Maternal ethanol administration increases BLBP mRNA in NEP+mHYP more strongly in female than male embryos
This experiment tested using qRT-PCR the effects of maternal intraoral ethanol administration compared to Control and Untreated control groups on gene expression of BLBP in the NEP+mHYP of female and male embryos. There was a significant main effect of ethanol treatment (F(2,36) = 39.36, p < 0.001), in addition to an effect of sex (F(1,36) = 20.73, p < 0.001) and a sex x treatment interaction (F(2,36) = 9.96, p < 0.001) (Fig. 2). While there were no differences between respective control groups of females and males, the BLBP expression was higher in ethanol-exposed females compared to ethanol-exposed males (p < 0.001). Furthermore, ethanol significantly increased BLBP mRNA levels in both sexes compared to the Control (p < 0.001 for female and p = 0.004 for male) and Untreated (p < 0.001 for female and p = 0.018 for male) groups, with this effect significantly greater in females than males (t(12) = 3.49, p = 0.004). There was no significant main effect of maternal ethanol on the body weight of dams at E19 (F(2,18) = 0.456, p = 0.641) and their E19 embryos (F(2,18) =1.117, p = 0.349) and also no effect on dam’s daily chow intake (F(2,18) = 1.156, p = 0.337) and litter size (F(2,18) = 0.405, p = 0.405) (Table 4). Together, these results show that maternal ethanol administration at a moderate dose has a stimulatory effect on the expression of radial glia progenitor cells in the NEP+mHYP, with female embryos exhibiting greater sensitivity to ethanol.
Table 4 Body weights of embryos and dams and the chow intake and litter size of dams.
Ethanol
|
|
|
|
|
Untreated
|
Control
|
Ethanol
|
Body weight – E19 (g)
Body weight – Dams (g)
|
6.670 ± 0.39
295.5 ± 8.5
|
6.120 ± 0.16
285.0 ± 12.3
|
6.310 ± 0.19
282.1 ± 10.3
|
Chow intake (kcal/day)
|
71.90 ± 2.9
|
70.70 ± 3.2
|
65.40 ± 3.4
|
Litter size
|
11.70 ± 1.0
|
12.40 ± 0.7
|
12.70 ± 0.6
|
CCL2
|
|
|
|
|
Untreated
|
Control
|
CCL2
|
Body weight – E19 (g)
Body weight – Dams (g)
|
6.200 ± 0.28
292.1 ± 7.4
|
6.000 ± 0.17
277.3 ± 5.0
|
6.040 ± 0.18
279.1 ± 6.5
|
Chow intake (kcal/day)
|
66.70 ± 4.1
|
68.40 ± 3.2
|
64.10 ± 4.4
|
Litter size
|
11.70 ± 1.2
|
12.40 ± 0.7
|
12.70 ± 0.6
|
Ethanol + INCB
|
|
|
|
|
Control
|
Ethanol
|
Ethanol + INCB3344
|
Body weight – E19 (g)
Body weight – Dams (g)
|
6.290 ± 0.23
280.3 ± 8.7
|
6.070 ± 0.23
273.9 ± 11.0
|
6.160 ± 0.13
282.7 ± 9.1
|
Chow intake (kcal/day)
|
75.30 ± 4.7
|
72.20 ± 3.0
|
69.50 ± 2.7
|
Litter size
|
11.30 ± 1.0
|
11.60 ± 1.3
|
11.00 ± 1.2
|
Data are mean ± SEM. n = 7/group, *p < 0.05 versus control. Two-way ANOVA was used to compare means between groups and showed no significant effect of maternal administration of ethanol, CCL2 or CCR2 antagonist, INCB3344, on these measures. Abbreviations: kcal/day, kilocalories per day; g, grams; INCB3344, CCR2 antagonist.
