Neuroanatomical and Functional Consequences of Oxytocin Treatment at Birth in Prairie Voles

Abstract

Birth is a critical period for the developing brain, a time when surging hormone levels help prepare the fetal brain for the tremendous physiological changes it must accomplish upon entry into the „extrauterine world’. A number of obstetrical conditions warrant manipulations of these hormones at the time of birth, but we know little of their possible consequences on the developing brain. One of the most notable birth signaling hormones is oxytocin, which is administered to roughly 50% of laboring women in the United States prior to / during delivery. Previously, we found evidence for behavioral, epigenetic, and neuroendocrine consequences in adult prairie vole offspring following maternal oxytocin treatment immediately prior to birth. Here, we examined the neurodevelopmental consequences in adult prairie vole offspring following maternal oxytocin treatment prior to birth. Control prairie voles and those exposed to 0.25 mg/kg oxytocin were scanned as adults using anatomical and functional MRI, with neuroanatomy and brain function analyzed as voxel-based morphometry and resting state functional connectivity, respectively. Overall, anatomical differences brought on by oxytocin treatment, while widespread, were generally small, while differences in functional connectivity, particularly among oxytocin-exposed males, were larger. Analyses of functional connectivity based in graph theory revealed that oxytocin-exposed males in particular showed markedly increased connectivity throughout the brain and across several parameters, including closeness and degree. These results are interpreted in the context of the organizational effects of oxytocin exposure in early life and these findings add to a growing literature on how the perinatal brain is sensitive to hormonal manipulations at birth.

1. Introduction:

Oxytocin (OXT) is a potent and pleiotropic hormone that surges at birth to help facilitate the tremendous changes that both mammalian mothers and their offspring must accomplish upon delivery (Kenkel, 2020; Kenkel et al., 2014; Kingsbury and Bilbo, 2019). As of 2019, 29.4% of laboring women in the U.S. received OXT to induce labor (Martin et al., 2021), and according to available survey data, this figure raises to ~50% of birthing women in America when considering OXT used to either induce and/or augment labor (Declercq et al., 2014). This obstetric practice is of interest to neuroscience because there is evidence OXT can cross the placenta (Malek et al., 1996) and a growing literature suggests the neonatal brain is particularly sensitive to OXT around the time of birth, when OXT receptor (Oxtr) expression begins to accelerate (Rokicki et al., 2022) and OXT neurons in the brain undergo intense remodeling (Madrigal and Jurado, 2021). Indeed, the long-term, developmental effects of OXT manipulations in early life are well-documented in animal models (Bales and Perkeybile, 2012; Hammock, 2015), which suggests that the perinatal period may be a sensitive period with regard to the impact of OXT.

Some initial studies suggested higher rates of autism spectrum and attention deficit / hyperactivity disorder amongst children born to women whose labors were induced with OXT; however, meta-analysis of these findings suggest that any such conclusions remain premature (Lønfeldt et al., 2019) and more recent work examining nationwide registries suggest that such associations likely arose from confounding factors (Lønfeldt et al., 2020; Stokholm et al., 2021). While there have been conflicting reports as to whether OXT administered to induce / augment labor is associated with increased rates of autism spectrum disorder or autistic-like behavior in offspring, considerations of dose add an important degree of nuance to this topic (Guastella et al., 2018; Soltys et al., 2020). Thus, regardless of whether the consequences of obstetrically administered OXT raise to the level of a neurodevelopmental disorder, the question of whether OXT affects offspring neurodevelopment is of great public health relevance given its widespread use.

Previously, we investigated the impact of maternally administered OXT on offspring neurodevelopment and behavior using the socially monogamous prairie vole (Kenkel et al., 2019). We found that fetal physiology was indeed sensitive to maternally administered OXT and that, in the fetal brain, such OXT dose-dependently increased methylation of the Oxtr promoter. In adulthood, OXT-exposed offspring of both sexes were found to demonstrate a broadly gregarious phenotype such that they exhibited more spontaneous alloparental care toward unrelated pups and spent more time in close social contact with opposite-sex adults. Male voles exposed to OXT also showed increased density of OXT receptor in the central amygdala, insular cortex, and parietal cortex, while showing decreased vasopressin receptor 1a density in the ventral pallidum. These findings built upon a substantial body of research, carried out largely in prairie voles, that established long-lasting, often sexually-dimorphic and dose-dependent organizational effects on both brain and behavior following direct treatment of neonatal vole pups with OXT on the first postnatal day (Bales and Carter, 2003; Kramer et al., 2006; Yamamoto et al., 2004).

Because OXT is a pleiotropic hormone, we opted for a broad survey of the brain in the present study, carrying out whole-brain resting functional connectivity to further characterize the scope of the neurodevelopmental consequences of OXT exposure at birth. Here, we used magnetic resonance imaging (MRI) to scan the brains of adult male and female prairie vole offspring originally born to pregnant females treated with 0.25 mg/kg OXT on the expected day of delivery. We examined both anatomical measures (voxel-based morphometry (VBM) and diffusion-weighted imaging (DWI)) as well as functional measures (resting state functional connectivity (rs-fMRI) and several graph theory measures (detailed below).

2. Materials and Methods:

2.1. Subjects

Prairie vole offspring (Microtus ochrogaster) were generated as previously described (Kenkel et al., 2019). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Northeastern University. On the expected day of delivery, pregnant females were either injected intraperitoneally with OXT (0.25 mg/kg, ‘OXT’) or left undisturbed (‘Control’). Administrations of this dose of OXT to term pregnant females is beneath the threshold to induce precipitous labor. At 0.25 mg/kg, this dose is equivalent to a 5 IU dose in humans, which is within the range of reported doses used clinically (1.8 – 5.5 IU) (Grotegut et al., 2017; Guastella et al., 2018; Soltys et al., 2020).

Offspring were only included if they were delivered within 24 hours of OXT treatment. Offspring were raised by their birth parents as we have previously observed no effect of maternal-OXT treatment on offspring-infant interaction of a similar maternal treatment OXT (Kenkel et al., 2019). At 20 days of age, OXT and Control offspring were weaned into same-sex sibling pairs and were left to mature. Upon reaching adulthood (postnatal days 60–70), OXT and Control offspring underwent three neuroimaging scans: aT1-weighted anatomical scan for voxel based morphometry scan (VBM), an awake, resting state functional scan (rs-fMRI), and an anesthetized diffusion-weighted imaging scan (DWI), as detailed below. Subject offspring consisted of 17 Control females, 19 Control males, 17 OXT-exposed females, and 16 OXT-exposed males. From these, 7 Control females, 12 Control males, 13 OXT-exposed females and 13 OXT-exposed males were ultimately included in the rs-fMRI analyses after removing subjects due to motion artefact or technical difficulties.

2.2. Neuroimaging

All neuroimaging measures were collected using a Bruker BioSpec 7.0T/20-cm Ultra Shield Refrigerated horizontal magnet (Bruker, Billerica, MA). A 20-G/cm magnetic field gradient insert (inner diameter 12 cm) was used to scan anesthetized subjects using a quadrature transmit/receive volume coil (inner diameter 38 mm). Imaging sessions began with an anatomical scan with the following parameters: 20 slices; slice thickness, 0.70 mm; field of view, 2.5 cm; data matrix, 256 x 3 x 256; repetition time, 2.5 seconds; echo time (TE), 12.0 ms; effective TE, 48 ms; number of excitations, 2; and total acquisition time, 80 seconds. VBM yields measures of the volume of each brain region scaled, while DWI yields several measures related to the diffusivity of water within gray matter microarchitecture. The DWI measure apparent diffusion coefficient (ADC) reflects the impedance of water diffusion through Brownian movement in any direction, where high values indicate restricted diffusion. On the other hand, fractional anisotropy (FA) represents the directionality of water diffusion within brain tissue, with low values suggesting less restrictions on water’s movement. Finally, rs-fMRI reports the degree of correlation between brain regions in the absence of any stimuli beyond the conditions of scanning.

2.2.1. Voxel Based Morphometry (VBM)

The following procedures were adapted for use in the vole from those described previously for rats (Lawson et al., 2020). For each subject, the atlas (image size 256 x 256 x 63) (H × W × D) was warped from the standard space into the subject image space (image size 256 × 256 × 40) using the nearest-neighbor interpolation method. In the volumetric analysis, each brain region was therefore segmented, and the volume values were extracted for all 111 regions of interest (ROIs), calculated by multiplying unit volume of voxel (in mm³) by the number of voxels using an in-house MATLAB script. To account for different brain sizes, all ROI volumes were normalized by dividing each subjecťs ROI volume by their total brain volume.

