Fibroblast growth factor 2: Role in prenatal alcohol-induced stimulation of hypothalamic peptide neurons

Abstract

Prenatal alcohol exposure (PAE) increases alcohol consumption and risk for alcohol use disorder. This phenomenon in rodents is suggested to involve a stimulatory effect of PAE, in female more than male offspring, on neurogenesis and density of neurons expressing neuropeptides in lateral hypothalamus (LH), including melanin-concentrating hormone (MCH), known to promote alcohol intake. With evidence suggesting a role for fibroblast growth factor 2 (FGF2) and its receptor FGFR1 in stimulating neurogenesis and alcohol drinking, we investigated here whether the FGF2-FGFR1 system is involved in the PAE-induced increase in MCH neurons, in postnatal offspring of pregnant rats given ethanol orally (embryonic day 10-15) at a low-moderate (2 g/kg/day) or high (5 g/kg/day) dose. Our results demonstrate that PAE at the low-moderate but not high dose stimulates FGF2 and FGFR1 gene expression and increases the density of MCH neurons co-expressing FGF2, only in females, but FGFR1 in both sexes. PAE induces this effect in the dorsal but not ventral area of the LH. Further analysis of FGF2 and FGFR1 transcripts within individual MCH neurons reveals an intracellular, sex-dependent effect, with PAE increasing FGF2 transcripts positively related to FGFR1 in the nucleus as well as cytoplasm of females but transcripts only in the cytoplasm of males. Peripheral injection of FGF2 itself (80 μg/kg. s.c.) in pregnant rats mimics these effects of PAE. Together, these results support the involvement of the FGF2-FGFR1 system in mediating the PAE-induced, sex dependent increase in density of MCH neurons, possibly contributing to increased alcohol consumption in the offspring.

1. Introduction

Alcohol use disorder (AUD) is a major health problem, impacting over 15 million adults in the USA and 76 million worldwide at great cost (WHO, 2014; Bose et al., 2016). The risk for alcohol-related behaviors and AUD is markedly increased by maternal consumption of alcohol during pregnancy (Malone et al., 2010; Caputo et al., 2016; Gupta et al., 2016). This phenemenon in the offspring, similarly evident in rodents as shown in studies from this laboratory (Chang et al., 2015; Chang et al., 2018) and others (Fabio et al., 2015; Wille-Bille et al., 2020), is believed to involve disturbances in neuropeptide (Poon et al., 2016) and neuroimmune (Drew et al., 2015; Terasaki and Schwarz, 2016; Coleman and Crews, 2018) systems in the lateral hypothalamus (LH). Investigations of the neuropeptide, melanin-concentrating hormone (MCH), which is densely expressed in neurons of the LH (Skofitsch et al., 1985) and positively linked to alcohol consumption and related behaviors (Duncan et al., 2005; Gonzalez-Burgos et al., 2006; Cippitelli et al., 2010; Morganstern et al., 2020), show that prenatal alcohol exposure (PAE) at low-moderate doses increases neurogenesis and density of MCH-immunoreactive neurons in the LH (Chang et al., 2012; Chang et al., 2018; Chang et al., 2020b). These effects of PAE, observed in the embryo as well as in postnatal and adolescent offspring, are accompanied by an increase in alcohol drinking, and this behavioral change similar to the neuronal effects is sex related, consistently stronger in females than males, suggesting a possible mechanism mediating specific sex differences in the fetal alcohol spectrum disorder (Baer et al., 2003; Alati et al., 2006; Malone et al., 2010). The ethanol-induced stimulation of MCH neurons is associated with an increase in proinflammatory factors, chemokines and their receptors (Chang et al., 2015; Terasaki and Schwarz, 2016; Chang et al., 2018; Chang et al., 2020a; Chang et al., 2020b), known to stimulate the motility and migration of neurons (Poon et al., 2016; Watson et al., 2020).

With recent evidence relating growth factors to alcohol drinking and behaviors associated with AUD (Liran et al., 2020) but little information available on how they respond to PAE and possibly mediate ethanol’s stimulatory effects on neuronal development (Singh et al., 1996; Feng et al., 2005), we focused here on fibroblast growth factor 2 (FGF2) and its receptor, fibroblast growth factor receptor 1 (FGFR1), which are localized in neuronal as well as non-neuronal cells in various brain regions including the LH (Gómez-Pinilla et al., 1994; Gonzalez et al., 1995; Ford-Perriss et al., 2001) and stimulate cell proliferation and differentiation during brain development (Dono et al., 1998; Ford-Perriss et al., 2001; Reuss and und Halbach, 2003). There is recent evidence positively relating this FGF2-FGFR1 system to alcohol drinking (Even-Chen et al., 2017; Even-Chen and Barak, 2019a, b; Even-Chen et al., 2021). In adult rodents, alcohol administration increases gene expression of FGF2 and its receptor FGFR1 in forebrain areas, the drinking of alcohol is stimulated by peripheral and forebrain injection of FGF2 in close association with dopamine D2-like receptor activation, and FGF2 deficiency and systemic administration of an FGFR1 antagonist or striatal infusion of an anti-FGF2 neutralizing antibody reduce alcohol consumption. While there are no animal studies of the FGF2-FGFR1 system involving in utero exposure to ethanol, there is a clinical report showing that PAE elevates circulating levels of FGF2 in children (Bodnar et al., 2020) and evidence in rats that FGF2 and FGFR1 colocalize with neuropeptides in hypothalamic neurons (Gonzalez et al., 2004; Rodrigo et al., 2010). Moreover, as with MCH, sex is shown to play a significant role in regulating FGF2 in the periphery (Larsson et al., 2002; Mao et al., 2010), with FGF positively related to estrogen (Zhang et al., 2016).

Building on evidence linking FGF2 to AUD (Even-Chen and Barak, 2019b), we investigate here in rats the involvement of the FGF2-FGFR1 system in mediating the stimulatory and sex-dependent effects of PAE on the development of MCH neurons in the LH.

2. Materials and Methods

All procedures were conducted in a fully accredited AAALAC facility, in accordance with protocols approved by The Rockefeller University Animal Care and Use Committee and consistent with the NIH Guide to the Care and Use of Laboratory Animals.

2.1. Animals

Adult female and male Sprague-Dawley rats, obtained from Charles River Breeding Laboratories, were maintained under standard lighting conditions (22 °C, 12:12-h light–dark cycle with lights off at 8 am), with food (LabDiet Rodent Chow 5001) and filtered water available ad libitum. The breeding of rats was performed to strictly control the prenatal environment, and all timed pregnant rats were subsequently housed individually, weight matched, and randomly assigned to experimental or control groups. After birth, the sex of postnatal day 5 (P5) offspring was determined through visual inspection of a larger genital papilla and longer ano-genital distance in male versus female offspring as described in (Liu et al., 2008), followed by sacrifice. In all experiments, 1 male and 1 female P5 offspring were taken from each litter, with the number of rats/sex/group (n = 4-6) equal to the number of litters/group for all experiments.

2.2. Maternal administration of ethanol

Pregnant rats (n = 4-6/experiment) were intraorally administered, from E10-E15 when MCH neurons develop in the hypothalamus (Brischoux et al., 2001), with either ethanol (30% v/v) at a low-moderate dose (2 g/kg/day) or high dose (5 g/kg/day) or a control solution of maltose-dextrin made isocaloric to the ethanol solution (control), as previously described (Chang et al., 2012). The daily dose of ethanol was split in half with all rats gavaged twice daily, with the first gavage occurring 2 h after start of the dark cycle and the second gavage occurring 7 h later. As reported in our previous publications, examination of the ethanol-exposed and control rats in this study revealed no differences in measures of the dams’ chow intake and body weight and of the size and body weight of their litters, with no spontaneous abortions. Also, with our prior studies of untreated groups repeatedly showing no differences from the control offspring, these untreated controls were not tested in the present study.

2.3. Maternal administration of FGF2

To examine the effects of maternal administration of FGF2 itself on MCH neurons and on FGF2 and FGFR1 in neurons of the LH, pregnant rats at E14 (n = 4) were peripherally injected with 80 ug/kg (s.c.) of human recombinant FGF2 (Sigma-Aldrich, Cat #SRP4037) or vehicle (PBS containing 0.1% BSA), as described (Even-Chen et al., 2017). This daily dose was split in half, with the first injection occurring 2 h after start of the dark cycle and the second injection occurring 7 h later. This injection of FGF2 compared to its vehicle had no effect on the dams’ chow intake and body weight or the body weight of their litters.

2.4. Quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) was used to measure the gene expression of MCH, FGF2 and FGFR1 in the whole LH of P5 offspring after maternal administration of ethanol, as previously described in ours (Barson et al., 2013; Chang et al., 2018) and other (West et al., 2017; Adachi et al., 2018) publications. Briefly, the P5 offspring were sacrificed by an experimenter blind to the experimental condition, and their brains were removed and immediately placed in a matrix, with the ventral surface facing up. For dissection of the LH, we made a 0.6 mm coronal slice with the anterior edge 0.6 mm posterior to the middle optic chiasm, at level A2.9-A2.3 mm of the 10-day-old rat brain atlas (Sherwood and Timiras, 1970). With this brain slice placed on a glass slide with the posterior surface facing up, the LH was dissected bilaterally under a microscope, with the medial cut at the lateral border of fornix, the lateral and dorsal cuts ~0.4 mm lateral and dorsal, respectively, to the fornix, and the ventral cut 0.1 mm dorsal to the ventral edge of the hypothalamus.

