Journal: bioRxiv

Article Title: Sex-specific control of feeding and defensive behaviors by MC3R neurons in the bed nuclei of the stria terminalis

doi: 10.64898/2026.02.08.702109

Figure Lengend Snippet: a, b, c Low magnification confocal images in an MC3R-Cre::tdTomato mouse illustrating MC3R neurons are distributed in distinct patterns throughout the rostrocaudal extent of the BST. Scale bar, 100 µm. d No differences in the number of MC3R neurons was found in the BSTd between males and females (n=5-6/group, unpaired t-test). e-k High magnification confocal images depicting Mc3r neurons coexpress e Slc32a1 , f Slc17a6 , g Crh , h Prkcd , i Glp1r , j Th, and k Esr1 . Scale bar, 20 µm. l, m Percentage of Mc3r neurons expressing Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 . Mc3r neurons exhibit the highest coexpression with Slc32a1 . aco, anterior commissure; al, anterolateral; am, anteromedial; BST, bed nuclei of the stria terminalis; Crh, corticotropin-releasing hormone; Esr1, estrogen receptor 1; fx, fornix; fu, fusiform; Glp1r, glucagon-like peptide-1 receptor; ju, juxtacapsular; mg, magnocellular; ov, oval; pr, principal; Prkcd, protein kinase C delta; rh, rhomboid; Slc17a6, solute carrier family 17 member 6 (vesicular glutamate transporter 2 gene); Slc32a1, solute carrier family 32 member 1 (vesicular GABA Transporter gene); Th, tyrosine hydroxylase.

Article Snippet: Total numbers of labeled, Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 neurons that co-express Mc3r were counted manually in each BSTd image stack, aided by Imaris software (Bitplane, v9.5.1).

Techniques: Expressing

Journal: bioRxiv

Article Title: Sex-specific control of feeding and defensive behaviors by MC3R neurons in the bed nuclei of the stria terminalis

doi: 10.64898/2026.02.08.702109

Figure Lengend Snippet: a Schematic of experimental paradigm. AAV5.hM3Dq-mCherry was injected into the BSTd of male and female MC3R-Cre mice. Animals were allowed to recover for at least two weeks prior to any experiments being conducted. On the day of the experiment, mice received an injection of either CNO (2mg/kg) or saline 30 minutes prior to access to food that began at the start of the dark phase. Food intake was measured for 4 hours. b Low magnification confocal image depicting representative injection site of AAV5.hM3Dq- mCherry. Scale bar, 200 µm. c-f Chemogenetically exciting BST MC3R neurons in MC3R-Cre mice in a fed state blunts cumulative 4 hour feeding in c, d males but not e, f females (c, e: n=7-10/group, 2-way ANOVA, Sidaks multiple comparisons test; d, f: n=7-10/group, unpaired t-test). g Schematic of experimental paradigm. AAV5.hM3Dq-mCherry was injected into the BSTd of male and female MC3R-Cre mice. Animals were allowed to recover for at least two weeks prior to any experiments being conducted. Mice were fasted for 24 hours. On the day of the experiment, mice received an injection of either CNO (2mg/kg) or saline 30 minutes prior to access to food. Food intake was measured for 3 hours. h-k Chemogenetically exciting BST MC3R neurons in MC3R-Cre mice has no significant effect on refeeding after a fast in h, i males or j, k females (h, j: n=7-8/group, 2-way ANOVA, Sidaks multiple comparisons test; i, k: n=7-8/group, unpaired t-test). Aco, anterior commissure; fx, fornix. * p < 0.05, # p < 0.09.

Article Snippet: Total numbers of labeled, Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 neurons that co-express Mc3r were counted manually in each BSTd image stack, aided by Imaris software (Bitplane, v9.5.1).

Techniques: Injection, Saline

Journal: bioRxiv

Article Title: Sex-specific control of feeding and defensive behaviors by MC3R neurons in the bed nuclei of the stria terminalis

doi: 10.64898/2026.02.08.702109

Figure Lengend Snippet: a Schematic of experimental paradigm. AAV5.Syn.Flex.GCaMP7f was injected into the BSTd of male and female MC3R-Cre mice and a fiberoptic cannula was implanted immediately after injection. Animals were allowed to recover for at least two weeks prior to any experiments being conducted. On the day of the experiment, photometry signal and animal behavior was concurrently recorded to time-lock GCaMP signal with specific behaviors. b Low magnification confocal image depicting representative injection site of AAV5.Syn.Flex.GCaMP7f and fiberoptic cannula placement. Scale bar, 500 µm. c Average time-locked GCaMP signal while sniffing a normal chow pellet in males (blue) and females (red). d Individual calcium transient traces of the average z-score for each animal. e, f Both the e peak and f average z-score is significantly greater than baseline in both males and females (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). g Average time-locked GCaMP signal while sniffing a high fat chow pellet in males (blue) and females (red). h Individual calcium transient traces of the average z-score for each animal i The peak z-score is significantly greater than baseline in both males and females (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). j The average z-score is significantly greater in males only (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). k Average time-locked GCaMP signal while biting a high fat chow pellet in males (blue) and females (red). l Individual calcium transient traces of the average z-score for each animal. m, n Both the m peak and n average z-score is significantly greater than baseline in males only (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). o Average time-locked GCaMP signal while sniffing a novel object (battery) in males (blue) and females (red). p Individual calcium transient traces of the average z-score for each animal. q The peak z-score is significantly greater than baseline in both males and females (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). r The average z-score is significantly greater in females only (n=8-10/group, 2-way ANOVA, Sidaks multiple comparisons test). Aco, anterior commissure; fx, fornix. * p < 0.05, ** p < 0.01. *** p < 0.001, **** p < 0.0001.