2.2.2. Diffusion-weighted Imaging (DWI)

The following procedures were identical to those described previously (Ferris et al., 2019; Kulkarni et al., 2020). Diffusion-weighted imaging (DWI) was acquired with a spin-echo echo-planar imaging (EPI) pulse sequence with the following parameters: repetition time/TE, 500/20 ms; 8 EPI segments; and 10 noncollinear gradient directions with a single b-value shell at 1000 seconds/mm² and 1 image with a b-value of 0 seconds/mm² (referred to as b0). Geometrical parameters were as follows: 48 coronal slices, each 0.313 mm thick (brain volume) and with in-plane resolution of 0.313 x 3 x 0.313 mm² (matrix size, 96 x 3 x 96; field of view, 30 mm²). The imaging protocol was repeated 2 times for signal averaging. DWI acquisition took 35 to 70 minutes. DWI included diffusion-weighted three-dimensional EPI image analysis producing FA maps and apparent diffusion coefficient. DWI analysis was implemented with MATLAB (version 2017b) (The MathWorks, Inc., Natick, MA) and MedINRIA version 1.9.0 (http://www-sop.inria.fr/asclepios/software/MedINRIA/index.php) software.

Each brain volume was registered with the three-dimensional MRI Vole Brain Atlas template (Ekam Solutions LLC, Boston, MA) allowing voxel- and region-based statistics (Yee et al., 2016). In-house MIVA software was used for image transformations and statistical analyses. For each vole, the b0 image was coregistered with the b0 template (using a 6-parameter rigid-body transformation). The coregistration parameters were then applied on the DWI indexed maps for each index of anisotropy. Normalization was performed on the maps providing the most detailed and accurate visualization of brain structures. Normalization parameters were then applied to all indexed maps and then smoothed with a 0.3-mm Gaussian kernel. To ensure that preprocessing did not significantly affect anisotropy values, the nearest neighbor option was used following registration and normalization.

2.2.3. Resting State Functional MRI (rs-fMRI)

We used the same equipment and scanning protocols as in our recent work; for complete details see (Ortiz et al., 2020, 2022; Yee et al., 2016). Data were analyzed as 111 nodes corresponding to brain regions specified in a vole-specific atlas (Yee et al., 2016). Pearson’s correlation coefficients were computed per subject across all node pairs (6105), assessing temporal correlations between brain regions. Then, r-values’ (−1 to 1) normality were improved using Fisher’s Z-transform. For each group, 111×111 symmetric connectivity matrices were constructed, each entry representing the strength of edge. An |Z|=2.3 threshold was used to avoid spurious or weak node connections (Worsley, 2001).

2.2.4. Network Analyses

Graph theory network analysis was generated using Gephi, an open-source network and visualization software (Bastian et al., 2009). For all groups, the absolute values of their respective symmetric connectivity matrices were imported as undirected networks and a threshold of |Z|=2.3 was applied to each node’s edges to avoid spurious or weak node connections (Worsley et al., 1992).

2.2.5. Betweenness Centrality

Betweenness centrality analyzes occurrences where a node lies in the path connecting other nodes (Freeman, 1977). Let ${\text{n}}_{i,j}^{\text{k}}$ be the number of pathways from i to j going through k. Using these measures of connection, the betweenness of vertex k is:

$$B_k={\sum }_{ij}\frac{n_{i,j}^k}{n_{ij}}.$$

2.2.6. Degree Centrality

Degree centrality indicates the number of associations of a specific node (Freeman, 1978). Non-weighted, binary degree is defined as:

$$C_D\left(j\right)=\sum _{j=1}^n{A}_{ij}$$

where n is the number of rows in the matrix in the adjacency matrix A and the elements of the matrix are given by A_ij, the number of edges between nodes i and j.

2.2.7. Closeness Centrality

Closeness centrality measures the average distance from a given starting node to all other nodes in the network (Sabidussi, 1966). Closeness is defined as:

$$C\left(x\right)=\frac{N-1}{{\sum }_{y}d\left(y,x\right)}$$

where d(y,x) is the distance between vertices x and y and N is the number of nodes in the graph.

2.3. Statistics

Normality tests of control females, control males, OXT-exposed females and OXT-exposed males were performed to examine if parametric or non-parametric assumptions were required for future analysis. Shapiro-Wilk’s tests were performed to examine normality assumption for degree, closeness and betweenness centrality values. Regional p-values that were greater than 0.05 were assumed to be normal. A corresponding list of nodes that classified a region is detailed in table S1. After assumptions of normality were validated, one-way ANOVA tests were used to compare differences in degree, closeness and betweenness centralities between groups. When necessary, a nonparametric Kruskal-Wallis test was performed if there was evidence against normality assumption. Statistical differences between groups were determined using a Mann-Whitney U test (a = 5%). The following formula was used to account for false discovery from multiple comparisons:

$$P\left(i\right)\le \frac{i}{V}\frac{q}{c\left(V\right)}$$

P(i) is the p value based on the t test analysis. Each of 111 regions of interest (ROIs) (i) within the brain containing V ROIs. For graph theory measures, statistical analyses were calculated using GraphPad Prism version 9.0.0 for MacOS (GraphPad Software, San Diego, California USA, www.graphpad.com). For post-hoc analyses of graph theory parameters, Holm-Šídák test (for parametric) and Dunn’s (for nonparametric) were used after correction for multiple comparisons.

3. Results:

Detailed results can be found in the figures and tables; here, we will address broad patterns such as the number of brain regions affected for a given measure. We observed a number of differences in adult VBM measures after OXT treatment at birth; however, most were small to very small in effect size (Figure 1 and Tables 1–3). Accounting for sex differences was important because we previously found males to be more sensitive in terms to the neuroanatomical effects of OXT treatment at birth (Kenkel et al., 2019). Within the Control group, there was only a single sex difference in regional volume (the reticular nucleus); within the OXT group, however, OXT-exposed males had smaller volumes in 8 of 17 cortical regions and larger volumes in 11 brainstem / cerebellar regions compared to OXT-exposed females (Table 1). Similarly, OXT-exposed males had smaller volumes in 9 of 17 cortical regions and larger volumes in 9 brainstem / cerebellar regions compared to Control males (Table 2). Comparing within females, OXT treatment at birth resulted in smaller volumes in 4 of 17 cortical regions (Table 3). Control animals generally had larger amygdalar volumes than OXT animals (4 of 6 subregions in males; 2 of 6 in females). When morphometry data from all 111 brain regions were loaded into a PCA, the overall explanatory value of dimensions 1 and 2 was modest (29.4% and 15.9% respectively) and there were impacts of both sex and treatment on dimension 1, with male sex (F(1,69) = 4.98, p = 0.029) and OXT treatment (F(1,69) = 16.06, p < 0.001) leading to greater values (Figure 2).

Figure 1.

A 3D color coded reconstructions summarizing the significantly different brain areas with volumetric changes for each experimental condition. Details of these differences can be found in tables 1–3.

Table 1.

A) a list of the single brain area, reticular nucleus of the thalamus, that significantly differs (p=0.021, critical value p<0.05) in volume between adult female and male voles that were treated with saline vehicle within 24 hrs of parturition. Shown are the average and standard deviation in volume in mm3 and effects size (omega square ωSq). With a false discovery rate (FDR) p=0.0017 this singular finding can be dismissed concluding there is no sex difference in brain volumes between female and male voles resulting following the OXT manipulation used here. In contrast, Table 1B lists the brain areas that are significantly different in volume between adult female and male voles exposed to OXT during birth (FDR p=0.043). The brain areas are ranked in order of significance and are truncated from a larger list of 116 areas taken from the vole MRI atlas (see Supplementary Table S1).