Total RNA was extracted from each microdissected sample, cDNA was synthesized, and qRT-PCR was performed as previously described (Barson et al., 2009; Chang et al., 2012). The primers, designed with ABI Primer Express Version 3.0 software from published sequences, were:(1) cyclophilin:5′ TGTGCTGAATATTGGTGCTTGTAA-3′ (forward) and 5′-TTGTGC TTTTCGGTATAGTGCTTT-3′ (reverse); (2) MCH: 5′-CAAACAGGATGGCGAAGATGA-3′ (forward) and 5′-AGGCTTTCCCCATCCTGAAT-3′ (reverse); (3) FGF2: 5′-GCGACCCACACGTCAAACTA-3′ (forward) and 5′-CCATCTTCCTTCATAGCCAGGTA-3′ (reverse); and (4) FGFR1: 5′-GACTTTGGCCTAGCTCGAGACA-3′ (forward) and 5′-CTCAGGTGCCATCCACTTCA-3′ (reverse). Concentration of the cyclophilin, FGF2 and FGFR1 primers was 200 nM and of the MCH primer was 50 nM, as previously described in ours (Barson et al., 2013; Chang et al., 2018) and other (West et al., 2017; Adachi et al., 2018) publications. Messenger RNA levels of the target gene in each offspring were normalized within subject relative to mRNA levels of an internal housekeeping gene, cyclophilin, and this ratio of target gene expression to housekeeping gene expression was calculated in each offspring using the standard delta-delta Ct method. The average ratio of target gene in PAE groups was compared to the average ratio in control, yielding the measure of “mRNA fold change” as reported in the results.

2.5. RNAscope in situ hybridization

For RNA in situ hybridization, we used RNAscope Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics), according to the manufacturer's instructions and as described previously (Li et al., 2015; Rubio et al., 2015), to provide a precise analysis of individual MCH neurons and their co-expression of FGF2 and FGFR1 transcripts. Briefly, P5 offspring were perfused intracardially with 0.9% normal saline followed by 4% paraformaldehyde (PFA) in PB. Their brains were removed and post-fixed overnight in the same fixative at 4°C, cryoprotected in 25% sucrose at 4°C for 72–96 h, and then frozen and stored at −80°C. Serial coronal brain sections containing the LH, from coronal plates A2.9 to 2.3 mm (Sherwood and Timiras, 1970), were collected at 16 um using a cryostat. Following the standard RNAscope pre-treatment procedure for fixed, frozen tissue (Wang et al., 2015), we then applied target probes for MCH, FGF2, and FGFR1 designed by Advanced Cell Diagnostics (catalog numbers: 563851-C3, 435391-C1 and 426951-C2, respectively) within a HybEZ Oven (Advanced Cell Diagnostics) at 40°C for 2 hours. Brain sections were then incubated in a series of amplifier probes (AMP1 for 30 min, AMP2 for 15 min, AMP3 for 30 min), followed by incubation in AMP4c at 40°c for 15 min to detect MCH (Alexa Fluor 488), FGF2 (Atto 550) and FGFR1 (Atto 647). Lastly, sections were incubated with DAPI for 30 seconds at RT and coverslipped with Prolong Diamond Antifade Mountant (ThermoFisher Scientific). The LH area and MCH neurons examined in this study (described below) are illustrated in Fig. 1, and for each RNAscope experiment, a probe against the bacterial gene dapB (310043) was used as the negative control, and a 3-plex control probe (320891) was used as the positive control, as illustrated in Fig. 2.

Figure 1:

Representative photomicrograph illustrating, at low magnification (2.5 x) and the anterior-posterior level of bregma A2.6 mm (Sherwood and Timiras, 1970) in PAE female, the lateral hypothalamus (LH) which lies between the internal capsule (IC), zona incerta (ZI), perifornical hypothalamus (PeF) outlined by dashed lines, and also the MCH neurons (green) within the LH examined in this study using RNAscope in situ hybridization. Within the LH, the dorsal (dLH) and ventral (vLH) areas are outlined by a line extending from the dorsal edge of the fornix (F) laterally to the lateral edge of the LH. Cells are counterstained by DAPI (blue), which is also seen in the ventricles (V) and along the lateral edge of the brain. Scale bar, 300 μm. Additional abbreviations: DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus.

Figure 2:

Photomicrographs illustrating the RNAscope positive and negative controls, with DAPI staining shown in blue. A 3-plex control probe was used as the positive control (Box 1), and a probe against the bacterial gene dapB was used as the negative control (Box 2). Scale bars: low magnification, 50 μm; high magnification, 10 μm. Abbreviations: dLH, dorsal lateral hypothalamus; vLH, ventral lateral hypothalamus.

2.6. Image acquisition and analysis

RNAscope images were acquired with an inverted Zeiss LSM 780 laser scanning confocal microscope, with a 40x oil immersion (1.2 NA) objective (z step = 1 μm) using tiling to cover the whole LH or a 100x oil immersion (1.4 NA) to acquire high magnification images of individual MCH neurons within the LH (z step = 0.5 μm). Raw images were then converted into Imaris files using Imaris File Converter 9.7.2 (Bitplane) and analyzed using Imaris 9.7.2 software (Bitplane). We analyzed the dense population of MCH neurons (Fig. 1) within the LH area dissected for qRT-PCR, which we have shown using immunofluorescence histochemistry to be strongly stimulated by PAE in postnatal offspring, as well as in the embryo and adolescent offspring (Barson et al., 2013; Chang et al., 2018; Chang et al., 2020b). Based on the position of the fornix, this LH area was further separated into the dorsal (dLH) and ventral (vLH) areas, by a line from the dorsal surface of the fornix that extends dorsolaterally to the lateral border of the LH (Fig. 1), as outlined by Hahn (Hahn, 2010).

Quantification of the different measures used in the examination of MCH neurons in the LH was permitted using the following procedures with Imaris software. All images were processed and analyzed under experimenter-blind conditions.

Density of MCH neurons:

The ‘Spots’ operation in Imaris was used on the 40x tiled images first to count the number of MCH neurons in the LH (estimated XY diameter for an MCH neuronal soma set to 7 μm for automatic detection, followed by manual correction). Then, to convert the total number of MCH neurons into density within regions of interest, namely, the whole LH, dLH and vLH, the total number of MCH neurons within an image was divided by the volume of these regions (calculated by multiplying X, Y, and Z dimensions of each image exported from Imaris). Only MCH cells containing nuclear DAPI staining were counted.

Number of MCH transcripts within each individual MCH neuron:

The ‘Spots’ operation in Imaris was used on 100x images (estimated XY diameter set to 1 μm, followed by manual selection of MCH transcripts per individual neuron). While the volume of MCH neurons was also evaluated using the ‘Surface’ function, we observed no changes in this measure throughout any condition, leading us not to report these data.

Density of cells expressing FGF2 and FGFR1:

The ‘Spots’ operation in Imaris was used to first detect these transcripts throughout the LH (estimated XY diameter set to 1 μm with a consistent threshold across images and conditions). This was followed by automatic selection of nuclear transcripts by colocalizing the transcripts with a ‘Surface’ object of DAPI. Then, the number of DAPI nuclei was quantified that contained two or more colocalized nuclear FGF2 or FGFR1 transcripts, with these cells defined as being positive for FGF2 or FGFR1 as described (Hultman et al., 2019). The density of these FGF2 and FGFR1 cells in the whole LH, the dLH and vLH was then calculated by dividing the number of these positive cells by the volume of these areas.

Density of MCH neurons expressing FGF2 and/or FGFR1:

The ‘Spots’ object for the FGF2 and FGFR1 transcripts was colocalized with the ‘Surface’ object of MCH neurons, and the total number of MCH neurons positive for FGF2 and/or FGFR1 transcripts, defined by having two or more FGF2 and/or FGFR1 transcripts, were quantified in the whole LH in addition to the dLH and vLH and were divided by the volume of these areas.

Expression level of FGF2 and FGFR1 transcripts in MCH neurons:

The ‘Spots’ object for the FGF2 and FGFR1 transcripts was colocalized with the ‘Surface’ object of MCH neurons, and the total number of FGF2 and FGFR1 transcripts within an individual MCH neuron was manually selected to create a new ‘Spots’ object denoted as ‘total transcripts’ in Imaris to quantify the number of FGF2 or FGFR1 transcripts per MCH neuron in the whole LH.

Intracellular distribution of FGF2 and FGFR1 transcripts in MCH neurons:

The nuclear transcripts (defined as transcripts that directly colocalize with DAPI) within individual MCH neurons in the whole LH were manually selected to create a new ‘Spots’ object denoted as ‘nuclear transcripts’ in Imaris. The remaining transcripts were manually selected out to create a new ‘Spots’ object denoted as ‘cytoplasmic transcripts’.

2.7. Experimental design

Effect of PAE on mRNA of MCH, FGF2 and FGFR1.

To determine the effect of low-moderate and high doses of PAE on mRNA levels of MCH, FGF2 and FGFR1 in the whole LH of postnatal offspring, the dams received daily intraoral infusions (from E10-E15) of either ethanol at 2g/kg/day, ethanol at 5 g/kg/day, or an isocaloric control solution. The gene expression of MCH, FGF2 and FGFR1 of female and male offspring (n = 6/group/sex) was measured using qRT-PCR.

Effect of PAE on density and transcript expression of MCH neurons.

With high-dose PAE having no effect on mRNA levels of MCH, FGF2 and FGFR1, we next examined using RNAscope the effects of low-moderate dose of PAE on the density and transcript expression level of MCH neurons in the LH of postnatal offspring. The dams received daily intraoral infusions (from E10-E15) of either ethanol (2 g/kg/day) or an isocaloric control solution. The density of MCH neurons, in the whole LH or the dLH and vLH, of female and male offspring (n = 4-6/group/sex) was determined by dividing the MCH neuron number by the volume of the respective area. The MCH transcript expression in the LH was determined by counting the number of MCH transcripts per MCH neuron.

