Distribution and localization patterns of estrogen receptor-beta and insulin-like growth factor-1 receptors in neurons and glial cells of the female rat substantia nigra: localization of ERbeta and IGF-1R in substantia nigra

Abstract

Although several studies have focused on the neuroprotective effects of estrogen (E2) on stroke, there have been tantalizing reports on the potential neuroprotective role of E2 in degenerative neuronal diseases such as Alzheimer's and Parkinson's (PD). In animal models of PD, E2 protects the nigrostriatal dopaminergic (DA) system against neurotoxins. However, little is known about the cellular and molecular mechanism(s) involved by which E2 elicits its neuroprotective effects on the nigrostriatal DA system. A preferred mechanism for neuroprotection is the interaction of E2 with specific neuroprotective growth factors and receptors. One such neuroprotective factor/receptor system is insulin-like growth factor-1 (IGF-1). E2 neuroprotective effects in the substantia nigra (SN) DA system have been shown to be dependent on IGF-1. To determine whether E2 also interacts with the IGF-1 receptor (IGF-1R) and to determine the cellular localization of estrogen receptor (ER) and IGF-1R, we compared the distribution of ER and IGF-1R in the SN. Stereological measurements revealed that 40% of the subpopulation of tyrosine hydroxylase-immunoreactive (TH-ir) SN pars compacta (SNpc) DA neurons are immunoreactive for estrogen receptor-beta (ERbeta). No immunolabeling for ERalpha was observed. In situ hybridization and immunocytochemistry studies confirmed the expression of IGF-1R mRNA and revealed that almost all TH-ir SNpc DA neurons were immunoreactive for IGF-1R, respectively. Moreover, one-third of glial fibrillary acidic protein (GFAP-ir) cells in the SN were ERbeta-ir, and 67% of GFAP-ir cells expressed IGF-1R-ir. Therefore, the localization of ERbeta and IGF-1R on SNpc DA neurons and astrocytes suggests a modulatory role of E2 on IGF-1R, and this modulation may affect neuroprotection.

Fig 1

Immunoflourescent photomicrographs from substantia nigra pars compacta sections. (A) TH (DAB), black dashed lines depicts SNpc. (B) TH (Texas Red), white dashed lined depicts SNpc. (C) Fluorogold retrograde staining. (D) Immunoflourescent product for IGF-1R (green). (E) Immunoflourescent product for Z8P ERβ (Green). (F) Ab-1 ERβ (Green). (A) Scale bar = 200 µm. (B–F) Scare bar = 50 µm.

Fig 2

Darkfield photomicrographs of the substantia nigra of the adult OVX female rat showing hybridization signal for IGF-1R mRNA. (A) Panel represents sense probes. (B) Panel is the result with an antisense probe. (C) Representive photomicrograph depicting brightfield localization of IGF-1R mRNA (silver grains) counterstained with thioinin. Arrows point to spots of dense signal corresponding to the positive cells. Scale bar = 200 µm (A & B). Scale bar = 50 µm (C). Ventral Tegmental Area (VTA), red nucleus (R), medial lemniscus (ML), substantia nigra pars compacta (SNpc), pars lateral (SNpl), pars reticular (SNpr).

Fig 3

Immunoflourescent photomicrographs demonstrating differences in localization of ERβ-ir between Z8P antibody (A) and Ab-1 (E) on TH-ir SNpc DA neurons. (B–D) Enhanced magnification of ERβ and TH colocalization. TH (red, B), Z8P (green, C). Overlay (D) of B and C; arrow demonstrates nuclear labeling. (F–H) Enhanced magnification of ERβ and TH colocalization. TH (red, F), anjd Ab-1 antibody (green, G). Overlay (H) of F and G; arrow demonstrates cytoplasmic labeling. Scale bar = 10 µm.

Fig 4

Low (A) and high-power (B) photomicrographs illustrating the distribution of nickel DAB ERβ-ir on TH-ir SNpc DA neurons. (B) Arrowheads point to TH-ir neurons displaying nuclear ERβ-ir, arrows demonstrate no nuclear ERβ-ir. Low (C) and high-power (D) photomicrographs illustrating the distribution of nickel DAB IGF-1R-ir in TH-ir SNpc DA neurons. (D) Arrowheads, demonstrate IGF-1R-ir in TH-ir SNpc DA neurons, arrows demonstrate no IGF-1R-ir. Scale bar = 50 µm (A and C). Scale bar = 10 µm (B and D).

Fig 5

Colocalization of ERβ and FG labeled SNpc DA neurons. Confocal microscopy showing the fluorescent immunoreaction product for Fluorogold (red color substituted with Axivision 3.1 software, A). Immunoflourescent product for Z8P ERβ (green, B). 25 Overlay images (C) from A and B; Arrows indicate ERβ immunolabeling (yellow) in FG retrograde labeled DA neurons of the SNpc (Red). Scale bar = 200 µm (A–C).

Fig 6

Immunolocalization of IGF-1R in SNpc DA neurons. Confocal microscopy displaying fluorescent immunoreactivity for TH (red, A), and IGF-1R (green, B and E). Overlay images (C) from A and B; arrow indicates co-localization (yellow) of IGF-1R and TH in DA neurons of the SNpc. Arrowhead indicates TH-immunoreactivity alone. Fluorogold (D) (red color substituted with Axivision 3.1 software). Overlay images (F) from D and E, co-localization (yellow) in fluorogold containing neurons in the SNpc (Red). Scale bar = 50 µm (A–C) and 200 µm (D–F).

Fig 7

Confocal photomicrographs from substantia nigra sections showing immunofluorescence for GFAP (red, A and D), ERβ (green, B) or IGF-1R (green, E), and their colocalization (yellow, C and F). Magnified image in (C) displays nuclear, cytoplasmic and processes immunostaining for ERβ. Scale bar = 50 µm.

Fig 8

Proposed model for the neuroprotective interactions between E2 and IGF-1 on SNpc DA neurons. A) E2 and IGF-1 can directly protect the subset of SNpc DA neurons that express ERβ (green labeled nucleus) or IGF-1R (red dots). B) E2 and IGF-1 can stimulate secretion of neuroprotective factors (e.g. IGF-1; yellow filled circles) in glial cells that express ERβ, resulting in indirect neuroprotection of SNpc DA neurons that are ERβ negative.