# Imbalanced innervation of the hypothalamic paraventricular nucleus by the AgRP and α-MSH axonal projections in germ-free mice

Oarii Vahirua, Mouna El Mehdi, Emilie Lahaye, Aurélie Balvay, Sylvie Rabot, Sergueï O Fetissov

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Oarii Vahirua, Mouna El Mehdi, Emilie Lahaye, Aurélie Balvay, Sylvie Rabot, et al. Imbalanced innervation of the hypothalamic paraventricular nucleus by the AgRP and α-MSH axonal projections in germ-free mice. Developmental Neuroscience, 2025, pp.1-13. 10.1159/000549483. hal-05403527

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# Imbalanced Innervation of the Hypothalamic Paraventricular Nucleus by the AgRP and α-Melanocyte-Stimulating Hormone Axonal Projections in Germ-Free Mice

Oarii Vahirua<sup>a</sup> Mouna El Mehdi<sup>a</sup> Emilie Lahaye<sup>a</sup> Aurélie Balvay<sup>b</sup>

Sylvie Rabot<sup>b</sup> Sergueï O. Fetissov<sup>a</sup>

<sup>a</sup>Regulatory Peptides – Energy Metabolism and Motivated Behavior Team, Neuroendocrine, Endocrine and Germinal Differentiation and Communication Laboratory, Inserm UMR1239, University of Rouen Normandie, Rouen, France; <sup>b</sup>Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

## Keywords

Hypothalamus · Arcuate nucleus · Neuropeptides · Appetite · Gut microbiota-brain axis · Germ-free mice

## Abstract

**Introduction:** Innervation of the paraventricular nucleus of the hypothalamus (PVN) by the orexigenic agouti-related protein (AgRP) and anorexigenic α-melanocyte-stimulating hormone (α-MSH) neurons of the arcuate nucleus (ARC) is a key element in the appetite-regulating neuronal circuitry whose development is influenced by circulating metabolic signals. In the present work, we studied if PVN innervation by the AgRP and α-MSH fibers is influenced by gut microbiota.

**Methods:** To this aim, we compared, using immunohistochemistry, the innervation of PVN by AgRP and α-MSH fibers between germ-free and specific pathogen-free 7-week-old female mice. **Results:** We found that germ-free mice display an increased innervation of the PVN by both AgRP and α-MSH fibers, but also that the increase in AgRP fiber density was about twice as pronounced as that of α-MSH. **Conclusion:** These data reveal that gut microbiota plays a modulatory role in the development of the ARC/

PVN axonal projections. An imbalance between AgRP and α-MSH innervation in germ-free mice may contribute to their metabolic and behavioral alterations.

© 2025 The Author(s).
Published by S. Karger AG, Basel

## Introduction

Development of the axonal projections of agouti-related protein (AgRP)- and α-melanocyte-stimulating hormone (α-MSH)-synthesizing neurons of the arcuate nucleus (ARC) of the hypothalamus occurs in early ontogenesis, reaching the adult levels at about the postnatal day 21 [1–3]. AgRP is orexigenic by antagonizing the activation of melanocortin receptor type 4 (MC4R) by anorexigenic α-MSH [4]. AgRP is co-expressed in the ARC with neuropeptide Y (NPY), another key orexigenic mediator [5]. The paraventricular nucleus of the hypothalamus (PVN) is one of the main targets for the AgRP/NPY and α-MSH ARC projections involved in the integration of metabolic, autonomic, and stress-related signals, and it is an integral part of the brain appetite circuitry [6, 7]. Indeed, insufficient

<footnote>karger@karger.com
www.karger.com/dne</footnote>

<footnote>© 2025 The Author(s).
Published by S. Karger AG, Basel</footnote>

<footnote>This article is licensed under the Creative Commons Attribution 4.0 International License (CC BY) (https://karger.com/Services/OpenAccessLicense). Usage, derivative works and distribution are permitted provided that proper credit is given to the author and the original publisher.</footnote>

