Effects of chronic NMDA-NR2b inhibition in the median eminence of the reproductive senescent female rat

Abstract

Gonadotrophin-releasing hormone (GnRH) neurones of the hypothalamic-pituitary-gonadal (HPG) axis drive reproductive function and undergo age-related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b-containing receptors with ifenprodil, an NR2b-specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle-aged females. In the present study, we chronically administered ifenprodil, an NR2b-specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle-aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr-1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro-oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co-labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR-NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.

Keywords: NMDA receptor; gonadotrophin-releasing hormone; median eminence; menopause; reproductive senescence.

Figure 1. ME Cannula Placement

Cannula placements are indicated for vehicle (X) and ifenprodil (O) treated animals. For immunohistochemistry and microscopic analysis, we subdivided the ME into the pericapillary region of the external zone and the periventricular region of the internal zone (depicted in the medial ME section at A/P −2.6 mm). Abbreviations: ARC = arcuate nucleus, RCh = retrochiasmatic nucleus, ME = median eminence, int = internal zone, ext = external zone, Cap = capillary region, Inf = Infundibulum, third ventricle shown in black.

Figure 2. Effect of Ifenprodil Treatment in the ME on Vaginal Cytology

Daily vaginal cytology was used to monitor changes in oestrous cyclicity during chronic infusion with either Ifenprodil or Vehicle for ~28 days. A. Percentage of days spent in pro-oestrus during treatment, as determined by the presence of nucleated cells in the vaginal smear. B, C. Representative estrous cycles of a Vehicle (B) and an Ifenprodil (C) rat are shown. Abbreviations: E = oestrus, P = pro-oestrus, D = dioestrus. *** p ≤ 0.001.

Figure 3. Colocalisation of NR2b/GnRH and pNR2b/GnRH

Representative confocal micrographs in the ME of double-label immunofluorescence of A) NR2b (green) with GnRH (red); and B) pNR2b (green) with GnRH (red). No colocalisation of GnRH was seen with NR2b but a small amount was observed with pNR2b in the external zone of the ME (white arrowhead). A single confocal slice is shown in the inset of B demonstrating a higher resolution image of GnRH/pNR2b colocalisation in the external zone of the ME (small white arrowheads). Linear mixed modelling revealed GnRH/pNR2b colocalisation density and mean intensity levels were higher in the external zone. Raw data are graphed in C and D (not estimated means from model). Abbreviations: 3V = third ventricle, Cap = capillary region, *** p value ≤0.001. Scale bar = 20 μm for A and B, 4 μm for inset.

Figure 4. Effect of ME Zone and Ifenprodil Treatment in the ME on NR2b and pNR2b Expression

Imaris software was used to analyse punctate labeling of NR2b and pNR2b subunit expression in the ME. A, C) Density of NR2b and pNR2b punctate staining, respectively. B, D) Mean fluorescence intensity of NR2b and pNR2b, respectively. No effects of treatment were found. Linear mixed modelling revealed that NR2b and pNR2b densities were higher in the external zone of the ME, whereas NR2b fluorescence intensity was higher in the internal zone of the ME. Raw data are graphed here (not estimated means from model). * p ≤ 0.05, *** p ≤ 0.001.

Figure 5. Effect of ME Zone and Ifenprodil Treatment in the ME on GnRH Expression

Imaris software was used to analyse the A) density and B) mean fluorescence intensity of GnRH punctate staining in the ME. Linear mixed modelling revealed that GnRH density and fluorescence intensity were higher in the external zone of the ME. A Treatment x Zone interaction was found for GnRH intensity levels (p = 0.021), and a paired T-test revealed that GnRH fluorescence intensity was significantly increased by ifenprodil treatment in the external zone. Raw data are graphed here (not estimated means from model). *** p ≤ 0.001.

Figure 6. Network Analysis

A correlation network of the ME-cannula data was made for the ifenprodil and vehicle groups to examine the relationships among protein expression, hormone levels and other physiological endpoints. Networks were created separately for the external and internal zones of the ME and only significant correlations are shown (p < 0.05 after Benjamini-Hochberg False Discovery Rate). Positive Pearson’s r correlation coefficients are indicated with solid blue lines and negative Pearson’s r coefficients are indicated with dashed red lines. Thicker lines correspond to higher Pearson’s r coefficients. PSI = pituitary somatic index, E2 = oestradiol, P4 = progesterone