Effect of PAE on density of FGF2 and FGFR1 cells.

To determine the effect of low-moderate PAE on the density of FGF2 and FGFR1 cells in the FH of postnatal offspring, the dams received daily intraoral infusions (from E10-E15) of either ethanol (2 g/kg/day) or the control solution. The density of FGF2 or FGFR1 cells, in the whole LH or the dLH and vLH of female and male offspring (n = 4-6/group/sex), was determined by dividing the number of DAPI cells with FGF2 or FGFR1 transcripts by the volume of the areas studied.

Effect of PAE on density of double- and triple-labeled MCH neurons with FGF2 and FGFR1 transcripts.

To determine the effect of low-moderate PAE on the density of double-labeled MCH neurons with FGF2 or FGFR1 transcripts and triple-labeled MCH neurons with both FGF2 and FGFR1 transcripts, the dams received daily intraoral infusions (from E10-E15) of either ethanol (2 g/kg/day) or the control solution. The density of MCH, FGF2 or FGFR1 cells, in the whole LH or the dLH and vLH of female and male postnatal offspring (n = 4-6/group/sex), was determined by dividing the number of MCH neurons double- or triple-labeled with FGF2 and FGFR1 transcripts by the volume of the whole LH or the dLH and vLH.

Effect of PAE on the expression of FGF2 and FGFR1 transcripts and their intracellular distribution.

Expression level of FGF2 and FGFR1 transcripts in individual MCH neurons was determined in whole LH of female and male postnatal offspring (n = 4-6/group/sex) by counting FGF2 and FGFR1 transcript number per MCH neuron. To determine the intracellular distribution of the FGF2 and FGFR1 transcripts within MCH neurons, transcripts in the nucleus and cytoplasm were manually quantified in MCH neurons as described above. For correlations, FGF2 and FGFR1 transcripts were manually counted in the nucleus and cytoplasm of triple-labeled MCH neurons.

Effect of prenatal FGF2 injection on density and expression levels of MCH neurons, density of double- and triple-labeled MCH neurons with FGF2 and FGFR1 transcripts, and intracellular distribution of FGF2 and FGFR1 transcripts.

To determine the effect of prenatal FGF2 exposure by maternal administration on neuronal density and transcript expression of MCH, FGF2 and FGFR1 cells in the LH of postnatal female offspring (n = 4/group), the dams at E14 received subcutaneous injection of 80 ug/kg of human recombinant FGF2 or its vehicle. The density of MCH neurons alone and double-labeled with FGF2 or FGFR1 or triple labeled with both FGF2 and FGFR1 was determined as described above. The FGF2 and FGFR1 transcript expression in individual MCH neurons was determined by counting FGF2 and FGFR1 transcript number per MCH neuron. To evaluate the effects of FGF2 injection on the intracellular distribution of FGF2 and FGFR1 transcripts within MCH neurons, nuclear and cytoplasmic transcripts were manually quantified.

2.8. Statistical analysis

Data from the qRT-PCR analysis of expression of MCH, FGF2 and FGFR1 in whole LH, indicated by mRNA fold change, were analyzed using two-way ANOVA to test the main effects of PAE and sex and their interaction, followed by post-hoc Tukey’s multiple comparisons test. Data from RNAscope experiments, measuring in whole LH the density of MCH, FGF2 and FGFR1 cells and MCH transcript expression, were also analyzed using two-way ANOVA, which tested the main effects of PAE and sex and their interaction, followed by post-hoc Tukey’s multiple comparisons test. Data from RNAscope experiments, measuring the density of MCH, FGF2 and FGFR1 cells in the dLH and vLH areas or the FGF2 and FGFR1 transcripts in the nucleus and cytoplasm, were analyzed using three-way ANOVA, which tested the main effects of PAE, sex, and either LH areas (dLH/vLH) or intracellular distribution (nucleus/cytoplasm) and their interactions. To further investigate significant interactions, subsequent two-way ANOVAs were performed, followed by post-hoc Tukey’s multiple comparisons test. The RNAscope data for the FGF2- vs vehicle-injection groups, measuring neuronal density of MCH, FGF2 and FGFR1 in LH of female offspring, were analyzed by two-tailed, unpaired t tests, with the intracellular distribution (nucleus vs cytoplasm) of transcripts analyzed using two-way ANOVA testing the main effects of PAE and intracellular distribution and their interaction, followed by post-hoc Tukey’s multiple comparisons test. Pearson’s correlation was performed to analyze the relationship between total number of FGF2 and FGFR1 transcripts per MCH neuron and also the numbers of transcripts specifically in the nucleus vs cytoplasm. For all tests, significance was determined at p ≤ 0.05, with Bonferroni adjusted alpha levels used when appropriate. All data are presented as mean ± SEM and were analyzed using Prism (GraphPad Prism, version 9).

2.9. Data availability

All data described in this study are available from the corresponding author upon request.

3. Results

3.1. PAE has dose-, sex- and area-dependent effects on MCH neurons in LH of the offspring

Building on our published results showing PAE to stimulate neurogenesis and increase the density of MCH-immunoreactive neurons in female more strongly than male offspring (see Introduction), we examine here in the LH of female and male postnatal offspring the effects of PAE at two doses, low-moderate (2 g/kg/day) and high (5 g/kg/day), on mRNA levels of MCH measured using qRT-PCR. Finding the high-dose to have no effect on MCH, we then tested the effect of PAE at the low-moderate dose on the density and expression level of MCH neurons measured using RNAscope. These experiments demonstrate that PAE stimulates MCH-expressing neurons in the LH in a dose-, sex-, and area-dependent manner.

Specifically, the dose- and sex-dependent nature of this effect is demonstrated by analysis of MCH mRNA levels in whole LH (Fig. 3A). PAE at the low-moderate dose increases MCH gene expression in females more strongly than males, while at the high-dose it has no effect in either females or males (two-way ANOVA; main effect of PAE (F_(2,18) = 29.16, p = 0.0001) and sex (F_(1,18) = 6.03, p = 0.024); interaction between PAE and sex (F_(2,18) = 4.58, p = 0.024); post-hoc, low-moderate PAE females vs control females (p = 0.0001), low-moderate PAE males vs control males (p = 0.041), low-moderate PAE females vs PAE males (p = 0.011), high-dose PAE females vs control females (p = 0.912), high-dose PAE males vs control males (p = 0.883), high-dose PAE females vs PAE males (p = 0.99)).

Figure 3:

Prenatal alcohol exposure (PAE) relative to control group, at low-moderate (2 g/kg/day) or high dose (5g/kg/day) from embryonic day 10 (E10) to E15, affects mRNA expression and neuronal density of MCH in lateral hypothalamus (LH) of postnatal day 5 (P5) offspring (n = 4-6/group/sex), as measured using qRT-PCR and RNAscope. A, PAE at 2 g/kg/day but not 5 g/kg/day increases mRNA expression (fold change) of MCH in LH, more strongly in females than males. B, PAE at 2 g/kg/day increases density of MCH neurons in LH, again more strongly in females than males. C, This PAE-induced, sex-dependent increase in density of MCH neurons is illustrated in representative photomicrographs. Scale bar, 100 μm. D, PAE at 2 g/kg/day increases the number of MCH transcripts per MCH neuron, in females but not males. E, PAE at 2 g/kg/day increases density of MCH neurons in dLH of females and males, with no effect in vLH of either sex. Bar graphs indicate SEM ± mean. *p ≤ 0.05 versus control group, #p ≤ 0.05 versus PAE males. Abbreviations: MCH, melanin-concentrating hormone; dLH, dorsal lateral hypothalamus; vLH, ventral lateral hypothalamus.

A sex-dependent, stimulatory effect of PAE is also observed with the measurement of density of MCH neurons in whole LH (Fig. 3B), as illustrated in the photomicrographs (Fig. 3C). At the low-moderate dose, PAE increases their density in females more strongly than males (two-way ANOVA; main effect of PAE (F_(1,12) = 76.85, p = 0.0001) and sex (F_(1,12) = 8.46, p = 0.013); interaction between PAE and sex (F_(1,12) = 8.05, p = 0.014); post-hoc, PAE females vs control females (p = 0.0001), PAE males vs control males (p = 0.005), PAE females vs PAE males (p = 0.007)). Analysis of the expression level of individual MCH neurons in whole LH (Fig. 3D) reveals a PAE-induced increase in the number of MCH transcripts per neuron that is also sex dependent, evident in females but not in males (two-way ANOVA; main effect of PAE (F_(1,115) = 8.61, p = 0.004) and sex (F_(1,115) = 14.63, p = 0.0002); interaction between PAE and sex (F_(1,115) = 4.72, p = 0.031); post-hoc, PAE females vs control females (p = 0.002), PAE males vs control males (p = 0.951)).