<footnote>Correspondence to:
Sergueï O. Fetissov, serguei.fetissov@univ-rouen.fr</footnote>
development of the AgRP/NPY ARC/PVN projections, even with the increased *agrp* gene expression, underlies anorexia in several genetically altered mouse strains [8, 9]. By contrast, a decrease of α-MSH PVN projections was found in diabetic mice displaying hyperphagia [10]. A relative abundance of the orexigenic AgRP/NPY and anorexigenic α-MSH innervation of the PVN may, hence, shift the regulation of appetite and energy metabolism toward anabolic or catabolic processes, respectively. Importantly, the development of the ARC/PVN appetite circuitry is influenced by circulating metabolic signals, including leptin, ghrelin, insulin, etc., produced in the context of maternal nutritional environment (for reviews, see [11, 12]). It suggests that such signals may have a long-term imprinting effect on the development of the appetite circuitry and establishment of the metabolic phenotype.

Recently, gut microbiota appeared as an integral part of the regulation of many physiological processes, including brain function and behavior, and appetite control [13, 14]. Some specific bacterial metabolites can activate the ARC neurons, while the absence of microbiota in germ-free (GF) mice alters expression of neuropeptide mRNA in the ARC in response to starvation [15–17]. The establishment of the infant gut microbiota occurs early postnatally, with the transfer of maternal microbiota coinciding with the development of different body systems, including the brain [18]. These data suggest that gut microbiota may also influence the development of the ARC/PVN appetite circuitry.

Thus, in this study, we explored whether the absence of microbiota in GF mice may modify the AgRP and α-MSH innervation of the PVN as compared to specific pathogen-free (SPF) mice of the same age. We selected female mice for this study, based on the fact that some eating disorders, such as anorexia nervosa, are about 10 times more frequent in females [19].

## Methods

Animal care and experimentation complied with both French and European Community regulations, and the study was approved by the Normandie Regional Ethics Committee (APAFIS #44684). SPF C3H/HeN 6-week-old female mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Before perfusion, they were housed for 1 week of acclimation with a 12-h light-dark cycle at 23°C in ventilated standard holding cages for mice (Tecniplast, Buguggiate, Italy), 3 animals per cage

with ad libitum access to a pelleted standard diet (Envigo, Paris, France) and drinking water.

GF C3H/HeN female mice were born in breeding isolators in the GF animal facility of ANAXEM (Micalis Institute, INRAE, Jouy-en-Josas, France). Mice had access to γ-irradiated (45 kGy) standard diet (R03; Scientific Animal Food and Engineering, Augy, France) and autoclaved tap water ad libitum. Mice were exposed to a 12-h light/dark cycle with ambient temperature between 20 and 24°C. The GF status of the animals was monitored every week by microscopic examination and aerobic and anaerobic cultures of drinking water and freshly voided fecal samples.

At the age of 7 weeks, both SPF (n = 6) and GF (n = 6) female mice were anaesthetized by an intraperitoneal injection of a lethal dose of Ketamine/Xylazine (3:1 vol, 0.1 mL) and perfused intracardially with 20 mL of PBS followed by 40 mL of 4% paraformaldehyde in PBS. Brains were dissected out and immersed in the same fixative overnight, then kept in PBS at 4°C until cut using a vibratome (Leica VT1000S, Leica Microsystems SA, Nanterre, France) to obtain 50 µm coronal sections from the middle part of the PVN, approximately –0.8–0.9 mm from Bregma [20]. Up to 4 adjacent sections were collected for AgRP and α-MSH staining. Floating sections were processed for immunohistochemical staining. Briefly, sections were permeabilized with PBS – Triton X100 0.3% NaAzide 0.01% at room temperature under agitation during 1 h, then incubated at 4°C during 24 h with primary polyclonal rabbit antibodies directed against AgRP (Phoenix Pharmaceuticals Inc, Burlingame, CA, USA) or against α-MSH (Peninsula Laboratories, Bachem AG, Bubendorf, Switzerland) both diluted 1/1000 in PBS. The sections were incubated with anti-rabbit fluorescein-conjugated secondary antibody (Alexa Fluor 488 Donkey-anti-Mouse, 1/1000e, Invitrogen A21202) for 2 h, at room temperature. Sections were mounted on Superfrost® Plus slides (Thermo Fisher Scientific, Waltham, MA, USA) in an anti-fading agent (Sigma Aldrich, L'Isle-d'Abeau Chesnes, Saint-Quentin-Fallavier, France) and coverslipped.

