The steroid hormone estradiol controls brain function by activating classical nuclear [estrogen receptor-alpha (ER$\alpha$); estrogen receptor-beta (ER$\beta$)] and membrane [G-protein-coupled ER (GPER/GPR30)] ERs. Estradiol acts by ER-dependent mechanisms to exert critical organizational, neurotrophic, regulatory, and neuroprotective actions over the lifespan [1, 2, 3, 4, 5, 6, 7]. During central nervous system (CNS) differentiation, ER stimulation causes gender-imprinting of distinct forebrain loci, resulting in sex-dimorphic cytoarchitectural composition and estradiol regulation of function of the anteroventral periventricular nucleus, medial preoptic area, medial amygdaloid nucleus, stria terminalis bed nucleus, and ventromedial hypothalamic nucleus (VMN) [8, 9, 10, 11, 12, 13, 14]. The ER-abundant VMN imposes sex-contingent regulation of arousal, maternal, sexual, aggressive, and defensive behaviors; energy balance; autonomic outflow; and thermogenesis [15, 16, 17, 18, 19, 20, 21, 22]. Estradiol shapes body-wide glucose homeostasis [23, 24, 25, 26, 27]. The VMN is a foremost sensory and integrative component of the brain glucose regulatory network [21, 28] and an important substrate for estradiol control of glycemic and counterregulatory neurochemical profiles [29, 30, 31].

Aging has broad-ranging effects on somatic and homeostatic bodily functions, including those governed by ER-regulated neural outflow [32, 33, 34, 35]. Indeed, the notion that aging may alter brain cell receptivity to estradiol has garnered ample, justifiable attention. Available studies provide valuable documentation of hypothalamic loci in which age-associated changes in ER expression occur [36, 37, 38, 39, 40]. Nevertheless, the unique cyto- and chemo-architectural heterogeneity of each structure accentuates the necessity for high-neuroanatomical resolution analytical techniques to determine if and how aging may affect estradiol receptivity of neurons of characterized functionality. Analysis of brain tissue samples corresponding to whole brain regions, nuclei, or areas poses a risk that averaged endpoint values may mask or obscure unique responses of individual neuron populations. Type 1 diabetes mellitus (T1DM) is a pervasive health concern in the elderly as acute and long-term complications of this condition exacerbate patient morbidity and mortality [41, 42, 43]. Older diabetic patients face an elevated risk of hypoglycemia-associated brain injury as counteractive hormone outflow and neurogenic awareness are impaired with age [44, 45, 46, 47, 48, 49]. There is an obvious need to identify the mechanism(s) that elicits aging-related counterregulatory collapse. VMN structure is altered by aging in each sex [50]; yet it is unclear if or how age affects ER expression in VMN neurons that engage in neural regulation of glucose homeostasis, and if estradiol-induced changes in cellular function are responsible for these changes.

The VMN functions to assimilate metabolic, hormonal, and neurotransmitter cues to shape glucose counterregulatory responses. Recent studies suggest that estradiol-sensitive dorsomedial VMN (VMNdm) growth hormone-releasing hormone (Ghrh) neurons are a common substrate for these diverse regulatory stimuli. Our work shows that this nerve cell type expresses mRNA that encodes the VMN-exclusive [51, 52] metabolic transcription factor steroidogenic factor-1 (SF-1) [53], which is implicated in regulation of systemic energy and glucose balance [54, 55, 56, 57, 58, 59]. In addition, SF-1 gene expression displays a differential response to insulin-induced hypoglycemia (IIH) [60]. VMNdm Ghrh neurons also express hypoglycemia-responsive genes that encode metabolic-sensory biomarkers (i.e., the glucose sensor glucokinase and catalytic subunit of the ultra-sensitive energy sensor 5^′-AMP-activated protein kinase [61]) and, in addition to Ghrh mRNA, transcripts for biosynthetic enzyme surrogates of characterized counterregulatory-constraining ($\gamma$-aminobutyric acid) or -enhancing (glutamate; nitric oxide) neurochemicals [60]. Evidence for concurrent expression of neurotransmitters of unique chemical structure, spatial, and temporal profiles that exert characteristic control of counterregulatory hormone secretion supports the provision of complex, coordinated multi-modal signal input by VMNdm Ghrh neurons to the glucostatic neural network.