Examination of MCH neurons separately in the dorsal (dLH) and ventral (vLH) areas of the LH (Fig. 3E), as described and outlined in the Materials and Methods (Fig. 1) and illustrated here in the photomicrographs (Fig. 3C), demonstrates that this PAE-induced increase in density of MCH neurons is area dependent (three-way ANOVA; main effect of PAE (F_(1,28) = 43.70, p = 0.0001), sex (F_(1,28) = 8.59, p = 0.007), and area (F_(1,28) = 3.36, p = 0.077); interaction between PAE and sex (F_(1,28) = 3.54, p = 0.070), PAE and area (F_(1,28) = 6.83, p = 0.014), sex and area (F_(1,28) = 0.717, p = 0.404), and PAE, sex and area (F_(1,28) = 4.79, p = 0.037)). Further analysis of these data independent of sex shows that PAE increases the density of MCH neurons in the dLH but not vLH (two-way ANOVA; main effect of PAE (F_(1,32) = 29.08, p = 0.0001) and area (F_(1,32) = 2.63, p = 0.114); interaction between PAE and area (F_(1,32) = 3.98, p = 0.054); post-hoc, PAE vs control in dLH (p = 0.0001) and vLH (p = 0.097)). In addition, analyses within each sex demonstrate a similar anatomical pattern in both sexes, with this PAE-induced increase in density of MCH neurons evident in the dLH but not vLH of females (two-way ANOVA; main effect of PAE (F_(1,12) = 29.35, p = 0.0002) and area (F_(1,12) = 0.396, p = 0.54); interaction between PAE and area (F_(1,12) = 9.39, p = 0.009); post-hoc, PAE females vs control females in dLH (p = 0.0003) and vLH (p = 0.382)), and also evident in the dLH but not vLH of males (two-way ANOVA; main effect of PAE (F_(1,16) = 13.66, p = 0.002) and area (F_(1,16) = 4.39, p = 0.050); interaction between PAE and area (F_(1,16) = 0.109, p = 0.744); post-hoc, PAE males vs control males in dLH (p = 0.050) and vLH (p =0.121)).

Together, these results provide new evidence that PAE increases mRNA levels of MCH in a dose- and sex-dependent manner, stimulates the expression level of individual MCH neurons in a similar sex-dependent manner, and increases in both sexes the density of these MCH neurons in an area-dependent manner, specifically in the dorsal but not ventral area of the LH.

3.2. PAE also has dose- and sex-dependent effects on FGF2 and FGFR1 in LH of the offspring

We next examined in female and male postnatal offspring the effects of PAE, at a low-moderate (2 g/kg/day) and high (5 g/kg/day) dose, compared to control on mRNA levels of FGF2 and FGFR1 in the LH measured using qRT-PCR. Again, finding the high-dose of PAE to have no effect on mRNA expression of either gene, we then tested using RNAscope the effect of PAE at low-moderate dose on the density of cells with FGF2 and FGFR1 transcripts. These experiments demonstrate that, similar to MCH, PAE increases FGF2 and FGFR1 mRNA in a dose- and sex-dependent manner and produces a sex-dependent increase in density of FGF2 cells, while unlike MCH PAE increases FGF2 and FGFR1 cells broadly throughout the whole LH.

Specifically, measurements of mRNA levels of FGF2 in whole LH (Fig. 4A) reveal a stimulatory effect in females and males that is dose dependent, with PAE increasing FGF2 gene expression at the low-moderate dose while having no effect at the high dose, and also that is sex dependent, with PAE increasing FGF2 mRNA in females more than males (two-way ANOVA; main effect of PAE (F_(2,18) = 97.79, p = 0.0001) and sex (F_(1,18) = 13.74, p = 0.001); interaction between PAE and sex (F_(2,18) = 6.09, p = 0.009); post-hoc, low-moderate PAE females vs control females (p = 0.0001), low-moderate PAE males vs control males (p = 0.0001), low-moderate PAE females vs PAE males (p = 0.001), high-dose PAE females vs control females (p = 0.994), high-dose PAE males vs control males (p = 0.981)). Whereas PAE also stimulates FGFR1 mRNA in a dose-dependent manner with effects at the low-moderate but not high dose (Fig. 4B), this effect on FGFR1 unlike FGF2 occurs independently of sex, similarly in females and males (two-way ANOVA; main effect of PAE (F_(2,18) = 29.62, p = 0.0001) and sex (F_(1,18) = 0.243, p = 0.627); interaction between PAE and sex (F_(2,18) = 0.17, p = 0.841); post-hoc, low-moderate PAE females vs control females (p = 0.004), low-moderate PAE males vs control males (p = 0.007), high-dose PAE females vs control females (p = 0.599), high-dose PAE males vs control males (p = 0.318)).

Figure 4:

Prenatal alcohol exposure (PAE) relative to control group, at low-moderate (2 g/kg/day) or high dose (5 g/kg/day) from E10-E15, affects mRNA expression and density of FGF2 and FGFR1 cells in lateral hypothalamus (LH) of P5 offspring (n = 4-6/group/sex), as measured using qRT-PCR and RNAscope. A, PAE at 2 g/kg/day but not 5 g/kg/day increases mRNA expression (fold change) of FGF2, more strongly in females than males. B, PAE at 2 g/kg/day but not 5 g/kg/day increases FGFR1 mRNA expression, in females similar to males. C, PAE at 2 g/kg/day increases the density of FGF2 cells, in females but not males. D, PAE at 2 g/kg/day increases the density of FGFR1 cells, similarly in females and males. E, This PAE-induced increase in density of FGF2 and FGFR1 cells in female P5 offspring is illustrated in the photomicrographs, showing FGF2 transcripts (red) and FGFR1 transcripts (white) in DAPI cells (blue) in the dLH and vLH, with FGF2-positive cells in top panel and FGFR1-positive cells in bottom panel. Scale bars: low magnification, 50 μm; high magnification, 10 μm. F, PAE at 2 g/kg/day increases density of FGF2-positive cells similarly in both dLH and vLH of females, with no effect in males. G, PAE at 2 g/kg/day increases density of FGFR1-positive cells similarly in both dLH and vLH of females and males. Bar graphs indicate SEM ± mean. *p ≤ 0.05 versus control females, #p ≤ 0.05 versus PAE males. Abbreviations: FGF2, fibroblast growth factor 2; FGFR1, fibroblast growth factor receptor 1; dLH, dorsal lateral hypothalamus; vLH, ventral lateral hypothalamus.

As with gene expression, PAE at the low-moderate dose (2 g/kg/day) increases in whole LH the density of cells with FGF2 transcripts examined with RNAscope (Fig. 4C) and produces this effect in a sex-dependent manner, in females but not males (two-way ANOVA; main effect of PAE (F_(1,12) = 21.79, p = 0.0005) and sex (F_(1,12) =13.57, p = 0.003); interaction between PAE and sex (F_(1,12) = 10.85, p = 0.006); post-hoc, PAE females vs control females (p = 0.0006), PAE males vs control males (p = 0.767), PAE females vs PAE males (p = 0.001)). In contrast, the density of cells with FGFR1 transcripts (Fig. 4D) is increased by PAE similarly in females and males (two-way ANOVA; main effect of PAE (F_(1,14) = 37.30, p = 0.0001) and sex (F_(1,14) = 4.75, p = 0.046); interaction between PAE and sex (F_(1,14) = 0.515, p = 0.484); post-hoc, PAE females vs control females (p = 0.002), PAE males vs control males (p = 0.006), PAE females vs PAE males (p = 0.217)). These effects of PAE on cells with FGF2 or FGFR1 transcripts in whole LH are illustrated in the photomicrographs for females (Fig. 4E) and males (Supplemental Fig. 4-1).

Separate analyses of the dorsal (dLH) and ventral (vLH) areas of the LH outlined in the photomicrographs show that the stimulatory effects of PAE on the density of FGF2 and FGFR1 cells are not area dependent, occurring uniformly throughout the whole LH. For FGF2 (Fig. 4F), they vary as a function of sex but not area (three-way ANOVA; main effect of PAE (F_(1,24) = 37.59, p = 0.0001), sex (F_(1,24) = 23.27, p = 0.0001), and area (F_(1,24) = 1.11, p = 0.303); interaction between PAE and sex (F_(1,24) = 18.65, p = 0.0002), PAE and area (F_(1,24) = 0.28, p = 0.596), sex and area (F_(1,24) = 0.062, p = 0.805), and PAE, sex and area (F_(1,24) = 0.42, p = 0.521)). Further analyses of these data within each sex show that this stimulatory effect of PAE on the density of FGF2 cells is observed similarly in the dLH and vLH of the female offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 36.93, p = 0.0001) and area (F_(1,12) = 0.57, p = 0.463); interaction between PAE and area (F_(1,12) = 0.004, p = 0.948); post-hoc, PAE females vs control females in dLH (p = 0.005) and vLH (p = 0.004)), while it is not evident in either area of the male offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 3.14, p = 0.101) and area (F_(1,12) = 0.619, p = 0.446); no interaction between PAE and area (F_(1,12) = 1.35, p = 0.267)).

For FGFR1 (Fig. 4G), the stimulatory effects of PAE are also not area dependent (three-way ANOVA; main effect of PAE (F_(1,24) = 62.32, p = 0.0001), sex (F_(1,24) = 15.68, p = 0.0006), and area (F_(1,24) = 7.43, p = 0.012); interaction between PAE and sex (F_(1,24) = 11.75, p = 0.002), PAE and area (F_(1,24) = 1.24, p = 0.277), sex and area (F_(1,24) = 0.339, p = 0.565), and PAE, sex and area (F_(1,24) = 1.375, p = 0.252)). Analyses of these data within each sex show that this stimulatory effect on FGFR1 cells is similarly evident in the dLH and vLH of both sexes, in female offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 41.15, p = 0.0001) and area (F_(1,12) = 3.51, p = 0.085); no interaction between PAE and area (F_(1,12) = 1.67, p = 0.219); post-hoc, PAE females vs control females in dLH (p = 0.007) and vLH (p = 0.016)) and also in male offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 22.55, p = 0.0005) and area (F_(1,12) = 5.19, p = 0.041); interaction between PAE and area (F_(1,12) = 0.004, p = 0.949); PAE males vs control males in dLH (p = 0.027) and vLH (p = 0.023)).