Sections were imaged using a Thunder Tissue 3D fluorescence microscope (Leica Microsystemes SAS, Nanterre, France) equipped with an HC PL APO 100×/1.40 oil immersion objective. Images from the PVN region were acquired at 0.0651 × 0.0651 × 0.2508 µm³ voxel size (X × Y × Z), 16-bit. Each stack comprised 21 Z-planes (0.2508 µm steps; total Z-depth 5.21 µm). Imaging was deliberately limited to the upper 5.21 µm of the section to minimize optical artifacts. Beyond this depth, we observed progressive degradation of image
quality caused by light scattering, refractive index mismatch, and spherical aberrations, resulting in a “halo” effect and loss of fine fiber detail. Because our aim was to quantify the relative coverage of labeled fibers with optimal structural preservation, we restricted acquisition within these 5.21 µm. The same fixed Z-depth and analysis pipeline were used for all samples and both experimental groups.

The images were analyzed using the public source “Image J” program (National Institute of Health, USA). In all analyses, the Z-stacks were first converted into mean-intensity projections to ensure that the entire fluorescent signal of the fibers within the sampled 5.21 µm depth was represented in a single 2D image. A semiautomated threshold was applied to separate fibers from the background. Threshold selection was guided by the image histogram and high signal noise ratio, which provided a clear intensity valley between background and fiber signal. We then employed the Fluorescence AnalysisIS Tool (FAST) plugin in ImageJ to automatically detect and extract multiple regions of interest (ROIs) corresponding to the fibers. These ROIs were subsequently merged into a single composite ROI to calculate the total fiber-covered area relative to the reference region surface.

For the quantification of PVN innervation by AgRP and α-MSH fibers, the same zone of interest delimiting the entire unilateral PVN as well as its subdivisions was applied on all the images containing these structures and aligned by the wall of the 3rd brain ventricle (Fig. 1, 2). The percentage of coverage by immunopositive peptide fibers within each zone toward the entire PVN (100%) was calculated.

Data were analyzed and graphs were plotted using the GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Each point on the graph corresponds to the data obtained from a section. Normality of distribution was evaluated by the Kolmogorov-Smirnov test. Group differences were analyzed using a two-tailed unpaired Student's t test, with *p* < 0.05 considered significant.

## Results

### Innervation of the PVN by AgRP Neurons

AgRP-positive fibers were detected in the PVN of all studied mice showing a typical triangular pattern observed on both sides of the 3rd ventricle of the coronal brain sections (Fig. 1a). By comparing the density of AgRP fibers in the PVN between SPF and GF mice, we found that the percentage of PVN coverage by these

fibers was increased in all the PVN subdivisions including both magnocellular and parvocellular parts (Fig. 1b–f) as well as in the entire PVN (Fig. 1f).

### Innervation of the PVN by α-MSH Neurons

α-MSH-positive fibers were present in the PVN of both SPF and GF mice, showing a less dense than AgRP staining pattern over the PVN (Fig. 2a). The comparison of the PVN coverage by α-MSH fibers between SPF and GF mice revealed a slight but significant increase in the GF group present in most of the PVN subdivisions. This includes both lateral and medial magnocellular parts (Fig. 2b, d) as well as the medial parvocellular part in which such an increase was the most prominent (Fig. 2e). Overall, the entire PVN showed higher α-MSH fiber coverage in GF than in SPF mice (Fig. 2f).