A	Female Vehicle vs Male Vehicle - Brain Volumes (mm3)
		Female			Male
	Brain Area	Ave	SD		Ave	SD	P-val	ω Sq
	reticular n.	2.6	0.4	>	2.3	0.3	0.021	0.167325

B	Female Oxytocin vs Male Oxytocin - Brain Volumes (mm3)
		Female			Male
	Brain Area	Ave	SD		Ave	SD	P-val	ω Sq
	frontal association ctx	12.4	3.1	>	9.7	2.8	0.003	0.168232
	ventral tegmental area	0.6	0.2	<	0.8	0.3	0.008	0.134096
	vestibular n.	1.5	0.6	<	2.2	0.8	0.009	0.12879
	agranular insular ctx	11.6	2.7	>	9.7	2.5	0.010	0.127444
	retrosplenial ctx	7.5	0.8	>	6.7	1.0	0.014	0.112513
	gigantocellular reticular n.	5.1	1.3	<	5.8	1.6	0.017	0.104148
	median raphe n.	0.5	0.2	<	0.8	0.4	0.019	0.100934
	secondary motor ctx	13.1	2.2	>	11.4	2.5	0.019	0.100645
	anterior hypothalamus	3.6	0.9	>	3.1	1.0	0.019	0.099491
	parasubiculum	1.4	0.4	<	1.8	0.5	0.021	0.096134
	paraflocculus cerebellum	5.4	1.7	<	6.1	1.6	0.022	0.094944
	cuneate n.	0.1	0.2	<	0.4	0.5	0.026	0.087519
	parietal ctx	2.5	0.7	>	2.1	0.6	0.029	0.083929
	7th cerebellar lobule	0.6	1.4	<	2.3	2.8	0.031	0.08128
	claustrum	1.9	0.5	>	1.5	0.6	0.031	0.080768
	medullary reticular n.	0.9	2.2	<	2.7	3.4	0.036	0.07577
	olivary n.	1.1	0.6	<	1.5	0.7	0.036	0.075573
	rostral piriform ctx	11.9	1.7	>	10.5	2.3	0.039	0.072415
	reticulotegmental n.	1.9	0.7	<	2.3	0.6	0.041	0.07042
	pontine reticular n. oral	2.0	0.8	<	2.7	1.2	0.042	0.069447
	anterior olfactory n.	14.1	2.1	>	12.2	2.8	0.045	0.067423
	anterior cingulate ctx	4.1	1.1	>	3.5	0.8	0.045	0.067423
	glomerular layer olfactory bulb	16.6	2.9	>	15.2	2.9	0.046	0.066422
	lateral preoptic area	1.4	0.3	>	1.2	0.4	0.052	0.061681
	caudal piriform ctx	9.0	1.4	>	8.3	1.2	0.052	0.061604

Table 3.

The list of brain regions that were significantly different in volume between OXT-exposed males and Control males (FDR p=0.06). Males were most affected by OXT a birth showing 35/116 brain regions with significant differences from vehicle controls. As in the case of the females exposed to OXT at birth, the majority of the affected brain regions in OXT-exposed males were smaller than vehicle controls. The regions most sensitive were many of the same for females e.g. olfactory system, limbic cortex, basal ganglia, striatum, amygdala, and hypothalamus. The brain regions that were significantly larger in volume with OXT exposure were in the cerebellum (5th, 7th,8th, 9th lobules) and brainstem (gigantocellularis, trigeminal complex, cuneate nuc., medullary reticular nuc. pontine nuc.).

Male Vehicle vs Male Oxytocin - Brain Volumes (mm3)
	Male Veh			Male OT
Brain Area	Ave	SD		Ave	SD	P-val	ω Sq
frontal association ctx	13.4	1.9	>	9.7	2.8	0.000	0.32943
agranular insular ctx	13.6	3.8	>	9.7	2.5	0.002	0.22789
claustrum	2.4	1.0	>	1.5	0.6	0.003	0.21273
rostral piriform ctx	13.2	2.4	>	10.5	2.3	0.003	0.20768
anterior olfactory n.	14.9	1.8	>	12.2	2.8	0.004	0.1931
5th cerebellar lobule	2.7	2.8	<	5.4	2.1	0.005	0.19098
endopiriform n.	4.6	1.4	>	3.5	0.8	0.006	0.17671
primary somatosensory ctx	25.4	3.3	>	22.1	3.2	0.006	0.17439
7th cerebellar lobule	0.3	0.9	<	2.3	2.8	0.007	0.17291
accumbens core	2.3	0.7	>	1.7	0.5	0.007	0.16991
granular cell layer olfactory bulb	11.4	3.5	>	9.0	1.2	0.008	0.16111
orbital ctx	15.7	6.0	>	11.0	2.8	0.011	0.14783
globus pallidus	3.5	0.6	>	2.8	0.6	0.012	0.14374
caudate putamen (striatum)	25.0	4.6	>	21.3	3.7	0.012	0.14359
glomerular layer olfactory bulb	18.9	4.9	>	15.2	2.9	0.013	0.13943
cortical amygdala	3.5	0.5	>	3.0	0.6	0.016	0.12943
retrosplenial ctx	7.6	1.1	>	6.7	1.0	0.016	0.12923
9th cerebellar lobule	0.1	0.4	<	1.4	1.9	0.018	0.12523
cuneate n.	0.0	0.1	<	0.4	0.5	0.018	0.12523
trigeminal complex	3.1	2.6	<	4.5	1.5	0.019	0.12132
secondary somatosensory ctx	6.2	1.0	>	5.2	1.0	0.019	0.1213
visual 2 ctx	9.0	1.0	>	8.1	1.4	0.020	0.11941
zona incerta	1.6	0.2	<	1.8	0.3	0.022	0.11573
dentate gyrus	8.1	0.4	>	7.5	1.1	0.022	0.11556
basal amygdala	5.2	0.7	>	4.5	0.9	0.025	0.10986
anterior cingulate ctx	4.2	0.9	>	3.5	0.8	0.026	0.10789
caudal piriform ctx	9.3	1.1	>	8.3	1.2	0.027	0.10603
anterior thalamus	2.0	0.8	>	1.3	0.4	0.029	0.10239
gigantocellular reticular n.	4.2	2.4	<	5.8	1.6	0.031	0.09872
central amygdaloid n.	2.3	0.3	>	2.0	0.5	0.032	0.09704
anterior hypothalamic area	3.9	1.3	>	3.1	1.0	0.034	0.09515
medullary reticular n.	0.7	2.5	<	2.7	3.4	0.038	0.08917
8th cerebellar lobule	0.3	1.1	<	1.6	1.9	0.039	0.08829
pontine n.	0.5	0.2	<	0.6	0.2	0.047	0.07992
infralimbic ctx	1.0	0.3	>	0.7	0.5	0.047	0.0797

Table 2.

The list of brain regions that were significantly different in volume between OXT-exposed females and Control females. OXT-exposed females showed smaller brain volumes in 15/21 of the affected regions (FDR p=0.036). The regions affected spread across the olfactory system (anterior olfactory nuc., piriform cortex), hypothalamus (paraventricular, anterior), amygdala (basal, extended), thalamus (anterior, paraventricular) and basal ganglia (nuc. accumbens, caudate putamen).

Female Vehicle vs Female Oxytocin - Brain Volumes (mm3)
	Female Veh			Female OT
Brain Area	Ave	SD		Ave	SD	P-val	ω Sq
tegmental n.	1.3	0.3	>	0.9	0.3	0.000	0.345836
paraventricular hypothalamus	0.7	0.2	>	0.4	0.2	0.001	0.284466
anterior thalamus	2.2	0.7	>	1.5	0.5	0.005	0.207551
anterior hypothalamus	4.5	0.9	>	3.6	0.9	0.007	0.187938
accumbens shell	4.5	1.5	>	3.3	1.0	0.009	0.174539
caudate putamen (striatum)	25.7	4.1	>	22.2	3.4	0.010	0.169285
solitary tract n.	0.2	0.3	<	0.5	0.4	0.012	0.157301
5th cerebellar lobule	2.3	2.5	<	4.8	2.3	0.015	0.144533
basal amygdala	5.7	0.8	>	4.9	0.8	0.016	0.141848
anterior olfactory n.	16.4	2.9	>	14.1	2.1	0.019	0.132333
paraventricular thalamus	1.2	0.3	>	1.0	0.3	0.022	0.125592
rostral piriform ctx	14.2	2.9	>	11.9	1.7	0.025	0.118632
extended amydala	2.4	1.0	>	1.8	0.6	0.026	0.116508
orbital ctx	17.7	5.9	>	12.6	3.9	0.026	0.116343
trigeminal complex medulla	2.6	1.8	<	4.1	1.7	0.035	0.101347
primary motor ctx	13.3	2.6	>	11.4	1.9	0.035	0.101255
vestibular n.	2.0	0.8	>	1.5	0.6	0.037	0.099219
diagonal band of Broca	0.4	0.3	<	0.6	0.3	0.038	0.097323
red n.	0.5	0.2	<	0.7	0.2	0.047	0.087375
primary somatosensory ctx	26.0	3.0	>	24.1	3.3	0.047	0.086965
habenula n.	0.5	0.2	<	0.7	0.2	0.049	0.085284

Figure 2.