Developmental organization of the sex-dimorphic VMN results in sex-specific estradiol regulation of VMN functions. VMNdm Ghrh neurons from young adult male and female express ER$\alpha$, ER$\beta$, and GPER mRNAs, but these ER variants exhibit sex-contingent Ghrh receptor (Ghrh-R)-dependent transcriptional responses to hypoglycemia [60]. Current research made use of in vivo gene silencing tools and combinative in situ immunocytochemistry, single-cell laser-catapult-microdissection, and single-cell multiplex qPCR techniques to examine the premise that aging may alter eu- and/or hypoglycemic patterns of VMNdm Ghrh neuron ER variant gene expression, and that such changes may correlate with age-associated adjustments in Ghrh-R control of distinctive ER mRNA profiles. The study design here incorporated animals of each sex to advance U.S. National Institutes of Health policy interest in evaluation of sex as an important biological variable. Firstly, present data depict and compare between sexes aging effects on the magnitude of baseline or IIH-associated patterns of expression of individual VMNdm Ghrh neuron ER mRNAs. A emerging study supports the intriguing prospect that age-associated shifts in ER$\alpha$/ER$\beta$ ratio may affect gene transcription and other nerve cell functions [62]. Therefore, age impact on the ratio of VMNdm Ghrh nerve cell ER$\alpha$, ER$\beta$, and GPER mRNA content during normo- and hypoglycemia was also examined in rats of each sex.

Central ERs are activated by ligand that is synthesized in the ovary or within the brain, where testosterone is metabolized to neuroestradiol by aromatase (CYP19A1) enzyme action [63, 64]. A small number of neural structures, including the VMN, are characterized by relatively high CYP19A1 gene and protein expression and enzyme activity profiles compared to other brain loci [65, 66, 67, 68, 69]. A recent study provides evidence to infer that VMN neuroestradiol influences neural governance of glucose counterregulation [70]. The cellular source(s) of neuroestradiol produced in the VMN or elsewhere remains to be characterized. The present study investigated the corollary hypothesis that VMNdm Ghrh neurons express CYP19A1, the gene that encodes CYP19A1 protein, and that eu- and/or hypoglycemia-associated transcription of this gene may be affected by age in one or both sexes.

Aging has documented effects on net ER variant mRNA and/protein content in distinct hypothalamic loci but understanding of how age may impact estradiol sensitivity of neurons that operate within the glucostatic neural circuitry is lacking. VMNdm Ghrh neurons likely function to integrate metabolic, endocrine, and neurochemical regulatory cues to shape counterregulatory neurotransmission. Here, single-cell laser-catapult-microdissection/multiplex qPCR analytical tools were applied to investigate whether aging perturbs, in one or both sexes, the magnitude and/or proportional expression of VMNdm Ghrh neuron ER variant mRNAs during eu- or hypoglycemia. Current research also examined the prospect that Ghrh neuromodulatory regulation of VMNdm Ghrh nerve cell ER gene transcription may change with aging in male and/or female rats using stereotactic delivery of in vivo Ghrh gene silencing tools.

Fig. 1 (Ref. [75]) illustrates effects of VMN Ghrh gene silencing on VMNdm Ghrh nerve cell ER$\alpha$/ESR1 gene expression in eu- and hypoglycemic aged rats. Data show that baseline ER$\alpha$ gene profiles were significantly decreased in old versus young animals of each sex [F_(7,88) = 10.88, p 0.001; Sex effect: F_(1.88) = 16.22, p 0.001; Treatment effect: F_(1,88) = 4.38, p = 0.039; Pretreatment effect: F_(1.88) = 31.52, p 0.001; Sex/treatment interaction: F_(1,88) = 1.03, p = 0.313; Sex/pretreatment interaction: F_(1,88) = 0.26, p = 0.605; Treatment/pretreatment interaction: F_(1,88) = 6.43, p = 0.013; Sex/pretreatment/treatment interaction: F_(1,44) = 0.83, p = 0.364]. Results also indicate that baseline ER$\alpha$ mRNA levels were suppressed by Ghrh gene silencing in old female, but not old male rats. This gene profile was refractory to hypoglycemia in both sexes, and patterns of ER$\alpha$ expression following INS injection were unaffected by Ghrh siRNA pretreatment.

Fig. 1.