Maternal ethanol administration stimulates radial glia cells in NEP and processes in mHYP more strongly in female than male embryos
We next tested using IF the effect of maternal administration of ethanol compared to Control and Untreated control groups on BLBP+ neuroprogenitor cells in the NEP and mHYP of female and male embryos. The BLBP+ radial glia cells were detected in the hypothalamic NEP and highly concentrated along the border of the third ventricle while very sparse in the mHYP. These cells have long, well-defined processes which project laterally through the mHYP in the direction of the LH. Maternal ethanol had strong stimulatory and sexually dimorphic effects on these cells and their processes. There was a significant main effect of ethanol treatment on the density of BLBP+ cells in the NEP (F(2,36) = 38.54, p < 0.001) and BLBP+ processes in the mHYP (F(2,36) = 34.69, p < 0.001), along with an effect of sex on BLBP+ cells (F(1,36) = 16.10, p < 0.001) and processes (F(2,36) = 34.69, p < 0.001) and a sex x treatment interaction for BLBP+ cells (F(2,36) = 5.18, p = 0.011) and processes (F(2,36) = 9.61, p < 0.001) (Fig. 3a), as illustrated in the photomicrographs (Fig. 3b). While there were no differences between respective control groups of females and males, ethanol-exposed females compared to ethanol-exposed males had a significantly higher density of the BLBP+ cells in the NEP (p < 0.001) and BLBP+ processes in the mHYP (p < 0.001). Also, maternal ethanol had a significant, stimulatory effect in both sexes on the density of BLBP+ cells in the NEP compared to the Control (p < 0.001 for female and p < 0.001 for male) and Untreated (p < 0.001 for female and p = 0.003 for male) groups and the density of BLBP+ processes in the mHYP compared to the Control (p < 0.001 for female and p = 0.013 for male) and Untreated (p < 0.001 for female and p = 0.036 for male) groups. However, the increase in density of BLBP+ cells was significantly greater in females than males compared to the Control (t(12) = 3.058, p = 0.010) and Untreated (t(12) = 5.0144, p = 0.021) groups, similar to the increase in density of BLBP+ processes compared to the Control (t(12) = 4.874, p < 0.001) and Untreated (t(12) = 3.353, p = 0.006) groups. Together, these results show that maternal ethanol administration at a moderate dose has a strong stimulatory effect on radial glia cells in the hypothalamic NEP and their processes in the mHYP, which are sexually dimorphic, consistently stronger in females than males.
Maternal ethanol administration stimulates CCL2 and CCR2 mRNA in NEP and mHYP predominantly in female embryos
Next we examined the CCL2/CCR2 system in the NEP and mHYP of female and male embryos and tested the effect of maternal ethanol administration compared to Control and Untreated control groups on gene expression of this chemokine and its receptor using qRT-PCR. There was a significant main effect of ethanol treatment on CCL2 (F(2,36) = 15.88, p < 0.001) and CCR2 (F(2,36) = 22.23, p < 0.001), along with an effect of sex on CCL2 (F(1, 36) = 9.78, p = 0.003) and CCR2 (F(2,36) = 17.99, p < 0.001) and a significant interaction between sex and ethanol treatment for CCL2 (F(2,36) = 4.73, p = 0.015) and CCR2 (F(2,36) = 4.22, p = 0.023) (Table 5). While again there were no differences between respective control groups of females and males, ethanol-exposed females exhibited a greater expression of CCL2 (p < 0.001) and CCR2 (p = 0.002) compared to males. Also, maternal ethanol administration compared to control groups significantly increased CCL2 mRNA levels in female embryos (p < 0.001 for Control and p < 0.001 for Untreated) but had no effect in male embryos (p = 0.067 for Control and p = 0.298 for Untreated). In addition, while ethanol significantly increased CCR2 mRNA levels in both sexes compared to their Control (p < 0.001 for female and p = 0.037 for male) and Untreated (p < 0.001 for female and p = 0.016 for male) groups, ethanol-induced increase in CCR2 mRNA in females was significantly greater than in males (t(12) = 3.738, p = 0.003) (Table 5). Together, these results demonstrate that maternal ethanol increases the expression of CCL2 only in female but not male embryos and of CCR2 in both sexes although more strongly in females.