(A) Voxel-based morphometry (VBM) measures from 111 brain regions were loaded into a principal component analysis. The overall explanatory value of dimensions 1 and 2 was 29.4% and 15.9%, respectively. (B) Both male sex and OXT treatment lead to greater values in dimension 1 (p < 0.029 for both comparisons). Post-hoc analyses revealed OXT-exposed males had significantly greater dimension 1 scores than Control males (* p = 0.017), while OXT-exposed females tended to be greater than Control females (# p = 0.079). (C) There were no effects in dimension 2.

Prairie voles from different populations that show differences in social behaviors regulated by OXT also show differences in functional connectivity and DWI measures of anatomical connectivity (Ortiz et al., 2020, 2022). Here, we observed a broad, albeit subtle pattern of effects in OXT-exposed males in DWI measures. In terms of FA, Control animals showed small but widespread sex differences, with females having greater FA than males across brain regions, a pattern not present in OXT animals due to increased FA among OXT-exposed males (Figure 3A, Tables 2 and 3).In contrast, there were no differences in either FA or ADC between OXT-exposed males and OXT-exposed females, whereas there were 62 and 48 such regions amongst Control animals. When FA data from all 111 brain regions were loaded into a PCA, there were no effects of either sex or treatment detected (Figure 3A). In terms of ADC, Control males had greater ADC values than OXT-exposed males across 84 brain regions (Table 5). While we observed widespread ADC differences between Control males and females, we observed no sex differences in ADC in the OXT-exposed condition. The PCA for ADC revealed a main effect of OXT exposure (F(1,65) = 5.80, p = 0.019) and a trend toward an interaction between sex and treatment (p = 0.087). Post-hoc analysis revealed that Control males had greater dimension 1 values than both OXT-exposed males (p = 0.026) and OXT-exposed females (p = 0.042). Thus, across the brain, OXT-exposed males’ ADC values more closely resembled Control females and OXT-exposed females than they did Control males.

Figure 3.

Diffusion-weighted imaging (DWI) measures for fractional anisotropy (FA, panels A and B) and apparent diffusion coefficient (ADC, panels C and D) from 111 brain regions were loaded into a principal component analysis. (B) There were no significant differences in FA. (D) Control males had greater dimension 1 scores than OXT-exposed males (* p = 0.026) and OXT-exposed females (* p = 0.042). in terms of ADC.

Table 5.

The list of brain regions that were significantly different in fractional anisotropy (FA) between OXT-exposed males and Control males. While widespread, differences were generally small in effect, with OXT-exposed males generally showing greater FA scores.

Fractional Anisotropy: Control Males vs Oxytocin Males
Brain Area	Males Control			Males Oxytocin
	Mean	SD		Mean	SD	P val	Effect
medial preoptic area	0.47	0.06	<	0.52	0.06	0.004	0.124903
pontine nuclei	0.50	0.07	<	0.53	0.04	0.001	0.091476
medial geniculate	0.55	0.06	<	0.59	0.05	0.024	0.07966
median raphe nucleus	0.52	0.05	<	0.56	0.06	0.015	0.079723
lateral geniculate	0.52	0.06	<	0.56	0.05	0.009	0.082289
parabrachial nucleus	0.51	0.03	<	0.53	0.05	0.012	0.058617
lateral dorsal thalamic nucleus	0.50	0.06	<	0.53	0.05	0.005	0.079003
reticular formation	0.52	0.05	<	0.54	0.05	0.002	0.067602
lemniscal nucleus	0.55	0.03	<	0.57	0.04	0.002	0.047455
anterior olfactory nucleus	0.48	0.07	<	0.51	0.03	0.023	0.071355
3rd cerebellar lobule	0.51	0.04	<	0.53	0.04	0.001	0.047575
ventral pallidum	0.46	0.04	<	0.49	0.06	0.026	0.071662
ventral medial nucleus	0.56	0.05	>	0.53	0.05	0.015	0.062455
reuniens nucleus	0.48	0.05	<	0.51	0.05	0.043	0.06446
trigeminal complex pons	0.51	0.06	<	0.53	0.06	0.007	0.066325
agranular insular ctx	0.55	0.06	<	0.57	0.04	0.014	0.052015
cortical amygdaloid nucleus	0.52	0.06	<	0.54	0.05	0.037	0.058075
auditory ctx	0.49	0.04	<	0.51	0.04	0.049	0.046308
medial amygdaloid nucleus	0.47	0.06	>	0.44	0.08	0.032	0.085575
periaqueductal gray	0.51	0.06	<	0.53	0.06	0.023	0.056552
ventral thalamic nuclei	0.50	0.05	<	0.53	0.07	0.027	0.058593
2nd cerebellar lobule	0.47	0.05	<	0.50	0.08	0.005	0.063622
posterior thalamic nucleus	0.52	0.06	<	0.54	0.04	0.006	0.043435
paraventricular nucleus, hypothalamus	0.48	0.06	<	0.50	0.07	0.009	0.055935
superior colliculus	0.45	0.04	<	0.46	0.06	0.012	0.050091
reticulotegmental nucleus	0.53	0.05	<	0.54	0.04	0.009	0.031722
frontal association ctx	0.53	0.05	<	0.55	0.05	0.049	0.036404
prelimbic ctx	0.53	0.05	<	0.54	0.04	0.047	0.028273
olivary nucleus	0.48	0.06	<	0.50	0.05	0.036	0.040708
central medial thalamic nucleus	0.52	0.05	<	0.54	0.06	0.017	0.038405
Ventricle	0.52	0.06	<	0.54	0.06	0.029	0.042143
reticular nucleus	0.48	0.07	<	0.50	0.05	0.033	0.044729
lateral preoptic area	0.52	0.05	<	0.53	0.04	0.001	0.030365
tegmental nucleus	0.50	0.05	<	0.52	0.07	0.011	0.03654
CA3	0.49	0.04	<	0.50	0.04	0.024	0.024621
lateral amygdaloid nucleus	0.50	0.04	<	0.51	0.07	0.028	0.036617
nucleus lateral olfactory tract	0.46	0.04	<	0.47	0.06	0.047	0.029911
medial dorsal thalamic nucleus	0.48	0.04	<	0.48	0.06	0.040	0.024701
subiculum	0.53	0.05	<	0.54	0.04	0.014	0.019047
8th cerebellar lobule	0.49	0.05	<	0.50	0.06	0.029	0.023925
CA1	0.48	0.03	<	0.49	0.06	0.042	0.019205
lateral septal nucleus	0.52	0.08	<	0.53	0.07	0.011	0.026644
accumbens shell	0.51	0.06	<	0.52	0.06	0.012	0.021319
entorhinal ctx	0.45	0.05	<	0.46	0.07	0.017	0.024468
paraflocculus cerebellum	0.50	0.03	>	0.49	0.07	0.047	0.019452
extended amydala	0.53	0.05	<	0.54	0.04	0.003	0.015815
inferior colliculus	0.47	0.04	<	0.47	0.07	0.008	0.020534
endopiriform nucleus	0.44	0.05	<	0.44	0.07	0.041	0.02301
visual 2 ctx	0.53	0.08	<	0.54	0.06	0.045	0.018208
substantia nigra	0.52	0.06	<	0.52	0.05	0.010	0.012212
central amygdaloid nucleus	0.45	0.04	<	0.45	0.06	0.037	0.012965
olfactory tubercles	0.49	0.08	<	0.50	0.07	0.034	0.011715
retrosplenial ctx	0.50	0.03	>	0.50	0.06	0.024	0.007609
7th cerebellar lobule	0.49	0.06	<	0.50	0.06	0.046	0.008682
lateral hypothalamus	0.45	0.05	<	0.45	0.07	0.033	0.007054
diagonal band of Broca	0.50	0.04	<	0.50	0.05	0.007	0.003926
mammillary nucleus	0.46	0.05	<	0.46	0.07	0.028	0.003179