### Relative Innervation of the PVN by AgRP and α-MSH Fibers

The mean percentage of the PVN innervation by AgRP and α-MSH fibers in SPF mice was 4.0% and 4.5%, respectively, without significant differences between the two peptides (Fig. 3a). However, in GF mice the mean percentage of the PVN innervation by AgRP fibers (12.4%) was higher than that of α-MSH fibers (5.9%), as shown in Figure 3b. Accordingly, the ratios of AgRP/α-MSH fibers' coverage of the PVN were 0.89, i.e., close to 1.0 (Fig. 1c), while in GF mice it was imbalanced with AgRP fibers about twice as more abundant as α-MSH fibers (Fig. 3d).

## Discussion

The results of this study demonstrate that the absence of microbiota in GF mice has an impact on the development of ARC AgRP and α-MSH axonal projections to the PVN. Considering that an increased PVN innervation by both AgRP and α-MSH was found in GF mice, we can conclude on the general inhibitory influence of microbiota on the development of the ARC/PVN projections. Such an inhibitory effect was nevertheless distinct between the AgRP and α-MSH neuronal populations, being more important for AgRP neurons, since the absence of microbiota in GF mice was accompanied by a more pronounced increase of AgRP than α-MSH PVN innervation. We cannot exclude either, that gut microbiota may produce both inhibitory and stimulatory growth factors, whose relative abundance may influence the development of the ARC/PVN projections both
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Innervation of the PVN by AgRP fibers. **a** Immunohistochemical visualization of AgRP-containing neuronal fibers in the PVN of SPF (left side) and GF (right side) mice. Different parts of the PVN are surrounded by dashed lines and referenced according to the corresponding number. Percentage of AgRP-positive fiber coverage of the PVN in different parts of the PVN: (b) PaLM, (c) PaDC, (d) PaMM, (e) PaMP, and (f) the entire PVN. Student's *t* test, \*p < 0.05, \*\*\*p < 0.0001. 3v, third ventricle, scale bar, 200 µm.</figcaption></figure>

directly and indirectly, e.g., via gut hormones and immune system [21].

A balance between AgRP and α-MSH projections should underlie an equilibrium in the capacity of corresponding neurons to regulate the activation of MC4R in the PVN target neurons [22]. Such a balance is necessary for a physiological regulation of appetite and energy metabolism in response to circulating metabolic factors such as leptin and insulin, as well as

gastrointestinal hunger/satiety hormones, such as ghrelin, peptide YY (PYY), etc. [23]. Indeed, the present work reveals that SPF mice display almost equal intensity of PVN innervation by AgRP and α-MSH fibers, with their mean ratio of the percentage of PVN coverage 0.89, i.e., close to 1.0. The situation is different in GF mice, indicating that mice of the same genotype but housed under GF conditions exhibit AgRP innervation of the PVN that is approximately
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Innervation of the PVN by α-MSH fibers. **a** Immuno-histochemical visualization of α-MSH-containing neuronal fibers in the PVN of SPF (left side) and GF (right side) mice. Different parts of the PVN are surrounded by dashed lines and referenced according to the corresponding number. Percent-</figcaption></figure>

age of α-MSH-positive fiber coverage of the PVN in different parts of the PVN: (b) PaLM, (c) PaDC, (d) PaMM, (e) PaMP, and (f) the entire PVN. Student's *t* tests, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, ns, non-significant, 3v, third ventricle, scale bar, 200 µm.

twice as abundant as that of α-MSH fibers. Such an imbalance in favor of the orexigenic pathway suggests that hyperphagia might be expected in GF mice. Indeed, earlier studies demonstrated that food intake was about 30% higher in GF than in conventionally raised mice [24]. A similar imbalance of AgRP and α-MSH PVN innervation was observed in mice deficient for amylin, an anorexigenic peptide produced by pancreatic β-cells [25].