Thus, similar to MCH, these results demonstrate that PAE, while having no effect at the high dose, has a stimulatory effect at the low-moderate dose on FGF2 mRNA levels and on the density of FGF2 cells in LH that is sex dependent, stronger in females than males. However, PAE’s effects on FGFR1 mRNA and cell density are not sex dependent, occurring similarly in females and males, and its stimulatory effect on the density of FGF2 and FGFR1 cells, unlike MCH neurons, occurs uniformly throughout the LH, similarly in both the dorsal and ventral areas.

3.3. PAE increases the density of MCH neurons with FGF2 and/or FGFR1 transcripts in LH of the offspring

This experiment examined in female and male offspring whether FGF2 and/or FGFR1 colocalize with MCH neurons in the LH. Finding that they do, we tested whether PAE at the low-moderate dose compared to control offspring affects the density of MCH neurons co-expressing FGF2 and/or FGFR1 transcripts and also if these effects are sex and/or area dependent, as indicated by our analysis above of MCH neurons.

Our results show that the MCH neurons do, in fact, co-express FGF2 and FGFR1 and that PAE alters their density in the LH. Quantification of these double-labeled neurons in whole LH shows that PAE increases the density of MCH neurons co-expressing FGF2 transcripts and produces this effect in a sex-dependent manner (Fig. 5A), in females but not males (two-way ANOVA; main effect of PAE (F_(1,13) = 8.06, p = 0.013) and sex (F_(1,13) = 5.34, p = 0.037); interaction between PAE and sex (F_(1,13) = 5.71, p = 0.032); post-hoc, PAE females vs control females (p = 0.010), PAE males vs control males (p = 0.982)). Whereas PAE also increases the density of MCH neurons co-expressing FGFR1 transcripts (Fig. 5B), this effect in contrast to FGF2 occurs independent of sex, similarly in females and males (two-way ANOVA; main effect of PAE (F_(1,15) = 33.60, p = 0.0001) and sex (F_(1,15) = 5.22, p = 0.037); interaction between PAE and sex (F_(1,15) = 1.71, p = 0.209); post-hoc, PAE females vs control females (p = 0.001), PAE males vs control males (p = 0.023), PAE females vs PAE males (p = 0.106)). Further, PAE increases in whole LH the density of triple-labeled MCH neurons that express both FGF2 and FGFR1 transcripts in a sex-dependent manner (Fig. 5C), in females but not males (two-way ANOVA; main effect of PAE (F_(1,13) = 8.31, p = 0.012) and sex (F_(1,13) = 5.26, p = 0.039); interaction between PAE and sex (F_(1,13) = 5.87, p = 0.031); post-hoc, PAE females vs control females (p = 0.009), PAE males vs control males (p = 0.988)). These effects of PAE on double- and triple-labeled neurons are illustrated in the photomicrographs for females (Fig. 5D) and males (Supplemental Fig. 5-1).

Figure 5:

Prenatal alcohol exposure (PAE) at 2 g/kg/day compared with control group affects the density of MCH neurons that co-express FGF2, FGFR1 or both FGF2 and FGFR1 transcripts in lateral hypothalamus (LH) of P5 offspring (n = 4-6/group/sex), as measured using RNAscope. A, PAE increases density of double-labeled MCH neurons co-expressing FGF2, in females but not males. B, PAE increases the density of double-labeled MCH neurons co-expressing FGFR1, similarly in females and males. C, PAE increases the density of triple-labeled MCH neurons expressing both FGF2 and FGFR1 transcripts, in females but not males. D, These effects of PAE on double-labeled and triple-labeled neurons are illustrated in the photomicrographs of females, showing in dorsal LH double-labeled MCH neurons (green) with FGF2 transcripts (red, top panel) and FGFR1 transcripts (white, middle panel) and triple-labeled MCH neurons with both FGF2 and FGFR1 transcripts (bottom panel). Scale bars: low magnification, 50 μm; high magnification, 10 μm. E, PAE increases the density of MCH neurons co-expressing FGF2 in the dLH but not vLH of females, while having no effect in either dLH or vLH of males. F, PAE increases the density of double-labeled MCH neurons co-expressing FGFR1 in the dLH but not vLH of both females and males. G, PAE increases the density of triple-labeled MCH neurons with both FGF2 and FGFR1 transcripts in dLH but not vLH of females, with no effect in either area of males. Bar graphs indicate SEM ± mean. *p ≤ 0.05 versus control groups. Abbreviations: MCH, melanin-concentrating hormone; FGF2, fibroblast growth factor 2; FGFR1, fibroblast growth factor receptor 1; dLH, dorsal lateral hypothalamus; vLH, ventral lateral hypothalamus.

Separate analyses of the dorsal (dLH) and ventral (vLH) areas of the LH identify the dLH as being most responsive to PAE’s stimulatory effect on the density of MCH neurons co-expressing FGF2 or FGFR1 transcripts, similar to that described above for the density of all MCH neurons but not of all LH cells with transcripts. Examination of the double-labeled neurons demonstrates that the PAE-induced increase in the density of MCH neurons with FGF2 transcripts (Fig. 5E) varies in relation to sex and area (three-way ANOVA; main effect of PAE (F_(1,28) = 10.85, p = 0.002), sex (F_(1,28) = 7.601, p = 0.010), and area (F_(1,28) = 4.13, p = 0.050); interaction between PAE and sex (F_(1,28) = 7.66, p = 0.009), PAE and area (F_(1,28) = 1.18, p = 0.285), area and sex (F_(1,27) = 0.224, p = 0.639), and PAE, sex and area (F_(1,28) = 1.46, p = 0.236)). Separate analyses within each sex of these double-labeled neurons show that the PAE-induced increase in density of MCH neurons with FGF2 transcripts is observed in the dLH but not vLH area of female offspring (two-way ANOVA; main effect of PAE (F_(1,16) = 15.25, p = 0.001) and area (F_(1,16) = 2.61, p = 0.125); interaction between PAE and area (F_(1,16) = 2.19, p = 0.158); post-hoc, PAE females vs control females in dLH (p = 0.007) and vLH (p = 0.348)), while it is not evident in either area of male offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 0.23, p = 0.636) and area (F_(1,12) = 2.07, p = 0.174); interaction between PAE and area (F_(1,12) = 0.01, p = 0.914)).

Similarly, the PAE-induced increase in density of MCH neurons with FGFR1 transcripts (Fig. 5F) also varies in relation to sex (three-way ANOVA; main effect of PAE (F_(1,24) = 35.84, p = 0.0001), sex (F_(1,24) = 8.37, p = 0.008), and area (F_(1,24) = 4.67, p = 0.041); interaction between PAE and sex (F_(1,24) = 4.92, p = 0.036), PAE and area (F_(1,24) = 3.52, p = 0.073), sex and area (F_(1,24) = 0.001, p = 0.975), and PAE, sex and area (F_(1,24) = 1.36, p = 0.254)). A separate analysis within each sex of these double-labeled neurons demonstrates that this stimulatory effect on MCH neurons with FGFR1 transcripts, as shown for FGF2, occurs in the dLH but not vLH, but in contrast to FGF2, this effect is detected in both sexes, in the female offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 22.79, p = 0.0005) and area (F_(1,12) = 1.53, p = 0.238); interaction between PAE and area (F_(1,12) = 3.13, p = 0.101); post-hoc, PAE females vs control females in dLH (p = 0.003) and vLH (p = 0.201)) and also in the male offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 13.58, p = 0.003) and area (F_(1,12) = 4.62, p = 0.051); interaction between PAE and area (F_(1,12) = 0.483, p = 0.50); post-hoc, PAE males vs control males in dLH (p = 0.040) and vLH (p = 0.203)).

Further analyses of the triple-labeled neurons in these two areas of the LH (Fig. 5G) demonstrate again that the PAE-induced increase in density of MCH neurons with both FGF2 and FGFR1 transcripts varies across sex (three-way ANOVA; main effect of PAE (F_(1,26) = 13.96, p = 0.0009), sex (F_(1,26) = 8.12, p = 0.008), and area (F_(1,26) = 7.05, p = 0.013); interaction between PAE and sex (F_(1,26) = 9.96, p = 0.004), PAE and area (F_(1,26) = 2.86, p = 0.102), sex and area (F_(1,26) = 1.02, p = 0.322), and PAE, sex and area (F_(1,26) = 3.44, p = 0.075)). Analyses within each sex of these triple-labeled neurons shows that the PAE-induced increase in their density is evident in the dLH but not vLH of female offspring (two-way ANOVA; main effect of PAE (F_(1,14) = 18.89, p = 0.0007) and area (F_(1,14) = 5.34, p = 0.036); interaction between PAE and area (F_(1,14) = 5.006, p = 0.042); post-hoc, PAE females vs control females in dLH (p = 0.001) and vLH (p = 0.468)), while it is not seen in either area of male offspring (two-way ANOVA; main effect of PAE (F_(1,12) = 0.25, p = 0.620) and area (F_(1,12) = 2.08, p = 0.174); interaction between PAE and area (F_(1,12) = 0.019, p = 0.89)).

These results demonstrate that MCH neurons in the LH co-express FGF2 and FGFR1 and that PAE increases the density of MCH neurons with FGF2 and FGFR1 transcripts and produces this effect in an area-dependent manner, in the dorsal but not ventral LH. Moreover, this stimulatory effect on MCH neurons with FGF2 transcripts or both FGF2 and FGFR1 transcripts occurs only in females, while the effect on MCH neurons with FGFR1 transcripts occurs similarly in both sexes, specifically in the dorsal LH.