Table 5 Effects of maternal ethanol administration on mRNA of CCL2 and CCR2 in NEP+mHYP area in E19 embryos
mRNA
|
Female
|
|
|
|
Male
|
|
|
Expression
|
Untreated
|
Control
|
Ethanol
|
|
Untreated
|
Control
|
Ethanol
|
CCL2
CCR2
|
1.2E-3 ± 5.7E-5
4.7E-3 ± 2.3E-4
|
1.2E-3 ± 5.1E-5
4.5E-3 ± 2.1E-4
|
1.9E-3 ± 4.0E-5*#
6.7E-3 ± 3.1E-4*#
|
|
1.3E-3 ± 6.1E-5
4.6E-3 ± 4.0E-4
|
1.3E-3 ± 5.5E-5
4.2E-3 ± 2.3E-4
|
1.4E-3 ± 5.7E-5
5.0E-3 ± 2.1E-4*
|
Effect of maternal administration of ethanol (2 g/kg/day, E10-E15) on mRNA levels of CCL2 and CCR2 was measured using qRT-PCR. Data are mean ± SEM. (n = 7/group/sex, *p < 0.05 versus Control). Two-way ANOVA was used to compare means between groups, simple main effect analyses to test differences between sexes as well as differences within each sex, and paired t-tests to directly compare within each sex the effects of maternal treatment vs control groups. Abbreviations: NEP, hypothalamic neuroepithelium; mHYP, medial hypothalamus; qRT-PCR, real time quantitative PCR.
Maternal ethanol administration stimulates the density of CCL2 cells in NEP and processes in mHYP predominantly in female embryos
This experiment tested the effect of moderate maternal exposure to ethanol on the density of CCL2+ cells in E19 embryos. Using IF, we detected in E19 embryos a high concentration of CCL2+ cells in the NEP along the border of the third ventricle, with very few evident in the mHYP, and found these CCL2+ cells to be far denser in the NEP than the LH [23], with some having a distinct shape of short, lightly-stained processes projecting laterally into the mHYP. Maternal ethanol administration had sexually dimorphic, stimulatory effects on both the CCL2+ cells and processes (Fig. 4a), as illustrated in the photomicrographs (Fig. 4b). There was a significant main effect of ethanol treatment on the density of CCL2+ cells in the NEP (F(2,36) = 18.04, p < 0.001) and of CCL2+ processes in the mHYP (F(2,36) = 23.30, p < 0.001), along with an effect of sex on CCL2+ cells (F(1,36) = 28.50, p < 0.001) and processes (F(1,36) = 39.46, p < 0.001) as well as a significant sex x treatment interaction on CCL2+ cells (F(2,36) = 5.84, p < 0.001) and processes (F(2,36) = 10.62, p < 0.001). While there were no differences between respective control groups of females and males, ethanol-exposed females had a significantly greater density of CCL2+ cells in the NEP (p < 0.001) and CCL2+ processes (p < 0.001) in the mHYP. Ethanol treatment significantly increased in females the density of CCL2+ cells in the NEP compared to the Control (p < 0.001) and Untreated (p < 0.001) groups, while having no effect on CCL2+ cells in the NEP of male embryos compared to the Control (p = 0.732) and Untreated (p = 0.080) groups. Similarly, ethanol also increased in females the density of CCL2+ processes in the mHYP compared to the Control (p < 0.001) and Untreated (p < 0.001) groups, while having no effect on the processes in the mHYP of male embryos compared to the Control (p = 0.251) and Untreated (p = 0.108) groups. Together, these measurements of mRNA and protein show that exposure of the embryo to ethanol at a moderate dose, while increasing CCR2 mRNA in both sexes but more strongly in females, stimulates CCL2 in the NEP and mHYP only in the female embryos.