Effects of ventromedial hypothalamic nucleus (VMN) growth hormone-releasing hormone (Ghrh) gene knockdown on total estrogen receptor-alpha (ER$\mathrm{\alpha }$/ESR1) gene expression in old male versus female dorsomedial VMN (VMNdm) Ghrh-immunoreactive (-ir) neurons. Groups of old male and female rats (n = 4 males and n = 4 females per group) were pretreated by bilateral intra-VMN scramble (SCR) or Ghrh siRNA infusion seven days before subcutaneous (sc) injection of vehicle (V) or neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw). Young SCR siRNA-pretreated, V-injected male and female rats served as controls. Individual brains were collected one hour after injection and cut into 10 micron-thick fresh frozen sections through the VMN; tissues were processed by immunocytochemistry to identify VMNdm Ghrh-ir neurons for single-cell laser-catapult-microdissection/multiplex quantitative real-time polymerase chain reaction (qPCR) analysis. mRNA values were normalized to the housekeeping gene GAPDH by the 2^{-Δ⁢Δ⁢Ct} method [75]. Data depict mean normalized total VMNdm Ghrh neuron ER$\alpha$ mRNA measures + S.E.M. for male (five bars, at left) and female (five bars, at right) rat treatment groups. Old rat treatment groups (identified by the abbreviation ‘O’) are identified as follows: SCR siRNA/V (cross-hatched bars; male: gray, n = 12; female: white, n = 1); Ghrh siRNA/V (stippled bars; male: gray, n = 12; female: white, n = 12); SCR siRNA/INS (diagonal-striped bars; male: gray, n = 12; female: white, n = 12); Ghrh siRNA/INS (horizontal-striped bars; male: gray, n = 12; female: white, n = 12). Young male and female treatment groups (indicated by the abbreviation ‘Y’) are represented by solid gray (n = 12) or white (n = 12) bars respectively. For each panel, circles depict individual independent data points. mRNA and protein data were analyzed between old rat treatment groups by three-way ANOVA and Student-Newman-Keuls post-hoc test. Data from young SCR siRNA/V versus old SCR siRNA/V control groups were evaluated by t test. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p 0.05; **p 0.01.

Data in Fig. 2 depict patterns of ER$\beta$/ESR2 gene transcription in VMNdm Ghrh neurons collected from old male or female rats pretreated with Ghrh versus SCR siRNA before sc V or INS injection. ER$\beta$ mRNA levels were not significantly different in old versus young rats of either sex [F_(7,88) = 10.43, p 0.001; Sex effect: F_(1.88) = 12.19, p = 0.001; Treatment effect: F_(1,88) = 7.77, p = 0.006; Pretreatment effect: F_(1.88) = 13.76, p 0.001; Sex/treatment interaction: F_(1,88) = 1.78, p = 0.185; Sex/pretreatment interaction: F_(1,88) = 13.20, p 0.001; Treatment/pretreatment interaction: F_(1,88) = 13.41, p 0.001; Sex/pretreatment/treatment interaction: F_(1,44) = 0.65, p = 0.423]. Baseline ER$\beta$ gene transcription was decreased in old male, but not old female rats. Hypoglycemia diminished this gene profile in old female, but not old male rats; patterns of ER$\beta$ gene expression following INS injection were unaffected by prior Ghrh siRNA pretreatment.

Fig. 2.

Patterns of VMNdm Ghrh neuron estrogen receptor-beta (ER$\mathrm{\beta }$/ESR2) gene expression in old eu- or hypoglycemic male versus female rats. Results present mean normalized total VMNdm Ghrh nerve cell ER$\beta$/V mRNA values + S.E.M. for male (five bars, at left) and female (five bars, at right) rat treatment groups. Old rat treatment groups, indicated by the abbreviation ‘O’, are identified as follows: SCR siRNA/V (cross-hatched bars; male: gray, n = 12; female: white, n = 1); Ghrh siRNA/V (stippled bars; male: gray, n = 12; female: white, n = 12); SCR siRNA/INS (diagonal-striped bars; male: gray, n = 12; female: white, n = 12); Ghrh siRNA/INS (horizontal-striped bars; male: gray, n = 12; female: white, n = 12). Young male and female treatment groups, identified by the abbreviation ‘Y’, are represented by solid gray (n = 12) or white (n = 12) bars respectively. For each panel, circles depict individual independent data points. Normalized mRNA data were analyzed between old rat treatment groups by three-way ANOVA and Student-Newman-Keuls post-hoc test Data from young SCR siRNA/V versus old SCR siRNA/V control groups were evaluated by t test. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p 0.05; **p 0.01; ***p 0.001.