3.4. PAE alters the number and intracellular distribution of FGF2 and FGFR1 transcripts in MCH neurons of offspring

By analyzing individual MCH neurons, we measured here in whole LH the expression level and intracellular distribution of FGF2 and FGFR1 transcripts in each neuron and found that PAE compared to control, in addition to increasing the total number of FGF2 and FGFR1 transcripts per MCH neuron, has sex dependent effects on these transcripts within the nucleus and cytoplasm of the neuron.

Analysis of the FGF2 transcripts in each MCH neuron (Fig. 6A) demonstrates that PAE increases their total number per neuron in a sex-dependent manner, in females but not males (two-way ANOVA; main effect of PAE (F_(1,607) = 18.39, p = 0.0001) and sex (F_(1,607) = 20.91, p = 0.0001); interaction between PAE and sex (F_(1,607) = 5.97, p = 0.014); post-hoc, PAE females vs control females (p = 0.0001), PAE males vs control males (p = 0.568)). We find that this effect on the intracellular distribution of FGF2 transcripts varies across sex (Fig. 6B) (three-way ANOVA; main effect of PAE (F_(1,1214) = 22.44, p = 0.0001), sex (F_(1,1214) = 26.83, p = 0.0001), and intracellular distribution (F_(1,1214) = 98.96, p = 0.0001); interaction between PAE and sex (F_(1,1214) = 6.93, p = 0.008), PAE and intracellular distribution (F_(1,1214) = 6.52, p = 0.011), sex and intracellular distribution (F_(1,1214) = 9.38, p = 0.002), and PAE, sex and intracellular distribution (F_(1,1214) = 3.13, p = 0.077)). Further analysis of these data independent of sex shows that PAE increases the number of FGF2 transcripts in both the nucleus and cytoplasm (two-way ANOVA; main effect of PAE (F_(1,1218) = 37.43, p = 0.0001) and intracellular distribution (F_(1,1218) = 112.1, p = 0.0001); interaction between PAE and intracellular distribution (F_(1,1218) = 2.85, p = 0.091); post-hoc, PAE vs control in nucleus (p = 0.009) and cytoplasm (p = 0.0001)). Also, analysis independent of treatment shows a greater number of FGF2 transcripts in the nucleus and cytoplasm of females than males (two-way ANOVA; main effect of sex (F_(1,1218) = 58.32, p = 0.0001) and intracellular distribution (F_(1,1218) = 102.3, p = 0.0001); interaction between sex and intracellular distribution (F_(1,1218) = 12.24, p = 0.0005); post-hoc, nucleus vs cytoplasm in females (p = 0.001) and males (p = 0.001), female nucleus vs male nucleus (p = 0.001), female cytoplasm vs male cytoplasm (p = 0.018)). Examination of these data within each sex (Fig. 6B) demonstrates that PAE increases the number of FGF2 transcripts per MCH neuron in the nucleus as well as cytoplasm of females (two-way ANOVA; main effect of PAE (F_(1,722) = 23.18, p = 0.0001) and intracellular distribution (F_(1,722) = 72.23, p = 0.0001); interaction between PAE and intracellular distribution (F_(1,722) = 0.26, p = 0.608); post-hoc, PAE females vs control females in nucleus (p = 0.013) and cytoplasm (p = 0.001)). However, PAE increases the number of these transcripts only in the cytoplasm of males (two-way ANOVA; main effect of PAE (F_(1,492) = 3.04, p = 0.081) and intracellular distribution (F_(1,492) = 32.56, p = 0.0001); interaction between PAE and intracellular distribution (F_(1,492) = 12.84, p = 0.0004); post-hoc, PAE males vs control males in nucleus (p = 0.562) and cytoplasm (p = 0.001)). These stimulatory effects of PAE on the number of FGF2 transcripts in the nucleus and cytoplasm of MCH neurons in females and males are illustrated in the photomicrographs (Fig. 6C).

Figure 6:

Prenatal alcohol exposure (PAE) at 2 g/kg/day compared to control group affects the number and intracellular localization of FGF2 and FGFR1 transcripts per MCH neuron in lateral hypothalamus (LH) of female and male offspring (n = 4-6/group/sex), as measured using RNAscope. A, PAE increases total number of FGF2 transcripts per MCH neuron, in females but not males. B, PAE increases number of nuclear and cytoplasmic FGF2 transcripts per MCH neuron in females but only cytoplasmic FGF2 transcripts per MCH neuron in males. C, The effect of PAE on FGF2 (red) and FGFR1 (white) transcripts per MCH neuron (green) is illustrated in the photomicrographs of females (top panels) and males (bottom panels). Scale bar, 50 μm. D, PAE increases the total number of FGFR1 transcripts per MCH neuron, similarly in females and males. E, PAE increases the number of nuclear and cytoplasmic FGFR1 transcripts per MCH neuron in females and only cytoplasmic FGFR1 transcripts per MCH neuron in males. Bar graphs indicate SEM ± mean. *p ≤ 0.05 versus control groups. Abbreviations: MCH, melanin-concentrating hormone; FGF2, fibroblast growth factor 2; FGFR1, fibroblast growth factor receptor 1; Nuc, nuclear; Cyto, cytoplasmic.

Examination of the FGFR1 transcripts in each MCH neuron (Fig. 6D) shows that PAE increases their total number per neuron similarly in females and males (two-way ANOVA; main effects of PAE (F_(1,661) = 22.59, p = 0.0001) and sex (F_(1,661) = 16.26, p = 0.0001); interaction between PAE and sex (F_(1,661) = 0.17, p = 0.676); post-hoc, PAE females vs control females (p = 0.0003), PAE males vs control males (p = 0.025)). This effect on the intracellular distribution of FGFR1 transcripts varies with sex (Fig. 6E) (three-way ANOVA; main effects of PAE (F_(1,1322) = 33.60, p = 0.0001), sex (F_(1,1322) = 25.19, p = 0.0001), and intracellular distribution (F_(1,1322) = 226.8, p = 0.0001); interaction between PAE and sex (F_(1,1322) = 0.214, p = 0.643), sex and intracellular distribution (F_(1,1322) = 17.43, p = 0.0001), PAE and intracellular distribution (F_(1,1322) = 1.70, p = 0.193), and PAE, sex and intracellular distribution (F_(1,1322) = 0.49, p =0.479)). Analysis of these data independent of treatment reveals a greater number of FGFR1 transcripts in the nucleus but not cytoplasm of females than males (two-way ANOVA; main effect of sex (F_(1,1326) = 26.89, p = 0.0001) and intracellular distribution (F_(1,1326) = 176.8, p = 0.0001); interaction between sex and intracellular distribution (F_(1,1326) = 14.48, p = 0.0001); post-hoc, nucleus vs cytoplasm in females (p = 0.0001) and males (p = 0.0001), female nucleus vs male nucleus (p = 0.0001), female cytoplasm vs male cytoplasm (p = 0.762)). Further, analysis within each sex (Fig. 6E) demonstrates as with FGF2 that PAE increases the number of FGFR1 transcripts per neuron in both the nucleus and cytoplasm of females (two-way ANOVA; main effect of PAE (F_(1,822) = 20.40, p = 0.0001) and intracellular distribution (F_(1,822) = 192.5, p = 0.0001); interaction between PAE and intracellular distribution (F_(1,822) = 0.1855, p = 0.667); post-hoc, PAE vs control female in nucleus (p = 0.021) and cytoplasm (p = 0.003)), while PAE increases transcripts only in the cytoplasm of males (two-way ANOVA; main effect of PAE (F_(1,500) = 16.84, p = 0.0001) and intracellular distribution (F_(1,500) = 70.14, p = 0.0001); interaction between PAE and intracellular distribution (F_(1,500) = 2.39, p = 0.123); post-hoc, PAE males vs control males in nucleus (p = 0.271) and cytoplasm (p = 0.0004)). The effects of PAE on the number of FGFR1 transcripts in the nucleus and cytoplasm of MCH neurons are illustrated in the photomicrographs (Fig. 6C).

Further analysis of these FGF2 and FGFR1 transcripts in triple-labeled neurons using Pearson’s correlation reveals their close, intracellular relationship within MCH neurons. We find that the total numbers of FGF2 and FGFR1 transcripts per MCH neuron are highly correlated in PAE females (r = +0.739, p = 0.000), while weakly correlated in control females (r = +0.323, p = 0.004) and PAE males (r = +0.251, p = 0.004) and not correlated in control males (r = +0.182, p = 0.070). Also, a separate analysis of these transcripts in the nucleus or cytoplasm reveals in females strong, positive correlations between the numbers of FGF2 and FGFR1 transcripts in both the nucleus (r = +0.682, p = 0.000) and cytoplasm (r = +0.608, p = 0.000) but considerably weaker correlations in males in the nucleus (r = +0.264, p = 0.004) and cytoplasm (r = +0.431, p = 0.000).

Similar to the increased density of MCH neurons with FGF2 or FGFR1 transcripts, we demonstrate here with measurements of the total number of transcripts per MCH neuron that the stimulatory effect of PAE is sex related for FGF2 occurring in females but not males, while it occurs independently of sex for FGFR1. Further, with analyses of the intracellular distribution of transcripts, we show that the PAE-induced increase in FGF2 and FGFR1 transcripts is evident in both the nucleus and cytoplasm of females but only in the cytoplasm of males, and the numbers of FGF2 and FGFR1 transcripts per neuron are strongly, positively correlated in the nucleus and cytoplasm of PAE females, while weakly correlated in control females or in PAE and control males.

3.5. FGF2 injection mimics the stimulatory effects of PAE on FGF2 and FGFR1 in MCH neurons of female offspring

The purpose of this experiment was to determine in female offspring whether maternal administration of FGF2 compared to control vehicle also modulates these MCH neurons in the LH. Our results demonstrate that prenatal exposure to FGF2 has strikingly similar effects to PAE in females on the density of MCH neurons and their co-expression of FGF2 and FGFR1 in the LH.