Maternal administration of CCL2 stimulates BLBP+ and CCL2+ cells in the NEP and processes in the mHYP
In this experiment, the dams were either untreated (Untreated) or given daily injections (s.c., E10-E15) of CCL2 at 4 µg/kg/day (CCL2) compared to sterile water vehicle (Control). We tested in female E19 embryos using IF whether CCL2 administration, similar to ethanol as shown above, also stimulates radial glia in the NEP and mHYP along with endogenous CCL2/CCR2 signaling. There was a significant main effect of maternal CCL2 injection on the density of BLBP+ (F(2,20) = 23.41, p < 0.001) and CCL2+ (F(2,20) = 15.59, p < 0.001) cells in the NEP and of BLBP+ (F(2,20) = 7.525, p = 0.004) and CCL2+ (F(2,20) = 11.857, p < 0.001) processes in the mHYP (Fig. 5a), as illustrated in the photomicrographs (Fig. 5b). Maternal CCL2 significantly increased in the NEP the density of BLBP+ cells compared to Control (p < 0.001) and Untreated (p < 0.001) groups and of CCL2+ cells compared to Control (p < 0.001) and Untreated (p < 0.001) groups. It also increased the density of BLBP+ processes compared to Control (p < 0.001) and Untreated (p = 0.013) groups and of CCL2+ processes compared to Control (p = 0.004) and Untreated (p < 0.001) groups. Examination of the effect of maternal CCL2 on body weights revealed no significant main effect in the dams at E19 (F(2,18) = 1.921, p = 0.175) or the E19 embryos (F(2,18) = 0.251 p = 0.781) and also no effect on dam’s chow intake (F(2,18) = 0.309, p = 0.738) and litter size (F(2,18) = 0.405, p = 0.673) (Table 4). These results confirm that maternal administration of CCL2 mimics the effects of maternal ethanol, stimulating the cells and processes of both radial glia and CCL2 in the hypothalamic NEP and mHYP.
Maternal administration of CCR2 antagonist blocks ethanol’s stimulatory effects on BLBP and CCL2 mRNA
With CCR2 shown to be involved in the stimulatory effect of maternal ethanol administration on CCL2 neurons in the LH [14, 23], we next tested using qRT-PCR whether blockade of this receptor with maternal administration of the CCR2 receptor antagonist INCB3344 during the period of ethanol exposure also affects ethanol’s stimulatory effects on the radial glia and CCL2/CCR2 system in the NEP and mHYP. We examined female embryos at E19 in the following four groups: Isocaloric Control + Vehicle (Control); Ethanol + Vehicle (Ethanol); Ethanol + INCB3344; and Control + INCB3344. There was a significant main effect of treatment on mRNA levels of BLBP (F(3,27) = 9.051, p < 0.001) and CCL2 (F(3,27) = 14.795, p < 0.001) as well as CCR2 (F(3,27) = 6.690, p = 0.002) in the NEP+mHYP area (Table 6). Maternal ethanol compared to Control significantly increased the expression of BLBP (+43%, p < 0.001) and CCL2 (+57%, p < 0.001) as well as of CCR2 (+49%, p = 0.002), and these effects were blocked by maternal administration of the CCR2 antagonist, causing a decrease in mRNA expression of BLBP (p < 0.001), CCL2 (p < 0.001), and CCR2 (p = 0.006) to the same levels as those in the Control group (p = 0.336, p = 0.641, and p = 0.483, respectively). The antagonist alone was found to have no effect of its own on these measures, with no differences evident between the Control + INCB3344 and the Control groups in their expression of BLBP (p = 0.956), CCL2 (p = 0.912), and CCR2 (p = 0.513). Consistent with our previous findings [13], maternal administration of CCR2 antagonist had no significant main effect on the body weight of dams at E19 (F(3,24) = 0.315, p = 0.906) or their E19 embryos (F(3,24) = 0.184, p = 0.906) and also no effect on the dam’s chow intake F(3,24) = 0.372, p = 0.774) and litter size (F(3,24) = 0.405, p = 0.750) (Table 4). These findings support the involvement of the endogenous CCR2 receptor in mediating ethanol’s stimulatory effect on the radial glia and CCL2/CCR2 system in the NEP and mHYP.