Fig. 3 shows effects of VMN Ghrh siRNA administration on VMNdm Ghrh nerve cell GPER gene expression in eu- or hypoglycemia old male versus female rats. Data indicate that in each sex, baseline GPER mRNA content was significantly lower in old versus young animals [F_(7,88) = 22.84, p 0.001; Sex effect: F_(1.88) = 17.16, p 0.001; Treatment effect: F_(1,88) = 24.74, p 0.001; Pretreatment effect: F_(1.88) = 28.98, p 0.001; Sex/treatment interaction: F_(1,88) = 2.55, p = 0.113; Sex/pretreatment interaction: F_(1,88) = 22.16, p 0.001; Treatment/pretreatment interaction: F_(1,88) = 19.29, p 0.001; Sex/pretreatment/treatment interaction: F_(1,44) = 0.46, p = 0.501]. Hypoglycemia caused a significant reduction in this gene profiles in old male and female rats. This inhibitory response was exacerbated in old males but was reversed in old females.

Fig. 3.

Effects of VMN Ghrh Gene Silencing on Eu- and Hypoglycemic Patterns of VMNdm Ghrh neuron G-protein-coupled-estrogen receptor-1 (GPER/GRP31) mRNA expression in old male versus female rats. Data show mean total GPER mRNA values + S.E.M. for male (five bars, at left) and female (five bars, at right) rat treatment groups. Old rat treatment groups, indicated by the abbreviation ‘O’, are identified as follows: SCR siRNA/V (cross-hatched bars; male: gray, n = 12; female: white, n = 1); Ghrh siRNA/V (stippled bars; male: gray, n = 12; female: white, n = 12); SCR siRNA/INS (diagonal-striped bars; male: gray, n = 12; female: white, n = 12); Ghrh siRNA/INS (horizontal-striped bars; male: gray, n = 12; female: white, n = 12). Young male and female treatment groups, indicated by the abbreviation ‘Y’, are represented by solid gray (n = 12) or white (n = 12) bars respectively. For each panel, circles depict individual independent data points. Normalized mRNA data were analyzed between old rat treatment groups by three-way ANOVA and Student-Newman-Keuls post-hoc test. Data from young SCR siRNA/V versus old SCR siRNA/V control groups were evaluated by t test. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p 0.05; **p 0.01; ***p 0.001.

Patterns of VMNdm Ghrh nerve cell CYP19A1 gene expression in Ghrh versus SCR siRNA-pretreated, eu- or hypoglycemic old male and female rats are shown in Fig. 4. Data indicate that this gene profile was significantly up-regulated in old versus young animals of each sex [F_(7,88)= 25.39, p 0.001; Sex effect: F_(1.88)= 14.66, p 0.001; Treatment effect: F_(1,88) = 0.43, p = 0.512; Pretreatment effect: F_(1.88)= 116.62, p 0.001; Sex/treatment interaction: F_(1,88)= 3.66, p = 0.058; Sex/pretreatment interaction: F_(1,88)= 0.95, p = 0.332; Treatment/pretreatment interaction: F_(1,88)= 15.96, p 0.001; Sex/pretreatment/treatment interaction: F_(1,44)= 0.24, p = 0.625]. Ghrh gene silencing increased CYP19A1 mRNA levels in VMNdm Ghrh neurons from old male or female rats. Hypoglycemia alone did not modify CYP19A1 gene transcription; yet Ghrh siRNA pretreatment amplified this gene profile in INS-injected old rats of each sex.

Fig. 4.

VMNdm Ghrh neuron aromatase (CYP19A1) gene expression in old male versus female rat VMNdm Ghrh neurons. Data depict mean total CYP19A1 mRNA values $\pm$ S.E.M. for male (five bars, at left) and female (five bars, at right) rat treatment groups. Old rat treatment groups, indicated by the abbreviation ‘O’, are identified as follows: SCR siRNA/V (cross-hatched bars; male: gray, n = 12; female: white, n = 1); Ghrh siRNA/V (stippled bars; male: gray, n = 12; female: white, n = 12); SCR siRNA/INS (diagonal-striped bars; male: gray, n = 12; female: white, n = 12); Ghrh siRNA/INS (horizontal-striped bars; male: gray, n = 12; female: white, n = 12). Young male and female treatment groups, indicated by the abbreviation ‘Y’, are represented by solid gray (n = 12) or white (n = 12) bars respectively. For each panel, circles depict individual independent data points. Normalized mRNA data were analyzed between old rat treatment groups by three-way ANOVA and Student-Newman-Keuls post-hoc test. Data from young SCR siRNA/V versus old SCR siRNA/V control groups were evaluated by t test. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p 0.05; **p 0.01; ***p 0.001.