As demonstrated for PAE, maternal FGF2 injection increases the density of MCH neurons in the LH of the offspring (t test, p = 0.002) (Fig. 7A) as shown in the photomicrographs (Fig. 7B), while also increasing the number of MCH transcripts per neuron (control = 308.63 ± 46.9, FGF2-inj = 407.86 ± 24.26, t test, p = 0.044). Similarly, analyses of double-labeled neurons (Fig. 7A) demonstrate that maternal FGF2 administration increases the density of MCH neurons co-expressing FGF2 (t test, p = 0.002) or FGFR1 (t test, p = 0.001) transcripts and also the density of MCH neurons expressing both FGF2 and FGFR1 transcripts (t test, p = 0.001) as shown in the photomicrographs (Fig. 7C). These stimulatory effects of FGF2 administration on the density of double-or triple-labeled MCH neurons with FGF2 and/or FGFR1 transcripts, unlike the area-dependent effects of PAE, are detected in both the dLH and vLH. This is seen with measurements of the density of double-labeled MCH neurons containing FGF2 or FGFR1 transcripts (two-way ANOVA; main effect of FGF2 injection for FGF2 (F_(1,12) = 50.89, p = 0.0001) and FGFR1 (F_(1,12) = 52.02, p = 0.0001) and of area for FGF2 (F_(1,12) = 0.71, p = 0.417) and FGFR1 (F_(1,12) = 0.17, p = 0.682); interaction between FGF2 injection and area for FGF2 (F_(1,12) = 0.55, p = 0.471) and FGFR1 (F_(1,12) = 0.008, p = 0.929); post-hoc, for FGF2 in dLH (control = 0.189 ± 0.02, FGF2-inj = 0.906 ± 0.14, p = 0.0006) and vLH (control = 0.181 ± 0.056, FGF2-inj = 0.761 ± 0.094, p = 0.003) and for FGFR1 in dLH (control = 0.27±0.053, FGF2-inj = 0.999 ± 0.160, p = 0.001) and vLH (control = 0.22 ± 0.07, FGF2-inj = 0.965 ± 0.089, p = 0.001)). It is similarly observed with measurements of the density of triple-labeled MCH neurons containing both FGF2 and FGFR1 transcripts (two way ANOVA; main effect of FGF2 injection (F_(1,12) = 53.57, p = 0.0001) and area (F_(1,12) = 0.58, p = 0.459); interaction between FGF2 injection and area (F_(1,12) = 0.46, p = 0.506); post-hoc, for dLH (control = 0.19 ± 0.026, FGF2-inj = 0.9 ± 0.136, p = 0.0005) and vLH (control = 0.181 ± 0.056, FGF2-inj = 0.778 ± 0.099, p = 0.002)).

Figure 7:

Prenatal FGF2 injection (80 ug/kg, E14) compared to vehicle-injected control group affects the density of MCH neurons co-expressing FGF2, FGFR1 or FGF2 with FGFR1 transcripts in the LH of female offspring (n = 4/group), as measured using RNAscope. A, FGF2 injection increases the density of MCH neurons and density of MCH neurons with FGF2, FGFR1 and both FGF2 and FGFR1 transcripts. B, This effect of FGF2-injection on MCH neurons (green) in LH is illustrated in photomicrographs. Scale bar, 100 μm. C, This effect of FGF2-injection on triple-labeled MCH neurons in dorsal LH is illustrated in photomicrographs, showing a greater number of FGF2 (red) and FGFR1 (white) transcripts in MCH neurons (green) in dorsal LH of females. Scale bars: low magnification, 50 μm; high magnification, 10 μm. D, FGF2-injection increases the total number of FGF2 transcripts per MCH neuron and also in both the nucleus and cytoplasm. E, FGF2 injection also increases the total number of FGFR1 transcripts per MCH neuron and also in both the nucleus and cytoplasm. F, This effect of FGF2-injection on triple-labeled neurons is illustrated in the photomicrographs, showing a higher number of nuclear and cytoplasmic FGF2 (red) and FGFR1 (white) transcripts in MCH neurons (green) in LH of female offspring from the FGF2-injected group relative to control. Scale bar, 50 μm. Bar graphs indicate SEM ± mean. *p ≤ 0.05 versus control groups. Abbreviations: MCH, melanin-concentrating hormone; FGF2, fibroblast growth factor 2; FGFR1, fibroblast growth factor receptor 1; Nuc, nuclear; Cyto, cytoplasmic.

In addition to stimulating the density of MCH neurons, maternal injection of FGF2 increases in female offspring the total number of FGF2 transcripts per MCH neuron (Fig. 7D) (t test, p = 0.0001). This effect as with PAE is detected in both the nucleus and cytoplasm (two-way ANOVA; main effect of FGF2 injection (F_(1,606) = 34.91, p = 0.0001) and intracellular distribution (F_(1,606) = 10.28, p = 0.001); interaction between FGF2 injection and intracellular distribution (F_(1,606) = 2.94, p = 0.086); post-hoc, FGF2 injection vs control in nucleus (p = 0.016) and cytoplasm (p = 0.0001)). Moreover, FGF2 administration similarly increases the total number of FGFR1 transcripts per MCH neuron (Fig. 7E) (t test, p = 0.0001), and this effect is also evident in both the nucleus and cytoplasm (two-way ANOVA; main effect of FGF2 injection (F_(1,686) = 78.44, p = 0.0001) and intracellular distribution (F_(1,686) = 12.95, p = 0.0003); interaction between FGF2 injection and intracellular distribution (F_(1,686) = 0.50, p = 0.479); post-hoc, FGF2 injection vs control in both the nucleus (p = 0.0001) and cytoplasm (p = 0.0001)). These stimulatory effects of maternal FGF2 injection on endogenous FGF2 and FGFR1 transcripts in the nucleus and cytoplasm of MCH neurons are illustrated in the photomicrographs (Fig. 7F).

Further examination of the MCH neurons with both FGF2 and FGFR1 transcripts using Pearson’s correlation reveals in FGF2-injected females a strong, positive correlation between the number of FGF2 and FGFR1 transcripts per MCH neuron (r = +0.779, p = 0.00), with a weaker correlation observed in control females (r = +0.473, p = 0.0008). Analysis of these transcripts as a function of their intracellular distribution again shows after FGF2 injection strong, positive correlations between the number of FGF2 and FGFR1 transcripts in the nucleus (r = +0.678, p = 0.00) and cytoplasm (r = +0.742, p = 0.00), with a weaker or insignificant correlation in control offspring obtained in the nucleus (r = +0.503, p = 0.0003) and cytoplasm (r = +0.188, p = 0.209).

These results in females show that prenatal exposure to FGF2 after maternal administration has strong, stimulatory effects in LH very similar to PAE. It increases the density of MCH neurons that co-express FGF2 and/or FGFR1, with this effect evident throughout the whole LH, and it also increases the expression level or numbers of FGF2 and FGFR1 transcripts per MCH neuron in both the nucleus and cytoplasm, inducing strong, positive correlations between the numbers of FGF2 and FGFR1 transcripts per MCH neuron.

4. Discussion

Building on our prior studies showing PAE at low-moderate doses to stimulate neurogenesis and produce a sex-dependent increase in MCH-immunoreactive neurons in LH of the embryo, neonate and adolescent offspring (see Introduction), we provide evidence here in postnatal offspring supporting the involvement of the FGF2-FGFR1 system in mediating this stimulatory effect of PAE on MCH neuronal development.

We first demonstrate that PAE increases mRNA levels of FGF2 and FGFR1 in the EH, similar to MCH. This is consistent with evidence in adult rodents, showing acute or prolonged alcohol exposure and alcohol consumption to stimulate FGF2 and FGFR1 expression in forebrain areas (McBride et al., 2010; Even-Chen et al., 2017; Even-Chen and Barak, 2019a; Airapetov et al., 2020). Also like MCH, we find this effect in LH to be dose-dependent, with PAE at the low-moderate dose (2 g/kg/day) stimulating FGF2 and FGFR1 expression while having no effect at the high dose (5 g/kg/day). This dose-related pattern is consistent with evidence that FGF2 expression in chicken embryo is stimulated by low-dose alcohol while reduced by a high dose (Tufan and Satiroglu-Tufan, 2003). Our additional finding, that PAE’s stimulatory effect on FGF2 mRNA like MCH is sex-dependent, seen in females but not males, is consistent with sex differences observed in the regulation of FGF2 in the periphery (Larsson et al., 2002; Mao et al., 2010). This sex dependence of FGF2 contrasts with the stimulatory effect of PAE on FGFR1 mRNA that occurs similarly in females and males, consistent with other evidence showing FGFR1 to be regulated independently of sex (Sasaki et al., 2012; Yang et al., 2013; Even-Chen and Barak, 2019a; Bae et al., 2020). These stimulatory effects of PAE on mRNA levels are confirmed using RNAscope, revealing similarities that link the FGF2-FGFR1 system to MCH in the LH. As shown with MCH neurons, PAE at the low-moderate dose increases the density of cells expressing FGF2 or FGFR1, and this effect on FGF2 cell density is sex-dependent, evident in females but not males, while on FGFR1 cell density it occurs similarly in females and males.