Table 6 Effects of ethanol and CCR2 antagonist, INCB3344, on BLBP, CCL2, and CCR2 mRNA in NEP+mHYP area
mRNA
expression
|
Control
|
Ethanol
|
Ethanol+INCB3344
|
Control+INCB3344
|
BLBP
CCL2
|
2.3E-1 ± 9.4E-3
1.3E-3 ± 4.9E-5
|
3.3E-1 ± 2.2E-2*
2.0E-3 ± 1.5E-4*
|
2.5E-1 ± 1.9E-2
1.4E-3 ± 6.9E-5
|
2.3E-1 ± 6.4E-3
1.2E-3 ± 5.2E-5
|
CCR2
|
4.5E-1 ± 3.4E-3
|
6.7E-3 ± 5.4E-4*
|
4.8E-3 ± 4.1E-4
|
4.1E-3 ± 4.8E-4
|
Maternal ethanol administration group (2 g/kg/day, E10-E15) with and without INCB3344 treatment (1 mg/kg/day, i.p.) was compared to Untreated and isocaloric Control groups of embryos at E19. Data are mean ± SEM, n = 7/group, *p < 0.05 versus Control, Ethanol + INCB3344, and Control + INCB3344 groups. Difference between groups was analyzed using one-way ANOVA followed by SDS post-hoc test. Abbreviations: BLBP, brain lipid-binding protein; NEP, neuroepithelium.
Maternal administration of CCR2 antagonist blocks ethanol’s stimulatory effects on BLBP and CCL2 cells and processes.
Here we used single-label IF to test whether ethanol’s stimulatory effects on BLBP+ and CCL2+ cells in the NEP and their processes in the mHYP are blocked by maternal administration of the CCR2 receptor antagonist. We examined female embryos at E19 from dams in the following four groups: Isocaloric Control + Vehicle (Control); Ethanol + Vehicle (Ethanol); Ethanol + INCB3344; and Control + INCB3344. We found a significant main effect of treatment on the density of BLBP+ (F(3,27) = 10.228, p < 0.001) and CCL2+ (F(3,27) = 5.572, p = 0.005) cells and of BLBP+ (F(3,27) = 9.481, p < 0.001) and CCL2+ (F(3,27) = 18.543, p < 0.001) processes (Fig. 6a), as illustrated in the photomicrographs (Fig. 6b). Ethanol compared to Control group caused a significant increase in the density of BLBP+ (+36%, p < 0.001) and CCL2+ (+53%, p < 0.001) cells in the NEP and of BLBP+ (+31%, p < 0.001) and CCL2+ (+67%, p < 0.001) processes in the mHYP. These effects were reversed by INCB3344 treatment, causing a significant decrease in the Ethanol + INCB3344 compared to Ethanol group in the density of BLBP+ cells (p = 0.004) and processes (p = 0.002) and CCL2+ cells (p = 0.006) and processes (p< 0.001) that were reduced to the same levels as those in the Control group for BLBP+ cells (p = 0.471) and processes (p = 0.639) and CCL2+ cells (p = 0.561) and processes (p = 0.310). There were no differences between the Control + INCB3344 group and the Control group in the measures of BLBP+ (p = 0.820) and CCL2+ (p = 0.538) cells and BLBP+ (p = 0.406) and CCL2+ (p = 0.164) processes. These results demonstrate the importance of CCR2 in mediating ethanol’s stimulatory effects on the cells and processes of radial glia in the NEP and mHYP.