Functional connectivity is influenced by both sex and the OXT system in humans (Hernandez et al., 2020; Satterthwaite et al., 2015; Zhang et al., 2016). In the present analyses of voles’ functional connectivity (Figures 4–8), OXT-exposed males stood out as having a widespread pattern of greater connectivity. The vast majority of connections across all groups arose from positive correlations. Male sex and OXT treatment both increased the proportion of significant connections for both positive and negative connections (chi-square p < 0.001 for both effects). Whereas Control females were found to have significant functional connectivity in 5% of all possible connections, OXT-exposed females had significant connectivity in 7.2% of connections. Whereas Control males had connectivity in 6.3% of connections, OXT-exposed males had significant connectivity in 12.5% of connections (Figure 4A). Similarly, male sex and OXT treatment at birth both increased the strength of connectivity among region-region pairs whose activity was significantly correlated, but only for positively correlated pairs (p < 0.001 for both effects, Figure 4B). Thus, OXT led to more regions significantly functionally connected and stronger correlations in such regions among males and to a lesser extent among females (Figures 4,5). In examining the pattern of results within correlation matrices, we observed a concentration of stronger connectivity among regions of the same cluster (Figure 4D and E). This led us to examine the strength of intra- vs. inter-cluster connectivity as a function of sex and birth treatment, which revealed that male sex and OXT treatment increased intra-cluster connectivity (e.g. central amygdala and medial amygdala) to a greater degree than inter-cluster connectivity (e.g. central amygdala and dentate gyrus, Figure 4C).

Figure 4.

Resting-state functional connectivity from 111 brain regions. (A) Both OXT treatment and male sex increased functional connectivity, meaning OXT-exposed males had the greatest proportion of region-region pairs significantly functionally connected for both positive (dark fill) and negative (light fill) correlations. Significant group differences are indicated with different letters over top the bars. (B) The average strength of correlation among region-region pairs whose activity was significantly correlated. Both OXT treatment and male sex increased the strength of connectivity for positive connections (dark fill). There were no significant differences among negative connections (light fill). (C) Both OXT treatment and male sex increased the strength of connectivity among both intra- and inter-cluster, though intra-cluster connectivity was more sensitive to these effects. Significant group differences are indicated with different letters over top the bars. In panels (D) and (E), connectivity from males and females respectively, 111x111 cell matrices show the strength of connectivity for all possible pairs of brain regions. Reflected across the diagonal are opposing treatment conditions, with OXT on top and Control on bottom. Region-region pairs whose connectivity Z score was less than |2.3| were excluded.

Figure 8.

A map of degree averaged over regional clusters by group. Both OXT treatment and male sex increased closeness throughout the brain, with effects most apparent in OXT-exposed males. * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p <0.0001.

Figure 5.

A map of the strength of connectivity (i.e. correlations’ z-scores) averaged over regional clusters by group. For example, the ‘Hippocampus’ cluster includes the: CA1, CA3, Dentate gyrus, Subiculum and Parasubiculum. * p < 0.05, ** p < 0.01, *** p < 0.001.

Lastly, we averaged the functional connectivity results within regional clusters to condense and consolidate the large number of findings. As shown in Figure 5, when comparing the strength of connectivity at the level of regional clusters (e.g. all subregions of the amygdala), we observed a treatment effect in the medulla and olfactory system, however there were no significant post-hoc differences. There were also two treatment by sex interactions such that OXT-exposed males had weaker connectivity than OXT-exposed females across the thalamus (p < 0.002) and stronger connectivity than OXT-exposed females across the cortex (p < 0.001). We then examined three indices from analyses based in graph theory: betweenness, closeness, and degree (see above for definitions of each). In terms of betweenness, male sex and OXT treatment both increased betweenness in the basal ganglia (Figure 6, p < 0.01 for both effects). In terms of closeness, we observed similar albeit more widespread effects, with male sex and OXT treatment and both increasing closeness across several regional clusters (Figure 7). Lastly, similar effects were found in terms of degree, with OXT-exposed males having greater degree across a wide swath of regional clusters (Figure 8).

Figure 6.

A map of betweenness averaged over regional clusters by group. For example, the ‘Hippocampus’ cluster includes the: CA1, CA3, Dentate gyrus, Subiculum and Parasubiculum. * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p <0.0001.

Figure 7.

A map of closeness averaged over regional clusters by group. Both OXT treatment and male sex increased closeness throughout the brain, with effects most apparent in OXT-exposed males. * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p <0.0001.

4. Discussion:

Here we describe the effects of injection and exposure to exogenous OXT at birth on neurodevelopment in prairie voles, with consequences seen in neural anatomy and functioning into adulthood. Overall, anatomical differences, while widespread, were generally small, whereas differences in functional connectivity, particularly among OXT-exposed males, were larger. Anatomically, OXT at birth led to a slight reduction in amygdalar volume and OXT-exposed males in particular had slightly smaller cortices and slightly larger brainstem / cerebellums (Tables 1–3). OXT at birth led to males resembling females in terms of FA and ADC (Figure 3). However, functionally, OXT-exposed males showed marked differences from all other groups. OXT at birth led males to display robustly increased functional connectivity throughout the brain (Figures 4–8). This was particularly the case across the cortex in males in terms of the strength, closeness, and degree of connectivity (Figures 5, 7, and 8). Both the number (Figure 4A) and strength (Figure 4B) of positively correlated connections were greater in males and OXT-exposed animals. This was true both within cluster and, to a lesser extent, between clusters as well (Figure 4C). When these effects were examined regionally, few regions stood out (Figure 5); thus, we view these effects as reflecting broad, brain-wide differences. In analyses of brain regions as nodes within a graph, the basal ganglia stood out for both sex and OXT increasing betweenness, whereas such differences were more widespread for closeness and degree. What does this notably broad increase in functional connectivity mean for the OXT-exposed males? We have at present only a few hints.

Interestingly, the observed effects of OXT at birth extended beyond brain regions with dense expression of the OXT receptor. The robust and widespread changes in OXT-exposed males’ neural physiology (i.e. functional connectivity) were greater than the changes observed in neuroanatomy (i.e. VBM and DWI). If OXT-exposed males are continuously experiencing high levels of communication between brain regions, we would expect that to eventually produce changes in anatomical connectedness. Either our anatomical measures of connectivity were insufficiently sensitive to detect these changes, or the relatively small changes in anatomy we did detect are sufficient to produce functional changes that are comparatively more robust. The functional connectivity scans were undertaken with subjects lightly anesthetized, so it is unlikely that OXT-exposed males were responding differentially to the conditions of scanning. Furthermore, we have observed no evidence of stress reactivity being affected by perinatal OXT in our previous studies. OXT acts as a pleiotropic hormone around the time of birth to coordinate the transition from fetal to neonatal life (Kenkel et al., 2014), so by affecting neurodevelopmental trajectory, a single OXT exposure might produce widespread differences across the brain. In mice, seven days of repeated intranasal OXT administration leads to markedly widespread increases in functional connectivity (Pagani et al., 2020). This effect was specific to chronic OXT administration (not seen following acute OXT) and was particularly prominent in connections between cortical regions and the amygdala. When restricting our analysis to only cortico-amygdalar connections, we saw a similar pattern of more and stronger connections in OXT-exposed males (Figure S2). This similarity to our results raises the intriguing possibility that a single OXT administration in early life produces changes similar to chronic OXT administration in adulthood.

We highlight one set of findings in particular because of their relevance to our previous work. As adults, males exposed to OXT via maternal administration at birth had denser OXT receptor distributed along the extent of the agranular insular cortex (Kenkel et al., 2019). In the present study, OXT treatment at birth led to the agranular insular cortex registering as a smaller volume in the brains of adult males (Table 3), along with a slight increase in FA (Table 5) and decrease in ADC (Table 8). Moreover, OXT-exposed males’ agranular insular cortex had a greater number of functionally connected regions (35 vs. 19 for Control males) and stronger average connectivity among those regions (z-score 3.45 vs. 2.98 for Control males). Changes in the functioning of the agranular insular cortex correspond to the behavioral effects previously observed, such as increased alloparental caregiving (Kenkel et al., 2019).

Table 8.