A close, intracellular relationship between the FGF2-FGFR1 system and MCH is demonstrated by our finding that both FGF2 and FGFR1 co-localize with MCH in LH neurons and PAE at the low-moderate dose increases the density of MCH neurons co-expressing FGF2 and FGFR1. The well-known role of FGF2 in stimulating neurogenesis in newborn rats (Tao et al., 1996; Kuhn et al., 1997) and the proliferation and differentiation of neurons in adult rats (Kuhn et al., 1997; Jin et al., 2003) suggests the possibility that FGF2 in MCH neurons contributes to the PAE-induced increase in hypothalamic neurogenesis and density of MCH neurons. The dose-dependent nature of PAE’s stimulatory effect on FGF2 and MCH expression, occurring at the low-moderate but not high dose, is similarly observed with the FGF2-induced increase in cortical neurogenesis (Vaccarino et al., 1999; Ohmiya et al., 2001; Nelson and Svendsen, 2006) and ethanol-induced increase in hypothalamic neurogenesis (Livy et al., 2003; Gil-Mohapel et al., 2010; Chang et al., 2012).

Further, our evidence that PAE increases only in females the density of MCH neurons with FGF2 transcripts points toward a possible role of estrogen, which is elevated by PAE (Petridou et al., 1992; Nagata et al., 2007) and shown to stimulate FGF2 (Zhang et al., 2016) and positively regulate FGF2-induced activation in endothelial cell proliferation (Garmy-Susini et al., 2004). While testosterone is also positively related to FGF2 (Dandona et al., 2021), PAE suppresses testosterone levels in male offspring (Ward et al., 2003; Akison et al., 2019), possibly explaining why PAE has little effect in males on FGF2 in MCH neurons. With PAE increasing in both sexes MCH neurons containing FGFR1 transcripts, this receptor likely functions differently in females and males. Whereas FGF2 in MCH neurons may be the prominent ligand for FGFR1 in females, FGFR1 stimulated by PAE in males may function in a ligand-independent manner (Sarabipour and Hristova, 2016), through different fibroblast growth factors that bind to FGFR1 (Zhang et al., 2006), or independently of changes in gonadal steroids (Formisano et al., 2017; Gallo, 2021).

Whereas PAE stimulates FGF2 and FGFR1 cells uniformly and densely throughout the whole LH suggesting a broad function in multiple systems, we demonstrate that the PAE-induced increase in density of MCH neurons co-expressing FGF2 and/or FGFR1 is anatomically localized, evident in the dorsal but not ventral area of the LH. With the LH suggested to have diverse neuronal subpopulations controlling multiple physiological functions and behaviors including reward and ingestive behavior (Bonnavion et al., 2016), this evidence for MCH subpopulations responding differentially to ethanol’s proliferation-inducing effect may reflect the different functional roles suggested for neurons in these distinct areas. With the dorsal LH shown to be involved in reward seeking as well as cocaine self-administration and cocaine-induced conditioned place preference (Hurley and Johnson, 2014; Blacktop and Sorg, 2019), the MCH neurons in this area co-expressing FGF2 and FGFR1 may act through these systems in mediating the increased alcohol consumption also induced by PAE (Chang et al., 2015; Chang et al., 2018). This contrasts with the ventral LH, which is implicated in different functions such as positive emotional behavior and arousal (Alvarez-Bolado and Celio, 2016; Venner et al., 2016).

Our detailed analyses of individual MCH neurons demonstrate that PAE also stimulates the expression level of FGF2 and FGFR1 transcripts within MCH neurons and produces this effect in a sex-dependent manner, increasing the total number of FGF2 transcripts per neuron only in females but the FGFR1 transcripts similarly in females and males. Further, PAE has differential, intracellular effects that are sex related, increasing the number of FGF2 and FGFR1 transcripts in the nucleus as well as cytoplasm of females but only in the cytoplasm of males. With studies showing that nuclear FGF2 promotes the proliferation and differentiation of different cell types (Moffett et al., 1996; Clarke et al., 2001; Dunham-Ems et al., 2009), FGF2 may function similarly within MCH neurons, consistent with our finding in females that PAE while increasing the transcripts induces in the nucleus a strong, positive correlation between the numbers of FGF2 and FGFR1 transcripts. A role of nuclear FGF2-FGFR1 signaling in the PAE-induced increase in neurogenesis and density of MCH neurons may again involve estrogen, which stimulates nuclear accumulation of both FGF2 (Garmy-Susini et al., 2004) and FGFR1 (Stachowiak et al., 2003). The stimulatory effect in both sexes of PAE on cytoplasmic FGF2 and FGFR1 transcripts within MCH neurons suggests that the transcriptional changes in the nucleus translate into an increase in protein levels (Liu et al., 2016), consistent with our evidence that PAE increases the density of neurons immunoreactive for MCH protein in embryos and postnatal offspring, in females more than males (Chang et al., 2015; Chang et al., 2018; Chang et al., 2020b).

Direct support for the role of the FGF2-FGFR1 system in mediating PAE’s stimulatory effects on development of MCH neurons is provided by our finding that maternal administration of FGF2 has strikingly similar effects to those of PAE in postnatal female offspring. Consistent with evidence that peripheral or centrally injected FGF2 stimulates the proliferation and differentiation of forebrain, hippocampal and cortical neurons (Vaccarino et al., 1999; Wagner et al., 1999; Turner et al., 2011), we find that maternal FGF2 administration at E14 increases the density of MCH neurons co-expressing FGF2 and FGFR1, a broader effect than PAE involving the entire LH, and FGF2 also increases the number of endogenous FGF2 and FGFR1 transcripts in the nucleus as well as cytoplasm of these MCH neurons. Consistent with evidence for its involvement in the neurogenesis-promoting effects in adult rodents of nicotine in the subventricular zone of the lateral ventricle (Mudò et al., 2007) and of an enriched environment in the hippocampus (Grońska-Pęski et al., 2021), we propose based on our new findings that the FGF2-FGFR1 system has a role in mediating PAE’s stimulatory effects on the development of peptide neurons and based on our recent studies (Chang et al., 2020a; Chang et al., 2020c) that these neurons originate from radial glia neuroprogenitor cells in the hypothalamic neuroepithelium. With the PAE-induced increase in MCH neurons found to be accompanied by an increase in alcohol drinking in adolescent offspring that is similarly sex dependent, stronger in females than males (Chang et al., 2018), this PAE-induced increase in FGF2-FGFR1 signaling within MCH neurons may also contribute to the escalation of this behavioral response. This is supported by studies showing FGF2 acting through FGFR1 to be a strong, positive regulator of alcohol drinking in adult rodents (Even-Chen and Barak, 2019b) and FGF2 knockout mice to drink less alcohol compared to wild-type littermates (Even-Chen 2021). PAE also stimulates other alcohol-related behaviors, such as anxiety and alcohol-induced conditioned place preference, which can contribute to the alcohol addiction phenotype exhibited by the offspring (Chang et al., 2018; Cheng et al., 2018), and these effects may also be regulated by the FGF2/FGFR1 system as suggested by the finding that FGF2 deficient mice exhibit reduced alcohol-induced conditioned place preference (Even-Chen et al., 2021). While PAE increases the susceptibility to various psychiatric disorders including depression (Brocardo et al., 2012) and schizophrenia (Stoos et al., 2015), trophic factors such as FGF2 shown to have a protective effect in these conditions (Dremencov et al., 2021) may be functioning to ameliorate the symptoms of these disorders.

While likely not the only mediator of PAE’s effect on MCH neurons, FGF2 is notable in being distinguished from other growth factors that negatively regulate alcohol intake (Liran et al., 2020) and are suppressed by PAE (Marjonen et al., 2018; Carito et al., 2019). Further studies involving genetic and direct manipulations of FGF2/FGFR1 during pregnancy that target these specific genes in the embryo are needed to elucidate the precise function of the FGF2-FGFR1 complex, within the hypothalamus as well as extra-hypothalamic areas, in mediating both these neuronal and behavioral effects of embryonic ethanol exposure.

5. Conclusion

Our evidence described here in postnatal offspring supports the involvement of the FGF2-FGFR1 system in mediating the stimulatory effect of PAE on neuronal development in the LH, with PAE markedly increasing the density and expression level of MCH neurons that co-express FGF2 and FGFR1. This effect is strongest in the dorsal area of the LH, which is involved in reward seeking and drug abuse. It is also sex related, with the PAE-induced increase in density of FGF2-expressing MCH neurons occurring only in female offspring and the increase in number of FGF2 transcripts within MCH neurons occurring in both the nucleus and cytoplasm of females but only in the cytoplasm of males. A role of the FGF2-FGFR1 system in PAE’s stimulatory effects on MCH neuronal development receives direct support from our finding in female offspring that maternal administration of FGF2 during pregnancy has strikingly similar effects to those of PAE. This increase in density of MCH neurons may be involved in promoting the increase in alcohol consumption shown to be produced by PAE, as well as by administration of FGF2 itself.

Highlights.

• Prenatal alcohol exposure (PAE) increases peptide neurons promoting alcohol intake

• PAE stimulates fibroblast growth factor 2 (FGF2) and its receptor in these neurons

• These PAE effects are stronger in female offspring, in areas involved in drug abuse

• PAE increases FGF2 in nucleus and cytoplasm of females, only cytoplasm of males

• Peripheral injection of FGF2 itself in pregnant rats mimics these effects of PAE

Abbreviations:

PAE

prenatal alcohol exposure

LH

lateral hypothalamus

MCH

melanin concentrating hormone

FGF2

fibroblast growth factor 2

FGFR1

fibroblast growth factor receptor 1

AUD

alcohol use disorder

E

embryonic day

PeF

perifornical hypothalamus

IC

internal capsule

ZI

zona incerta

F

fornix

V

ventricles

DMH

dorsomedial hypothalamus

VMH

ventromedial hypothalamus

dLH

dorsal lateral hypothalamus

vLH

ventral lateral hypothalamus

qRT-PCR

Quantitative real-time PCR

Nuc

nuclear

Cyto

cytoplasmic