Maternal ethanol stimulates co-labeling of CCL2 in radial glia cells and neurons
Building on our published evidence that maternal ethanol stimulates CCL2 in the LH predominantly in neurons but not in
microglia or astrocytes [23], we tested here using double-labeling IF whether CCL2 in the NEP and mHYP stimulated by ethanol are also in neurons as well as radial glia. Using BLBP as the marker of radial glia, NeuN to label neurons, and Iba-1 to label microglia, we used double-labeling IF to examine in female E19 embryos the co-labeling in the NEP and mHYP of CCL2 with BLBP, NeuN and Iba-1. Maternal ethanol exposure stimulated all three cell types, radial glia, neurons, and microglia, in the NEP and mHYP (Table 7). Confirming the above results, analyses of single-labeled cells and processes
showed that maternal administration of ethanol compared to the Control group significantly increased the density of CCL2+ cells in the NEP (p = 0.014) and processes in the mHYP (p < 0.001) and of BLBP+ cells in the NEP (p = 0.002) and processes in the mHYP (p = 0.016). They further revealed a stimulatory effect of ethanol on the density of NeuN+ neurons, which were most concentrated along the periventricular region of the NEP (p = 0.010) while scattered throughout the mHYP (p = 0.026), and also of Iba-1+ microglia, which were detected along the lateral border of the NEP (p = 0.009) as well as scattered in the mHYP (p <0.001) (Table 7).
Analysis of the double-labeled cells and processes show CCL2 to colocalize with BLBP in radial glia and NeuN in neurons and maternal ethanol to stimulate their colocalization but fail to reveal any colocalization of CCL2 with Iba-1 in microglia under any condition (Fig. 7a), as illustrated in the photomicrographs (Fig. 7b). Ethanol compared to Control significantly increased the percentage of double-labeled CCL2+/BLBP+ cells in the NEP relative to single-labeled BLBP+ (t(10) = -32.05, p < 0.001) and CCL2+ (t(10) = -10.21, p < 0.001) cells and of double-labeled CCL2+/BLBP+ processes in the mHYP relative to single-labeled BLBP+ (t(10) = -10.374, p < 0.001) and CCL2+ (t(10) = -8.359, p < 0.001) processes. These effects on double-labeled radial glia, evident in the 20x photomicrographs of Ethanol compared to Control groups, are more clearly illustrated in the 40x images to the right with arrowheads identifying the radial glia cells and processes (Fig. 7b). In addition, as described in the LH [14, 23], Ethanol compared to Control significantly increased the percentage of double-labeled CCL2+/NeuN+ neurons in the NEP relative to single-labeled NeuN+ neurons (t(10) = -32.046, p < 0.001) and CCL2+ cells (t(10) = -10.220, p < 0.001), and these double-labeled neurons were concentrated along the periventricular border of the NEP while very sparse in the mHYP, as illustrated in the 20x photomicrographs and identified by arrowheads in 40x images to the right (Figs. 7b). This is in contrast to the Iba-1+ microglia, which while stimulated by ethanol showed no double-labeling of CCL2 as illustrated by the representative 20x images (Fig. 7b). This analysis of CCL2+ cells in the hypothalamic NEP and mHYP demonstrates that, while few of the radial glia, neurons and microglia contain CCL2 under control conditions, ethanol exposure stimulates all three cell types in the NEP and mHYP and increases the colocalization of CCL2 in ~60% of the radial glia and neurons while producing no colocalization in the microglia.