The list of brain regions that were significantly different in apparent diffusion coefficient (ADC) between OXT-exposed males and Control males. While widespread, differences were generally small in effect, with Control males generally showing greater ADC scores. Note: there were no regions where OXT-exposed females and Control females differed in terms of ADC.

Apparent Diffusion Coefficient: Control Males vs Oxytocin Males
Brain Area	Males Control		Males Oxytocin
	Mean	SD		Mean	SD	P val	Effect
periaqueductal gray	1.55	0.22	>	1.31	0.14	0.000	0.252665
tegmental nucleus	1.49	0.16	>	1.26	0.13	0.000	0.243266
vestibular nucleus	1.48	0.16	>	1.26	0.15	0.000	0.228493
dorsal raphe	1.49	0.20	>	1.28	0.14	0.000	0.224489
parafascicular thalamic nucleus	1.41	0.18	>	1.22	0.12	0.000	0.221093
reuniens nucleus	1.42	0.18	>	1.22	0.14	0.000	0.221224
paraventricular nuclus	1.49	0.16	>	1.29	0.13	0.000	0.210742
posterior thalamic nucleus	1.39	0.17	>	1.21	0.13	0.000	0.20552
superior colliculus	1.45	0.19	>	1.26	0.13	0.000	0.203265
ventral thalamic nuclei	1.41	0.18	>	1.23	0.14	0.000	0.198616
CA3	1.53	0.21	>	1.33	0.15	0.000	0.201625
central amygdaloid nucleus	1.49	0.18	>	1.31	0.14	0.000	0.19427
central medial thalamic nucleus	1.40	0.18	>	1.22	0.12	0.000	0.198094
lateral amygdaloid nucleus	1.56	0.20	>	1.39	0.13	0.000	0.174127
medial dorsal thalamic nucleus	1.40	0.19	>	1.22	0.14	0.000	0.200707
pretectal nucleus	1.42	0.18	>	1.25	0.12	0.000	0.182947
secondary somaotsensory ctx	1.42	0.17	>	1.27	0.13	0.000	0.16995
medial geniculate	1.40	0.17	>	1.24	0.14	0.000	0.181864
bed nucleus stria terminalis	1.43	0.17	>	1.26	0.13	0.000	0.184272
accumbens core	1.40	0.15	>	1.24	0.12	0.000	0.178726
CA1	1.53	0.19	>	1.36	0.15	0.000	0.177719
lateral dorsal thalamic nucleus	1.48	0.18	>	1.32	0.13	0.000	0.166541
parabrachial nucleus	1.46	0.17	>	1.30	0.16	0.000	0.163127
lateral posterior thalamic nucleus	1.43	0.17	>	1.26	0.14	0.000	0.178745
red nucleus	1.44	0.19	>	1.27	0.14	0.000	0.187887
primary somatosensory ctx	1.55	0.21	>	1.39	0.17	0.001	0.150456
reticular nucleus	1.46	0.18	>	1.29	0.13	0.001	0.178808
inferior colliculus	1.50	0.21	>	1.30	0.14	0.001	0.204884
habenula nucleus	1.52	0.24	>	1.35	0.19	0.001	0.166456
medial septum	1.46	0.14	>	1.29	0.13	0.001	0.173046
paraventricular nucleus	1.40	0.19	>	1.23	0.17	0.001	0.189772
White Matter	1.51	0.20	>	1.35	0.15	0.001	0.160272
lateral geniculate	1.42	0.18	>	1.25	0.13	0.001	0.17768
2nd cerebellar lobule	1.47	0.20	>	1.30	0.21	0.001	0.174388
caudate putamen (striatum)	1.39	0.18	>	1.23	0.12	0.001	0.169379
claustrum	1.36	0.17	>	1.21	0.11	0.001	0.167135
anterior thalamic nuclei	1.40	0.18	>	1.24	0.15	0.001	0.174358
median raphe nucleus	1.48	0.21	>	1.33	0.17	0.001	0.148878
reticular formation	1.47	0.19	>	1.31	0.19	0.001	0.162688
accumbens shell	1.43	0.16	>	1.26	0.14	0.002	0.181389
auditory ctx	1.42	0.19	>	1.27	0.16	0.002	0.1609
globus pallidus	1.37	0.17	>	1.21	0.14	0.002	0.171928
orbital ctx	1.48	0.21	>	1.31	0.14	0.003	0.173424
infralimbic ctx	1.62	0.18	>	1.44	0.16	0.003	0.162521
7th cerebellar lobule	1.29	0.28	>	1.08	0.18	0.003	0.263639
visual 1 ctx	1.50	0.25	>	1.34	0.19	0.003	0.158911
anterior olfactory nucleus	1.57	0.30	>	1.35	0.22	0.003	0.213927
10th cerebellar lobule	1.62	0.25	>	1.43	0.17	0.003	0.180961
dentate gyrus	1.60	0.22	>	1.41	0.19	0.003	0.180572
agranular insular ctx	1.45	0.17	>	1.30	0.23	0.004	0.157405
5th cerebellar lobule	1.25	0.24	>	1.07	0.19	0.004	0.229309
extended amydala	1.44	0.17	>	1.29	0.17	0.004	0.152473
3rd cerebellar lobule	1.30	0.22	>	1.13	0.21	0.004	0.203067
endopiriform nucleus	1.40	0.18	>	1.26	0.18	0.005	0.153927
6th cerebellar lobule	1.24	0.23	>	1.03	0.18	0.005	0.262008
parasubiculum	1.57	0.28	>	1.42	0.21	0.005	0.149793
subiculum	1.55	0.20	>	1.40	0.24	0.007	0.137179
frontal association ctx	1.58	0.24	>	1.43	0.19	0.008	0.150833
zona incerta	1.47	0.21	>	1.31	0.22	0.011	0.17364
temporal ctx	1.45	0.21	>	1.30	0.22	0.012	0.158125
lateral septal nucleus	1.60	0.18	>	1.48	0.12	0.012	0.109379
primary motor ctx	1.60	0.27	>	1.49	0.19	0.014	0.094467
visual 2 ctx	1.66	0.31	>	1.52	0.20	0.015	0.122483
ventral pallidum	1.47	0.18	>	1.32	0.18	0.015	0.151041
pontine reticular nucleus oral	1.48	0.22	>	1.36	0.27	0.016	0.122237
solitary tract nucleus	1.42	0.20	>	1.28	0.21	0.016	0.148623
retrosplenial ctx	1.77	0.33	>	1.64	0.22	0.016	0.106864
basal amygdaloid nucleus	1.45	0.18	>	1.30	0.23	0.022	0.162508
anterior hypothalamic area	1.55	0.23	>	1.39	0.27	0.023	0.159099
9th cerebellar lobule	1.52	0.28	>	1.33	0.19	0.024	0.188268
posterior hypothalamic area	1.44	0.18	>	1.28	0.16	0.024	0.170827
medial preoptic area	1.57	0.25	>	1.41	0.23	0.024	0.157191
diagonal band of Broca	1.53	0.22	>	1.40	0.21	0.025	0.134138
pontine reticular nucleus caudal	1.49	0.22	>	1.38	0.34	0.025	0.1033
perirhinal ctx	1.52	0.24	>	1.36	0.31	0.028	0.166175
anterior cingulate ctx	1.75	0.24	>	1.58	0.20	0.028	0.14404
crus ansiform lobule	1.36	0.24	>	1.23	0.23	0.029	0.151202
8th cerebellar lobule	1.38	0.29	>	1.23	0.24	0.034	0.162496
gigantocellular reticular nucleus	1.53	0.26	>	1.43	0.38	0.036	0.10108
granular cell layer olfactory bulb	1.56	0.30	>	1.40	0.16	0.040	0.160613
cuneate nucleus	1.48	0.29	>	1.34	0.25	0.042	0.140914
dorsal medial nucleus	1.64	0.25	>	1.51	0.20	0.046	0.11454
medial amygdaloid nucleus	1.68	0.25	>	1.52	0.34	0.049	0.135506

In our previous work using the same paradigm of OXT administration to term pregnant prairie voles, we found a dose-dependent increase in the methylation of the OXT receptor promoter within the fetal brain 90 minutes after maternal OXT administration (Kenkel et al., 2019). Importantly, this effect and those discussed above all took place in the context of a cross-fostered experimental design, which ruled out direct effects of OXT on maternal behavior and strongly suggested a direct effect of OXT on the pups. The present findings suggest that there may be long-term consequences of this epigenetic change in OXT receptor regulation at the time of birth, though clearly more work is needed. One intriguing possibility is that by disrupting the inflammatory processes and oxidative stress that normally occur at birth (Kingsbury and Bilbo, 2019), OXT could lead to the rewiring we have seen in this study. Owing to sex differences in susceptibility to oxidative stress at the time of delivery (Minghetti et al., 2013), this could explain the male-biased effects observed here.