Fig. 5 (Ref. [60]) presents VMNdm Ghrh neuron ER variant and CYP19A1 gene expression ratios for SCR versus Ghrh siRNA-pretreated sc V- (Fig. 5A,B) or INS- (Fig. 5C,D) injected old male rats. Each panel shows average ratios of individual target genes, identified by number, versus Ghrh mRNA; ratio values are depicted in graphical (above) and tabular (below) formats. An insert figure in the upper right-hand corner of each panel illustrates expression ratios for VMNdm Ghrh neuron target genes for comparable young male rat treatment groups. Data in Fig. 5A show that under euglycemic conditions, this nerve cell type exhibits differential magnitude of ER variant gene transcription, as indicated by dissimilar average ratio values, with GPER gene expression evidently predominating compared to nuclear ER gene profiles. This gene ratio reflects a change in proportionate transcription from young animals which are characterized by higher relative ER$\beta$ mRNA levels versus ER$\alpha$ and GPER. Fig. 5B depicts Ghrh gene knockdown effects on individual gene expression ratios and provides numerical notation of change in ratio values versus SCR siRNA/V controls. Data infer that this treatment respectively diminishes or augments proportionate GPER and CYP19A1 gene transcription in both old and young male rats. Comparison of VMNdm Ghrh/SF-1 neuron relative target gene expression in hypo- (Fig. 5C) versus euglycemic (Fig. 5A) old male rats indicates that glucose deficiency causes inverse changes to proportionate GPER (decreased) and CYP19A1 (increased) gene expression. Young male rats exhibit a similar negative change in relative GPER mRNA profiles, alongside decreased ER$\alpha$, ER$\beta$, and CYP19A1 gene expression. Ghrh control of proportional GPER and CYP19A1 transcription is comparable in eu- versus hypoglycemic old male rats, whereas young hypoglycemic male rats exhibit a gain of negative Ghrh control of relative ER$\beta$ gene expression and switch in direction of control of GPER (stimulatory to inhibitory) gene ratio compared to euglycemic animals. Data in Fig. 5 also show that Ghrh gene silencing increased CYP19A1 gene relative expression ratios in old and young eu- or hypoglycemic male rats, whereas hypoglycemia caused divergent changes in this ratio, i.e., increased in old versus decreased in young animals.

Fig. 5.

Mean ER variant and CYP19A1 mRNA relative expression ratios for eu- and hypoglycemic old male rat VMNdm Ghrh/SF-1 neurons. Multiplex single-cell qPCR data acquired from laser-catapult-microdissected Ghrh–ir neurons here were used to establish mean ratios for target gene expression levels relative to Ghrh mRNA. Averaged ratio values were derived from n = 12 laser-catapult-microdissected VMNdm Ghrh-ir neurons from old male rats treated as follows: SCR siRNA/V (A), Ghrh siRNA/V (B), SCR siRNA/INS (C), Ghrh siRNA/INS (D). In each figure panel, mean relative gene expression data are depicted in graphical (above; bars with accompanying S.E.M.) and tabular (below; top row: gene name; bottom row: average proportionate expression ratio value) formats. The insert to each panel depicts proportionate expression of ER variant and CYP19A1 transcripts relative to Ghrh in young male rats; these data were mined from the data set from which information on Ghrh gene knockdown effect on absolute target gene expression in young rats was previously reported [60].

Data shown in Fig. 6 (Ref. [60]) illustrate target gene versus Ghrh mRNA ratios in VMNdm Ghrh/SF-1 neurons from SCR siRNA/V (Fig. 6A), Ghrh siRNA/V (Fig. 6B), SCR siRNA/INS (Fig. 6C) and Ghrh siRNA/INS (Fig. 6D) old female rats. Like old males, euglycemic old female rats exhibit divergent target gene expression relative to Ghrh, with GPER mRNA expressed at the higher proportion. However, in young females, as in young males, ER$\beta$ is evidently the prevalent ER variant gene expressed in this neuron population. In VMNdm Ghrh neurons collected from old females, Ghrh gene silencing amplified each target gene profile relative to Ghrh and increased proportional ER$\beta$ expression (Fig. 6B). Young females, in contrast, exhibited down-regulated ER$\alpha$ and GPER mRNAs in response to this gene knockdown paradigm. Old hypoglycemic female rats exhibited increased relative ER$\alpha$ and ER$\beta$ gene transcription, which altered proportionate levels of these variants mRNAs versus GPER (Fig. 6C). In young animals, relative ER$\alpha$ and GPER gene expression was down-regulated due to hypoglycemia. Data in Fig. 6D indicate that in old and young hypoglycemic females, Ghrh gene knockdown substantially elevates the relative expression of each individual target gene. Results also show that VMN Ghrh gene silencing increased proportional CYP19A1 gene transcription V- or INS-injected old and young female rats, and that in contrast to old male rats, hypoglycemia up-regulated this relative expression ratio in old females.

