficial layers of the dorsal horn in the lumbar region of the spinal cord. (B and C) In E14.5 lumbar (B) and thoracic (C) spinal cords, ERβ is strongly expressed in the laminae I-II. There is also distinct nuclear staining in the lateral region of lamina V and near the central canal. (D and E) In E15.5 thoracic (D) and cervical (E) spinal cords, ERβ is found in laminae I–II; some ERβ-positive cells are also localized in laminae III–V. (F) In the sagittal section of lumbo–sacral spinal cords at E15.5, dense and deep ERβ staining is seen in the superficial layers. (G and H) In sagittal sections of thoracic spinal cords at E15.5, there are some ERα-positive cells in the dorsal horn (H), and the number of ERβ-positive cells far exceeds that of ERα-positive cells (G). (I) At E17.5 in the lumbar spinal cord, more ERβ-labeled cells appear in lamina II. (J–L) At E16.5 in the dorsal horn of the lumbar spinal cord, most of the ERα-positive cells also express ERβ. CC, central canal. (Scale bar: 20 μm.)

Fig. 2.

ERβ and ERα expression in the postnatal spinal cord. (A, B, D, and E) At P0 in lumbar (A and D) and sacral (B and E) regions of the spinal cord, ERβ-labeled neurons (A and B) appear less numerous in the outer dorsal horn but more numerous in the deeper layers of the spinal cord than ERα-labeled neurons (D and E). (C and F) In 10-month-old mouse spinal cords, more ERβ-stained cells (C) are localized in lamina II, whereas ERα-stained cells (F) are mainly localized in lamina I. (Scale bars: A, D, C, and F, 50 μm; B and E, 20 μm).

Fig. 3.

Expression of NeuN and calretinin in the dorsal horn of WT and ERβ^−/− female mice at E17.5. (A, B, D, and E) At E17.5, in thoracic (A and D) and lumbar (B and E) dorsal horn, there are fewer NeuN-labeled neurons in ERβ^−/− mice (D and E) than in WT littermates (A and B). (C and F) Calretinin expression in the lumbar dorsal horn of ERβ^−/− mice (F) is markedly lower than in WT mice (C); the reduction is especially noticeable in the superficial layer. Arrows in A and D indicate laminae III–V of thoracic dorsal horn, and arrows in B, C, E, and F indicate laminae I–II of lumbar dorsal horn. (Scale bar: 20 μm.)

Fig. 4.

Expression of calretinin and calbindin in the superficial layers of the dorsal horn in the lumbar region in postnatal WT and ERβ^−/− female mice. (A–C) At P0, double staining for ERβ and calretinin shows that, in the dorsal horn, most of the calretinin-positive neurons also express ERβ. (D and G) There are fewer calretinin-positive cells in ERβ^−/− mice (G) than in WT mice (D). (E and H) At P2, in ERβ^−/− mice (H), there are fewer calretinin-positive cells in the medial part of lamina II compared with WT mice (E). (F and I–K) Expression of calretinin in lamina II is significantly lower in ERβ^−/− mice (I and K) than WT mice (F and J) at 3 (F and I) and 18 (J and K) months of age. (L) The average percentage of calretinin-labeled cells in laminae I and II of spinal cord dorsal horn at 3 and 18 months of age is shown (n = 3; error bar, SD; **, P < 0.01, Student's t test). BERKO, ERβ^−/−. (M) In 3-month-old WT mice, calbindin is mainly localized in laminae I–II of the dorsal horn. (N) Decreased calbindin-labeled cells are seen in the outer layer II (IIo) and lamina I of ERβ ^−/− mouse dorsal horn. (Scale bars: A–E, G, and H, 20 μm; F, I–K, M, and N, 50 μm).

Fig. 5.

Distribution of CGRP, SP, and IB4 in lumbar superficial dorsal horn of 3-month-old, female WT and ERβ^−/−mice. (A, D, G, and J) IB4-labeled afferents terminate in inner lamina II and show similar expression pattern in ERβ^−/− (D and J) and WT mice (A and G). (B, E, H, and K) Expression of CGRP and SP in superficial dorsal horn is higher in ERβ^−/− (E and K) than in WT mice (B and H). (C, F, I, and L) Double labeling of IB4 and CGRP or SP clearly shows more CGRP or SP afferent innervation detected in lamina IIi in ERβ^−/− mice (F and L) compared with WT mice (C and I). Arrows in C, F, I, and L indicate inner lamina II. (Scale bar: 20 μm.)

Fig. 6.

Expression of IB4, CGRP, and SP in the DRG of female WT and ERβ^−/−mice at 3 months of age. (A and D) The number of IB4-labeled cells in DRG is higher in ERβ^−/− mice (D) than in WT littermates (A). (B, C, E, and F) Expression of CGRP (B and E), and SP (C and F) is similar in ERβ^−/− (E and F) and WT mice (B and C). (G) The average percentages of DRG neurons expressing IB4 or CGRP in WT and ERβ^−/− female mice at 3 months of age are shown (n = 3; error bar, SD; **, P < 0.01, Student's t test). BERKO, ERβ^−/−. (Scale bar: 20 μm.)