Recently, Ortiz et al. employed many of the same measures as those used in the present study in comparing a relatively prosocial population of male prairie voles (deriving from Illinois) with males from a relatively less prosocial population (deriving from an Illinois sire and a less social Kansas dam) (Ortiz et al., 2020, 2022). If we consider the OXT group of the present study as being analogous to the prosocial Illinois voles, we can see similarities in functional connectivity. Both Illinois males and OXT-exposed males showed higher levels of the closeness and degree centrality, particularly within brain regions associated with prosocial behavior. When restricting our analysis to only the prosocial core (Ortiz et al., 2022), we observed a main effect of birth treatment such that OXT led to stronger connectivity, similar to the Illinois males (Figure S3). However, this study’s OXT-exposed males did not resemble the prosocial Illinois males anatomically, as the latter displayed higher ADC values in regions associated with prosocial behavior.

Although OXT has been proposed as a treatment in autism spectrum disorders (Alvares et al., 2017), OXT has also been implicated in the etiology of autism spectrum disorders. For example, while some epidemiological studies have suggested a link between autism spectrum disorders and perinatal OXT exposure (Lønfeldt et al., 2019), residual confounding by either genetic or environmental vulnerabilities, could explain this apparent association. One pattern generally observed in the brains of humans with autism spectrum disorders is diminished long-distance functional connectivity and increased short-distance functional connectivity (O’Reilly et al., 2017; Rane et al., 2015). We observed that both male sex and OXT treatment at birth led to increased intra-cluster connectivity (analogous to increased short-distance hyperconnectivity), but also to increased inter-cluster connectivity, though to a lesser extent. Thus, the present results do not entirely resemble the equivalent effects seen in humans with autism spectrum disorders. In our previous work, we observed a broadly gregarious phenotype in OXT-exposed voles (Kenkel et al., 2019); if such results translated to humans, they would support the contention that underlying vulnerabilities bring on both a need for OXT in the mother and susceptibility to autism spectrum disorders in the child. While our previous work found behavioral differences in both males and females exposed to OXT, which is somewhat in contrast to the present study’s findings, we also found males to be much more affected by perinatal OXT in terms of neuroanatomy, which was assessed as the density of OXT and AVP receptors -and that finding matches those of the present study. Numerous previous studies have found sex-, dose-, and region-dependent effects of early life OXT manipulation (Carter, 2003; Hammock, 2015).

In the present study, we observed greater intra-cluster as well as inter-cluster connectivity in male voles. In human neuroimaging, a consensus appeared to be forming such that males show greater inter-cluster connectivity whereas females show greater intra-cluster connectivity (Allen et al., 2011; Satterthwaite et al., 2015; Tomasi and Volkow, 2012). However, more recent work has challenged this, finding greater local clustering in males (Zhang et al., 2016). Among neurotypical individuals of both sexes, increased genetic risk load for autism spectrum disorders within the OXT receptor gene is associated with greater functional connectivity between the nucleus accumbens and prefrontal cortex (Hernandez et al., 2020). We observed very little functional connectivity between the prefrontal cortex and any region of the basal ganglia in any of the four groups of the present study. Thus, a floor effect may have prevented the detection of any OXT-induced consequences for prefrontal cortex / basal ganglia connectivity.

There have been very few studies on the impact of birth interventions and subsequent brain development (Kenkel, 2020; Kenkel et al., 2014). In the realm of neuroanatomy, Chiesa et al. (2021) recently reported that cesarean section results in smaller brain volumes in neonatal mouse pups (Chiesa et al., 2021). However, no such findings have been reported in human children or infants (Deoni et al., 2019). In terms of VBM and functional connectivity in the present study, OXT-exposed males showed a more masculinized phenotype. However, in terms of ADC and FA, OXT led males demonstrated a more feminized phenotype. Further work is needed to reveal the meanings of these differences.

Limitations:

In this study the un-treated “Control” group did not receive a vehicle treatment or other manipulations. Whether the stress of injection given to the mother has a significant consequence for these measures cannot be determined from the current study. However, in a small validation study, adult offspring of saline-treated dams (n = 3 female, 5 male adult offspring) were not found to have meaningful differences in DWI values compared to the un-treated Control animals of the present study (n = 17 female, 19 male adult offspring). It would be difficult to disentangle maternal stress from OXT exposure in humans, as labor augmentation with OXT is associated with a more negative subjective birth experience and increased rates of spinal epidural usage indicating increased need for pain management (Joensuu et al., 2022). That said, a more translationally relevant approach here would have been to maintain steady level of OXT infusion over a long period of time, as takes place in the clinical setting. Such an approach has recently been employed in rats using microinfusion pumps (Giri et al., 2022). Pharmacodynamic considerations are important because OXT administration leads to the internalization and downregulation of the OXT receptor. For instance, stimulating OXT receptors with administration of an OXT agonist leads to 50–60% of receptors internalized (Gimpl and Fahrenholz, 2001; Smith et al., 2006). In our previous study on the methylation of the OXT receptor promoter, we found no difference between saline-injected and non-injected pups (Kenkel et al., 2019).

The data from this study suggest that OXT experienced by a fetus during the perinatal period could have lasting neural consequences. The results also point to the importance of comparing males and females with brain-region specific analyses. Collectively, these findings support the hypothesis that the perinatal brain is sensitive to OXT administered indirectly to the pregnant female. However, it is not possible to determine whether the specific effects measured here are direct or perhaps indirect effects of exogenous OXT, for example via uterine contractions or maternal distress. Finally, in this study, we have also applied several metrics to our fMRI data that have not previously used in prairie voles.

5. Conclusion

As the first experiment of its kind, the outcome of the present study must be considered preliminary, but also suggestive of the need for future investigation of the possible development consequences of perinatal peptides, including those administered to the mother. We suggest that given the widespread use in obstetric care of OXT for labor induction / augmentation, currently found in the majority of births in the U.S. (Declercq et al., 2014; Martin et al., 2021), a more complete understanding of the neurodevelopmental effects of OXT exposure at delivery is imperative. It is also important to note that the vast majority of deliveries now use OXT during the third stage of labor to prevent postpartum hemorrhage (Miranda et al., 2013; Salati et al., 2019). Delayed cord clamping is becoming standard practice (“Committee Opinion No. 684 Summary,” 2017), and this procedure may extend both the duration and dose of potential OXT exposure by the neonate. Because the perinatal period is a sensitive period for brain development in general and possibly in terms of OXT exposure, these practices deserve further investigation. Our findings suggest that prairie voles could help to inform these clinical practices by offering an experimental platform for the examination of long-term consequences of manipulations such as these.

Supplementary Material

MMC1

Table S1. The list of brain regions and their corresponding regional cluster to which they were classified.

MMC4

Figure S3. Pro-social core connectivity. Among significantly correlated connections between the following brain regions: Olfactory Tubercles, Rostral Piriform Cortex, Tenia Tecta Cortex, Anterior Olfactory Nucleus, Caudal Piriform Cortex, Cortical Amygdaloid Nucleus, Endopiriform Nucleus, Entorhinal Cortex, Glomerular Layer Olfactory Bulb, Granular Cell Layer Olfactory Bulb, and the Nucleus Lateral Olfactory Tract, OXT-males showed the strongest connectivity. Analysis revealed a main effect of treatment (p < 0.007) as well as a trend toward an effect of sex (p < 0.09); post-hoc analysis revealed that OXT-exposed males had significantly stronger connectivity than control females (p < 0.01).

MMC3

Figure S2. Cortico-amygdalar connectivity. Among significantly correlated connections between cortical regions and the amygdala, OXT-exposed males showed the strongest connectivity. Analysis revealed a sex by treatment interaction effect (p = 0.05), though post-hoc comparisons revealed no significant differences.