Subdivisions of the PVN contain distinct neuronal populations producing different peptides involved in the regulation of homeostatic functions, stress response, and appetite. Both magnocellular and parvocellular populations of the PVN neurons express oxytocin and vasopressin [26]. PVN oxytocin neurons are one of the ARC anorexigenic targets while AgRP neurons mediate development of oxytocin connectivity [27, 28]. Corticotropin is another anorexigenic peptide produced by
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Comparison of PVN innervation between AgRP and α-MSH fibers in SPF (a, c) and GF (b, d) mice as shown by the percentage of AgRP and α-MSH PVN fiber coverage (a, b) and their ratios (c, d). Student's *t* test *p* < 0.05, ns, non-significant.</figcaption></figure>

the PVN neurons, which express MC4R [29]. In the present study, we did not find marked differences in the AgRP or α-MSH innervations between the PVN subdivisions, suggesting a nonselective effect of microbiota on the development of ARC projections toward different parts of the PVN. This is in contrast to brain-derived neurotrophic factor, which was shown to stimulate AgRP projections toward the lateral part of PVN [30]. In this context, it is of interest that the PVN of newborn GF mice was shown to contain more microglia and cell death markers than in conventional mice eventually leading to reduced neuronal cell number [31, 32].

Besides their implication in appetite regulation, the oxytocin neurons play a major role in enhancing social interactions. It is, hence, of relevance to this study that supplementation of *Lactobacillus reuteri* to the offspring of mice fed with high-fat diet increased oxytocin production in the PVN and improved social behavior [33]. Maternal high-fat diet is known to alter the development of the ARC/PVN projections [34]. It indicates that the ARC/PVN projections can be a valid target also for the correction of altered social behavior via the microbiome modulation, a new therapeutic approach which is currently actively explored [35].

It is important to mention that other than PVN targets of the ARC are involved in the regulation of appetite,

both hypothalamic and extrahypothalamic, e.g., amygdala and the dorsal raphe nucleus [36, 37]. It would be of interest to study the influence of microbiota on the development of ARC innervation of different brain structures. It will also be important to identify the key bacterial species and their derived specific metabolites underlying the neurotrophic effects of these bacteria and also considering the bidirectional nature of such effects [38].

In conclusion, the study revealed that gut microbiota plays a modulatory, mostly inhibitory role in the development of the ARC/PVN axonal projections. In particular, an imbalance between AgRP and α-MSH PVN innervation, showing increased AgRP fiber density in GF mice, indicates that it may contribute to the metabolic and behavioral alterations.

## Acknowledgments

We would like to acknowledge the contribution of all the members of research consortia EC H2020 "GEMMA," ERAnet "MIGBAN and PTM-2, Inserm, for their roles in funding acquisition. We although thank the Imaging platform PRIMACEN (INSERM US51), University of Rouen Normandie, France where the microscopy was performed.
**Statement of Ethics**

Animal care and experimentation complied with both French and European Community regulations and the study was approved by the Normandy Regional Ethics Committee (APAFIS #44684).

**Conflict of Interest Statement**

The authors have no conflicts of interest to declare.

**Funding Sources**

The study was supported by “GEMMA”- Genome, Environment, Microbiome and Metabolome in Autism (project ID 825033) funded by the European Commission in the frame of the Horizon 2020 program (call H2020-SC1-BHC-03-2018). ERA-NET Neuron MiGBAN consortium (01EW1906A-01EW1906B), “Microbiome Gut Brain Axis in Anorexia Nervosa,” funded by the European Union (EU), the German Ministry for Education and

Research (BMBF), and the French Agence Nationale de la Recherche (ANR). S.O.F. research was also supported by the PTM-2 program, Inserm, France.

**Author Contributions**

S.O.F.: conceptualized the study and wrote the manuscript draft. A.B. and S.R.: breed animals. S.O.F., A.B., and S.R.: made perfusions. O.V. and M.E.M.: cut the brains and performed immunohistochemistry and microscopy. O.V.: analyzed the data and prepared the figures. S.O.F. and S.R.: obtained funding. S.O.F. and E.L.: prepared the ethical permits. All authors contributed to the final manuscript.

**Data Availability Statement**

The data that support the findings of this study are not publicly available due to restricted access. These data can be available upon written request to the corresponding author.

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