Table 7 Effects of ethanol on density of cells or processes that label CCL2, BLBP, NeuN or Iba-1 in NEP and mHYP
Labeled Cells/Processes
|
Location
|
Control (cells,objects/µm2)
|
Ethanol (cells,objects/µm2)
|
CCL2+
|
NEP
|
6.30E-5 ± 2.67E-6
|
7.50E-5 ± 2.07E-6*
|
|
mHYP
|
9.90E-5 ± 7.30E-6
|
1.70E-4 ± 1.46E-5*
|
BLBP+
|
NEP
|
1.51E-4 ± 4.62E-6
|
1.83E-4 ± 5.67E-6*
|
|
mHYP
|
1.59E-4 ± 1.32E-5
|
2.31E-4 ± 2.13E-5*
|
NeuN+
|
NEP
|
5.75E-5 ± 2.77E-6
|
7.92E-5 ± 7.24E-6*
|
|
mHYP
|
4.28E-5 ± 2.68E-6
|
5.17E-5 ± 2.64E-6*
|
Iba-1+
|
NEP
|
3.79E-5 ± 1.84E-6
|
4.73E-5 ± 1.79E-6*
|
|
mHYP
|
4.47E-5 ± 2.14 E-6
|
6.06E-5 ± 4.27E-6*
|
Maternal administration of ethanol (2 g/kg/day, E10-E15) was compared to an isoaloric maltose-dextrin control solution (Control) group in the NEP and mHYP areas of female E19 embryos, measured using single-labeling IF. Data are mean ± SEM, n = 6/group, *p < 0.05 versus Control. The difference between groups was analyzed using student t-test. Abbreviations: BLBP, brain lipid-binding protein; CCL2, C-C motif ligand 2; Iba-1, microglial marker; mHYP, medial hypothalamus; NEP, hypothalamic neuroepithelium; NeuN, neuronal marker.
Maternal ethanol increases MCH-expressing neurons in the NEP and mHYP of female more than male embryos
To investigate whether ethanol’s stimulatory effects on these CCL2 cells in the NEP are related to its effects on MCH neurons in the LH that colocalize CCL2 and CCR2 [13, 14, 23], E19 embryos from two separate sets of dams were either untreated (Untreated) or received daily intraoral administration (from E10-E15) of either 2 g/kg/day of ethanol (Ethanol) or an isocaloric maltose-dextrin control solution (Control) were examined. Measurements of MCH expression in the NEP+mHYP area of female and male embryos from the first set of dams revealed a significant increase in mRNA levels using qRT-PCR, albeit at a much lower levels than found in the LH [23]. There was a significant main effect of ethanol treatment on MCH mRNA (F(2,36) = 29.913, p < 0.001), in addition to an effect of sex (F(1,36) = 9.933, p = 0.003) and a sex x ethanol treatment interaction (F(2,36) = 5.018, p = 0.012) (Fig. 8a). While there were no differences between respective control groups of females and males, the ethanol-exposed females exhibited a significantly greater expression of MCH than the males (p < 0.001). Further, while ethanol significantly increased MCH mRNA levels in both sexes compared to their Control (p < 0.001 for female and p = 0.014 for male) and Untreated (p < 0.001 for female and p = 0.005 for male) groups, direct comparisons between females and males showed this effect of ethanol on MCH expression to be significantly greater in female than male embryos compared to the Control (t(12) = 2.802, p = 0.016) and Untreated (t(12) = 4.152, p < 0.001) groups (Fig. 8a). Analysis of these MCH+ neurons using DIG-ISH in the Ethanol compared to Control female embryos from the second set of dams revealed a significant increase in their density in the NEP (t(10) = -9.76, p < 0.001) and mHYP (t(10) = -2.69, p = 0.023) (Fig. 8a), as illustrated in the photomicrographs (Fig. 8b). A few MCH+ neurons were detected in the NEP, although only in ethanol-exposed embryos with a denser concentration of these neurons in the mHYP. Thus, similar to its effect in the LH [14, 23], maternal ethanol significantly increased in the embryo mRNA expression of MCH, more strongly in females than males, and the density of MCH neurons in both the NEP and mHYP of females.