Fig. 6.

Mean ER variant and CYP19A1 mRNA relative expression ratios for eu- and hypoglycemic old female rat VMNdm Ghrh/SF-1 neurons. Multiplex single-cell qPCR data acquired from laser-catapult-microdissected Ghrh–ir neurons here were used to establish mean ratios for target gene expression levels relative to Ghrh mRNA. Averaged ratio values were derived from n = 12 laser-catapult-microdissected VMNdm Ghrh-ir neurons from old female animals grouped as follows: SCR siRNA/V (A), Ghrh siRNA/V (B), SCR siRNA/INS (C), Ghrh siRNA/INS (D). In each figure panel, mean relative gene expression data are depicted in graphical (above; bars with accompanying S.E.M.) and tabular (below; top row: gene name; bottom row: average proportionate expression ratio value) formats. The insert to each panel depicts relative ER variant and CYP19A1 transcripts compared to Ghrh mRNA in young female rats; these data were mined from the data set from which information on Ghrh gene knockdown effect on absolute target gene expression in young female rats was previously reported [60].

Estradiol regulation of brain function requires insight into how normal- and patho-physiological processes affect brain cell sensitivity to this hormone signal. Aging is a natural, continuous process of change that affects all organ systems, including the brain, and purportedly alters ER variant gene and/or protein profiles in distinct brain regions or nuclei. The phenotypic and operational heterogeneity of cells that comprise these notable structures bolsters the need for continuing work to discern how aging affects ER expression in resident cell populations of known function. Current research illustrates the utility of multiplex qPCR technology, namely the capability for quantification of absolute and proportional expression profiles of diverse receptor targets for this hormone signal at the single-cell level, for research on the aging brain.

A Study has reported here examined how aging may affect ER variant gene expression in VMNdm Ghrh neurons appear to provide a complex, multi-modal neurochemical input to the counterregulatory neural network [60]. Present research also addressed the correlated premise that characteristic sex differences in ER transcription patterns in young animals may be likewise impacted by age. Results here document, for each sex, age-related absolute reductions in Ghrh neuron ER$\alpha$ and GPER gene expression levels, along with loss of ER$\alpha$ transcriptional reactivity to hypoglycemia. Effects of hypoglycemia on ER$\beta$ mRNA profiles varied between old male versus female rats, indicative of altered responsiveness in each sex due to age. The neuropeptide transmitter Ghrh regulates VMNdm Ghrh neuron ER variant transcript levels in young rats; data here document loss or change in direction of Ghrh control of distinctive ER mRNA profiles in old animals. Outcomes provide novel evidence that these neurons contain mRNA that encodes aromatase protein and that baseline expression of these transcripts is higher in old versus young rats of either sex, inferring that VMN Ghrh neuron neuroestradiol production may be enhanced with age, but cellular sensitivity to this local hormone signal may change during eu- and/or hypoglycemia. Current findings justify new efforts to ascertain how putative age-associated changes in absolute and proportionate signaling by ER variants may affect Ghrh neuron counterregulatory neurotransmission in each sex.

It is worthwhile considering that evidence for age-associated alterations in ER gene expression described here does not serve as conclusive confirmation that corresponding gene protein product production is likewise changed in a similar direction and magnitude. The plausible assumption that aging may elicit tandem adjustments ER gene versus protein expression under baseline and/or hypoglycemic conditions, will require application of proteomic analysis methods of requisite sensitivity for quantification of ER protein levels in single brain cell samples.

Administration of Ghrh siRNA to the old male rat VMN disclosed a loss of Ghrh neuromodulatory stimulation of VMNdm Ghrh nerve cell ER$\alpha$, but not ER$\beta$ or GPER gene expression. Age-associated reductions in baseline ER$\alpha$, but not GPER gene profiles in this sex may reflect Ghrh-dependent and -independent mechanisms, respectively. As Ghrh and Ghrh receptor mRNAs in this nerve cell type are respectively unaltered or decreased with age, attenuation of Ghrh control of ER$\alpha$ mRNA content may involve reduced Ghrh receptor activation of signaling pathways that regulate this ER gene profile. On the other hand, old female rats exhibit loss of Ghrh regulation of VMdm Ghrh ER$\beta$ and GPER gene profiles, but not ER$\alpha$ gene expression. As Ghrh and Ghrh receptor gene transcription are correspondingly diminished or refractory to aging, maintenance of ER$\beta$ gene profiles at levels observed in young animals may involve substitution of an alternative stimulus. Age-related down-regulated ER$\alpha$ and GPER gene expression in this sex may reflect loss of Ghrh positive tone in the former instance and age-associated emergence of a dominant inhibitory input in the latter scenario.