Article Snippet: Total numbers of labeled, Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 neurons that co-express Mc3r were counted manually in each BSTd image stack, aided by Imaris software (Bitplane, v9.5.1).

Techniques: Injection, Battery

Journal: bioRxiv

Article Title: Sex-specific control of feeding and defensive behaviors by MC3R neurons in the bed nuclei of the stria terminalis

doi: 10.64898/2026.02.08.702109

Figure Lengend Snippet: a Low magnification confocal image depicting representative injection site of AAVDJ-hSyn-FLEx-mGFP-2A-Synaptophysin-mRuby in the BST of an MC3R-cre mouse. Scale bar, 100 µm. b-n BST MC3R neuronal projections are present in the b ACB, c LPO, c MEPO, c, d MPO, e LS, e MS, f MPN, f PVpo, g AHN, g PVH, h, i LHA, i DMH, j VMHvl, k MEA, l VTA, m PAG, and n PBmm. o There were no sex differences in terminal density in any of the areas measured (n=3-6/group, unpaired t-test). ACB, nucleus accumbens; aco, anterior commissure; AHN, anterior hypothalamic nucleus; AVPV, anteroventral periventricular nucleus; AQ, cerebral aqueduct; BSTd, dorsal bed nuclei of the stria terminalis; DMH, dorsomedial hypothalamic nucleus; fx, fornix; LHA, lateral hypothalamic area; LPO, lateral preoptic area; LS, lateral septal nucleus; MEA, medial amygdalar nucleus; MEPO, median preoptic nucleus; MEV, midbrain trigeminal nucleus; ML, medial septal nucleus; MPN, medial preoptic nucleus; MPO, medial preoptic area; mtt, mammillothalamic tract; OPT, optic tract; OV, vascular organ of the lamina terminalis; PBmm, parabrachial nucleus, medial division, medial medial part; PAG, periaqueductal gray; PVH, paraventricular hypothalamic nucleus; PVpo, periventricular hypothalamic nucleus, preoptic part; scp, superior cerebellar peduncles; V3, third ventricle; VL, lateral ventricle; VMHvl, ventromedial hypothalamic nucleus, ventrolateral part; VTA, ventral tegmental area.

Article Snippet: Total numbers of labeled, Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 neurons that co-express Mc3r were counted manually in each BSTd image stack, aided by Imaris software (Bitplane, v9.5.1).

Techniques: Injection

Journal: bioRxiv

Article Title: Sex-specific control of feeding and defensive behaviors by MC3R neurons in the bed nuclei of the stria terminalis

doi: 10.64898/2026.02.08.702109

Figure Lengend Snippet: a Schematic of whole brain analysis pipeline. AAV1.FLEX.splitTVA.EGFPtTA and AAV1.TREtight.m.TagBFP2.B19G were injected into the right BSTd of male and female MC3R-Cre mice. One week later, EnvA-RVdg.mcherry was injected into the same area. Tissue was collected one week later, cleared, imaged and registered to the Allen Common Coordinate Reference Framework. b Three-dimensional rendering of a unilateral monosynaptic rabies injection into the BST of an MC3R-cre mouse. Scale bar, 1mm. c-f Coronal view of monosynaptic inputs to BST MC3R neurons in the same brain at the level of the c prelimbic and insular cortex, d paraventricular thalamus and hypothalamus, e lateral hypothalamic area and central amygdala, and f hippocampus and periaqueductal gray. Scale bar, 500 µm. g Average cell density of all animals (n=7) quantified by registering cell locations to the Allen Common Coordinate Reference Framework. h Top 50 areas of total average cell density in all animals (n=7) quantified by registering cell locations to the Allen Common Coordinate Framework. Over half of the top 50 areas that send inputs to BST MC3R neurons originate in the hypothalamus. i, j Representative images of rabies labeling in the PVpo of a i female and j male MC3R-cre mouse. Females have significantly more PVpo neurons sending inputs to BST MC3R neurons than males (n=3-4/group, unpaired t-test). Scale bar, 100 µm. k, l Representative images of rabies labeling in the PSTN of a k female and l male MC3R-cre mouse. Males have significantly more PSTN neurons sending inputs to BST MC3R neurons than females (n=3-4/group, unpaired t-test). Scale bar, 100 µm. m, n Comparison of average cell density between females and males quantified by registering cell locations to the Allen Common Coordinate Reference Framework (n=3-4/group, unpaired t-test). PSTN, parasubthalamic nucleus; PVpo, periventricular hypothalamic nucleus, preoptic part. # p < 0.06, * p < 0.05, **** p < 0.0001.

Article Snippet: Total numbers of labeled, Slc32a1 , Slc17a6 , Crh , Prkcd , Glp1r , Th , and Esr1 neurons that co-express Mc3r were counted manually in each BSTd image stack, aided by Imaris software (Bitplane, v9.5.1).

Techniques: Injection, Labeling, Comparison