Maternal ethanol increases the number and percentage of MCH neurons close to radial glia in NEP and mHYP of embryos
To determine whether the appearance and patterning of MCH+ neurons are linked to the ethanol-induced stimulation of radial glia cells and processes, we used a combination of DIG-ISH to label MCH+ neurons and IF to label BLBP+ radial glia in female embryos of dams that received daily intraoral administration (from E10-E15) of either 2 g/kg/day of ethanol (Ethanol) or an isocaloric maltose-dextrin control solution (Control). Replicating the results described above, we found Ethanol compared to Control to increase the density of MCH+ neurons in the NEP (p < 0.001) and the mHYP (p = 0.034) and also to increase the density of BLBP+ cells in the NEP (p = 0.026) and BLBP+ processes in the mHYP (p = 0.048) (Table 8).
With the combination of DIG-ISH and BLBP IF, we discovered in female embryos from a separate set of dams that the individual MCH+ neurons are closely related to the radial glia in both the NEP and mHYP, as illustrated most clearly in the photomicrographs of ethanol-exposed embryos (Fig. 9). In the NEP where the MCH+ neurons are relatively sparse, Ethanol compared to Control group significantly increased their number along the edge of this midline region, from 0.0 to 4.14 ± 0.34 cells ((12) = -12.182, p < 0.001), in the area just lateral to the radial glia cells. Moreover, in the mHYP where the MCH+ neurons are more abundant, ethanol also significantly increased their number, from 112.0 ± 3.53 to 134.0 ± 5.31 cells (t(12) = -3.359, p = 0.006), in the area of the radial glia processes. This increase occurred in both the number of MCH+ neurons, from 9.29 ± 0.61 to 21.00 ± 0.98 (t(12) = -10.20, p < 0.001), and the percentage of total MCH+ neurons, from 8.31 ± 0.53 to 16.02 ± 1.00% (t(12) = -6.638, p < 0.001), that are positioned close to and along the BLBP+ processes as they project laterally through the mHYP toward the LH (Fig. 9a). These effects of ethanol are clearly evident in the enlarged image showing three MCH+ neurons (white arrowheads) along a single radial glia process (white arrows) in the mHYP (Fig. 9b). We confirmed this anatomical relationship between MCH+ neurons and a distinct BLBP+ process using double-labeled IF for BLBP and MCH and found in the mHYP and LH of an ethanol-exposed embryo a rich concentration of the radial glia processes (but few cells) and the MCH+ neurons (Fig. 9c). Many of these MCH+ neurons (green) were located close to the radial glia processes (red), as illustrated by the six neurons (white arrowheads) along a single BLBP+ process (white arrows). These results suggest that the stimulatory effect of ethanol on the density of MCH+ neurons originates within the embryonic NEP in close relation to the radial glia cells, and it involves the radial glia processes, along which the MCH neurons are positioned as they project laterally through the mHYP and continue into the LH, their final destination.
Table 8 Effects of ethanol on MCH and BLBP cells and processes in NEP and mHYP of female embryos
Labeled Cells/Processes
|
Location
|
Control (cells,objects/µm2)
|
Ethanol (cells,objects/µm2)
|
MCH+
|
NEP
|
3.77E-6 ± 2.47E-6
|
2.58E-5 ± 2.18E-6*
|
|
mHYP
|
4.21E-5 ± 1.89E-6
|
4.92E-5 ± 2.31E-6*
|
BLBP+
|
NEP
|
1.09E-4 ± 7.76E-6
|
1.31E-4 ± 6.44E-6*
|
|
mHYP
|
1.37E-4 ± 6.99E-6
|
1.65E-4 ± 8.69E-6*
|
Maternal administration of ethanol (2 g/kg/day, E10-E15) was compared to isoaloric maltose-dextrin control solution (Control) group in the NEP and mHYP areas of female embryos, measured using DIG-labeled in situ hybridization and immunofluorescence histochemistry. Data are mean ± SEM. n = 6/group, *p < 0.05 versus Control. One-way ANOVA was used to compare group means followed by SDS post-hoc test. Abbreviations: BLBP, brain lipid-binding protein; MCH, mHYP, medial hypothalamus; NEP, hypothalamic neuroepithelium.