Co-expression of nuclear and membrane ER variants enables integrative estradiol regulation of VMNdm Ghrh neuron function [76, 77, 78]. It is well documented that the nuclear transcription factors ER$\alpha$ and ER$\beta$ play overlapping as well as unique roles in estradiol action in vivo as these variants exert dissimilar transcriptional activities in ligand-, cell type-, and promoter-specific contexts [79, 80, 81]. These ERs elicit nuclear or transcriptional effects by estrogen response element (ERE)-dependent and -independent mechanisms, which in the latter instance involves modulation of activities of other transcription factors such as activator protein-1, nuclear factor-kappa-B, and stimulating protein-1 [82]. Stimulation of membrane-anchored GPER causes cell type-specific rapid activation of signaling transduction cascades, namely phospholipase C/protein kinase, Ras/Raf/MAPK, phosphatidyl inositol 3 kinase/AKT, or cAMP/protein kinase A signaling cascades, which are capable of crosstalk with other signaling pathways [83, 84, 85, 86]. Assimilation of extra-nuclear and nuclear estradiol regulatory effects will plausibly depend upon which signaling pathway(s) is/are activated and resulting phosphorylation of co-activators [87, 88, 89, 90]. Fundamental questions that emerge from this and other recent work on VMNdm Ghrh neurons include the identity of GPER-activated signal transduction pathway(s) in these cells in young animals and potential effects of aging in each sex on pathway responsiveness to receptor stimulation.

Synchronized quantification of individual target gene profiles by multiplex qPCR analysis is a valuable tool for evaluation of relative expression of diverse ER transcripts within a common cellular source. Here, compilation of relative ER$\alpha$, ER$\beta$, and GPER mRNA expression ratios for VMNdm Ghrh neurons acquired from euglycemic, namely SCR siRNA-pretreated, V-injected old rats indicates that in each sex, GPER gene transcription in this nerve cell type is proportionately higher compared to ER$\alpha$ and ER$\beta$. This indicates change from young adulthood where this nerve cell type is primarily subject to ER$\beta$-mediated estradiol control. These data show that aging may significantly affect integration of estradiol input to VMNdm Ghrh nerve cells in each sex. Ghrh gene silencing here had profound sex-specific effects on mean ER variant gene expression ratios, as this genetic manipulation decreased proportional GPER gene expression in old males yet increased relative expression of all three ER variant mRNAs in the aged female while bringing ER$\beta$ and GPER expression ratios into closer alignment. These effects contrast with minimal effects of this treatment on relative ER variant gene expression in young animals of either sex. These results document an aging-associated gain in Ghrh inhibition of proportionate GPER gene expression (male) versus indiscriminate up-regulation of relative ER variant expression (female).

Data here show that IIH reduced proportionate GPER gene expression in old male rats, i.e., SCR siRNA/INS versus SCR siRNA/V treatment groups, without major effects on other ER variant mRNAs. This represents a change from young male rats, which exhibit pronounced down-regulated proportionate ER$\alpha$, ER$\beta$, and GPER gene expression. Thus, aging may cause a loss of hypoglycemic control of relative nuclear ER receptor gene transcription in this sex. Ghrh siRNA administration prior to hypoglycemia further reduced relative GPER gene expression in male rats. Results from old female rat VMNdm Ghrh neurons show that hypoglycemia elevated relative ER$\alpha$ and ER$\beta$ gene profiles, but did not alter the mean GPER expression ratio. Conversely, young INS-injected animals exhibit decreased proportionate ER$\alpha$ and GPER mRNA expression. Ghrh gene knockdown further augmented ER$\alpha$ and ER$\beta$ gene expression ratios and increased GPER relative expression compared to SCR siRNA-pretreated hypoglycemic old female rats. This pretreatment had similar effects on each ER variant relative expression in young female rats. Taken together, these results suggest that aging has differential effects on integrated estradiol regulation of VMNdm Ghrh neuron function during hypoglycemia, as relative ER variant gene expression is brought into alignment (as opposed to young animals) by reductions in proportional GPER gene expression in old males versus amplification of ER$\alpha$ and ER$\beta$ gene expression ratios in aged females.

The continuum of development over the male or female lifespan involves transition between distinct reproductive states, including reproductive senescence, which are typified by unique gonadal steroid hormone secretion patterns. Current findings show that VMNdm Ghrh neuron neuroestradiol production is also apparently age sensitive as CYP19A1 gene profiles are enhanced in old versus young rats of either sex. As noted above, further experimentation is required to verify that aging amplifies CYP19A1 protein and enzyme activity profiles in this neuron population in each sex. Interestingly, eu- and hypoglycemic patterns of CYP19A1 transcription are each up-regulated in response to VMN Ghrh gene silencing, which strongly suggests that this neuropeptide transmitter imposes a negative, inhibitory tone on this mRNA profile in each sex. The literature, as ably summarized by Cruz et al. [91], documents a progressive decline in female rat fertility between the ages of 8 to 12 months, characterized by reduced numbers of developing ovarian follicles and corpora lutea alongside increasing perturbances in estrous cyclicity, such as increased length, and development of constant estrous. Constant estrous, which occurs at 10 months and onward, is typified by low, unvarying circulating estradiol, estrone, testosterone, and progesterone levels, and an increase in estradiol/progesterone ratio. Female rats 12 months of age or older are reported to lack corpora lutea, a sign of absence of ovulation. While it is presumed that female subjects of similar age used in current work exhibited the attributes described above, the lack of direct verification of that supposition is a limitation. It is noted that potential changes in ovarian-derived estradiol signal volume to the aging female rat brain apparently coincide with possible augmentation of locally-generated neuroestradiol, as discussed above. Clearly, further studies are needed to quantify effects of age in both sex on net VMN tissue estradiol concentrations.

VMN is a known substrate for estradiol regulation of glucose homeostasis. Current work used combinative single-cell laser-catapult-microdissection/multiplex qPCR tools to establish if and how aging affects total and proportional VMN counterregulatory neuron ER variant gene expression in each sex. Results indicate that aging affects absolute ER$\alpha$ and GPER, but not ER$\beta$ mRNA levels in each sex, and abolished ER$\alpha$ (both sexes), ER$\beta$ (males), and GPER (males) transcriptional responses to hypoglycemia. Outcomes of VMN Ghrh gene silencing identify sex-specific age-related loss of neuropeptide control of total expression of discrete ER genes. Data also infer that aging likely affects integration of estradiol input to VMNdm Ghrh neurons during eu- and hypoglycemia. Euglycemic old animals, unlike young rats, exhibit higher relative baseline GPER gene transcription compared to ER$\alpha$ and ER$\beta$. Moreover, with age, hypoglycemia causes sex-specific equalization of proportionate ER variant gene expression, as males show reductions in proportional GPER gene expression occur whereas ER$\alpha$ and ER$\beta$ relative expression ratios are increased in females. Current evidence for possible age-associated increases in VMNdm Ghrh neuroestradiol synthesis infers that autocrine stimulation of ER$\beta$ may be amplified by age. Additional effort is needed to investigate how age-associated modifications in absolute and proportionate signaling by distinctive ER may affect Ghrh neuron into to the neural counterregulatory network.

CYP19A1, aromatase; ER$\alpha$/ESR1, estrogen receptor-alpha; ER$\beta$/ESR2, estrogen receptor-beta; Ghrh, growth hormone-releasing hormone; Ghrh-R, Ghrh receptor; GPER, G protein-coupled estrogen receptor-1; IIH, insulin-induced hypoglycemia; INS, insulin; sc, subcutaneous; VMN, ventromedial hypothalamic nucleus; VMNdm, VMN dorsomedial division.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

RS: conceptualization, investigation, formal analysis, validation, data curation, visualization, writing—original draft, writing—review and editing, visualization; SS: investigation, formal analysis, validation, data curation; KPB: conceptualization, writing—original draft, writing—review and editing, supervision, project administration, funding acquisition. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal experimentation was carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th Edition, as stated in the submitted manuscript. Study protocols and methods were approved by the University of Louisiana Monroe Institutional Committee on Animal Care and Use, Approval number 24NOV-KPB-01. Sex of animals used is included, along with discussion of sex impacts on study outcomes.

This research was supported by NIH grant DK-109382.

The authors declare no conflict of interest.