MMC2

Figure S1. The changes in brain volumes between sexes are not consistent, i.e. in some brain areas females are greater (black sign) and others less than males (red sign). The location of these different brain areas affected by OT at birth when comparing females and males can be seen in the probability heat maps in Fig 1. Brain regions that are greater in volume in females than males are organized around the olfactory system (A. glomerular layer; B. anterior olfactory n; B-D. piriform cortices) and prefrontal/limbic cortical areas (B. motor, insular; C. anterior cingulate; D. retrosplenial). Areas that are larger in males vs females are primarily associated with brainstem/cerebellum (E. reticulotegmental n., raphe, pontine reticular n; F. olivary n.; G. vestibular n., gigantocellularis, paraflocculus, F. cuneate n. 7th lobule, medullary reticular n.).

Table 4.

The list of brain regions that were significantly different in fractional anisotropy between OXT-exposed females and Control females.

Fractional Anisotropy: Control Females vs Oxytocin Females
Brain Area	Female Control		Female Oxytocin
	Mean	SD	Mean	SD	P val	Effect
3rd cerebellar lobule	0.4825	0.0609	0.5259	0.0582	0.0235	0.117822
5th cerebellar lobule	0.489	0.0585	0.5273	0.0573	0.0388	0.103671
anterior cingulate ctx	0.478	0.0515	0.5141	0.0609	0.044	0.100389
inferior colliculus	0.4405	0.0436	0.4718	0.0561	0.0492	0.094993
auditory ctx	0.4565	0.0437	0.4886	0.0457	0.025	0.094146

Table 6.

The list of brain regions that were significantly different in fractional anisotropy (FA) between Control females and Control males. While widespread, differences outside of the thalamus were generally small in effect, with females generally showing greater FA scores.

Fractional Anisotropy: Control Females and Males
Brain Area	Females (n=17)			Males (n=19)
	Mean	SD		Mean	SD	P val	Effect
parafascicular thalamic nucleus	0.48	0.06	>	0.39	0.07	0.000	0.324472
habenula nucleus	0.49	0.07	>	0.41	0.08	0.001	0.291157
lateral geniculate	0.56	0.05	>	0.47	0.07	0.000	0.261072
bed nucleus stria terminalis	0.48	0.04	>	0.41	0.05	0.000	0.260134
diagonal band of Broca	0.48	0.04	>	0.40	0.08	0.002	0.240761
claustrum	0.47	0.06	>	0.40	0.06	0.003	0.230304
lateral dorsal thalamic nucleus	0.58	0.05	>	0.51	0.10	0.005	0.210171
glomerular layer olfactory bulb	0.48	0.03	>	0.42	0.07	0.002	0.199748
lateral amygdaloid nucleus	0.50	0.08	>	0.43	0.08	0.020	0.198068
granular cell layer olfactory bulb	0.46	0.04	>	0.40	0.07	0.004	0.197001
central medial thalamic nucleus	0.51	0.06	>	0.44	0.08	0.010	0.191312
pretectal nucleus	0.48	0.04	>	0.42	0.06	0.001	0.190049
ventral tegmental area	0.57	0.06	>	0.50	0.07	0.003	0.186418
anterior olfactory nucleus	0.48	0.03	>	0.42	0.06	0.001	0.182102
medial geniculate	0.52	0.04	>	0.46	0.08	0.005	0.180481
olfactory tubercles	0.52	0.03	>	0.46	0.07	0.002	0.178895
6th cerebellar lobule	0.48	0.05	>	0.42	0.06	0.003	0.178003
medial preoptic area	0.51	0.03	>	0.45	0.08	0.006	0.177545
caudate putamen (striatum)	0.47	0.05	>	0.41	0.06	0.008	0.176083
posterior hypothalamic area	0.54	0.07	>	0.48	0.05	0.005	0.175232
orbital ctx	0.45	0.04	>	0.40	0.06	0.006	0.17508
tegmental nucleus	0.55	0.08	>	0.49	0.07	0.017	0.171393
reuniens nucleus	0.54	0.06	>	0.48	0.07	0.008	0.170836
dorsal raphe	0.52	0.07	>	0.46	0.08	0.024	0.170389
tenia tecta ctx	0.51	0.03	>	0.45	0.07	0.005	0.170225
red nucleus	0.53	0.06	>	0.47	0.07	0.010	0.168993
medial septum	0.48	0.07	>	0.43	0.07	0.035	0.168928
CA1	0.51	0.04	>	0.45	0.06	0.003	0.168383
posterior thalamic nucleus	0.52	0.03	>	0.46	0.07	0.004	0.166535
anterior thalamic nuclei	0.45	0.05	>	0.40	0.06	0.013	0.16469
substantia nigra	0.60	0.05	>	0.53	0.06	0.002	0.162592
secondary somaotsensory ctx	0.45	0.05	>	0.40	0.07	0.027	0.158391
lateral posterior thalamic nucleus	0.51	0.06	>	0.45	0.07	0.025	0.155251
CA3	0.53	0.04	>	0.47	0.07	0.007	0.152723
reticular formation	0.52	0.05	>	0.47	0.06	0.012	0.15052
dorsal medial nucleus	0.53	0.06	>	0.48	0.07	0.020	0.145566
accumbens core	0.53	0.04	>	0.48	0.06	0.003	0.144539
subiculum	0.49	0.04	>	0.45	0.06	0.011	0.139425
dentate gyrus	0.49	0.04	>	0.45	0.07	0.015	0.137887
ventral pallidum	0.53	0.04	>	0.48	0.06	0.008	0.137735
superior colliculus	0.44	0.04	>	0.40	0.06	0.019	0.13266
lateral preoptic area	0.56	0.04	>	0.51	0.08	0.023	0.132598
extended amydala	0.52	0.06	>	0.47	0.06	0.020	0.131951
parasubiculum	0.44	0.04	>	0.40	0.07	0.037	0.131038
median raphe nucleus	0.59	0.05	>	0.54	0.08	0.026	0.130939
interpeduncular nucleus	0.54	0.05	>	0.50	0.05	0.011	0.130127
7th cerebellar lobule	0.52	0.05	>	0.47	0.06	0.022	0.130134
accumbens shell	0.52	0.05	>	0.48	0.06	0.018	0.12372
zona incerta	0.64	0.05	>	0.58	0.07	0.018	0.119864
central amygdaloid nucleus	0.53	0.06	>	0.49	0.07	0.049	0.119507
8th cerebellar lobule	0.53	0.05	>	0.49	0.05	0.018	0.119381
endopiriform nucleus	0.49	0.06	>	0.45	0.05	0.046	0.11912
parabrachial nucleus	0.54	0.05	>	0.50	0.06	0.024	0.117299
paraventricular nuclus	0.55	0.06	>	0.50	0.07	0.040	0.116178
basal amygdaloid nucleus	0.56	0.05	>	0.51	0.07	0.046	0.115372
ventral thalamic nuclei	0.55	0.04	>	0.51	0.06	0.020	0.115201
9th cerebellar lobule	0.53	0.06	>	0.49	0.05	0.040	0.108169
pontine reticular nucleus oral	0.55	0.05	>	0.51	0.06	0.027	0.107503
cortical amygdaloid nucleus	0.53	0.04	>	0.50	0.06	0.040	0.101249
trigeminal complex medulla	0.53	0.02	>	0.49	0.05	0.007	0.099082
reticulotegmental nucleus	0.55	0.05	>	0.51	0.05	0.041	0.095013
crus ansiform lobule	0.50	0.04	>	0.47	0.05	0.040	0.089621

Table 7.

The list of brain regions that were significantly different in apparent diffusion coefficient (ADC) between Control females and Control males. While widespread, differences were generally small in effect, with males generally showing greater ADC scores.

Apparent Diffusion Coefficient: Control Females and Males
Brain Area	Females			Males
	Mean	SD		Mean	SD	P val	Effect
cuneate nucleus	1.27	0.11	<	1.48	0.29	0.008	0.191902
9th cerebellar lobule	1.31	0.15	<	1.52	0.28	0.009	0.186984
tegmental nucleus	1.29	0.15	<	1.49	0.16	0.000	0.184923
7th cerebellar lobule	1.13	0.09	<	1.29	0.28	0.025	0.172738
parafascicular thalamic nucleus	1.24	0.11	<	1.41	0.18	0.001	0.171383
central medial thalamic nucleus	1.22	0.09	<	1.40	0.18	0.001	0.170847
dentate gyrus	1.40	0.15	<	1.60	0.22	0.003	0.170166
red nucleus	1.26	0.14	<	1.44	0.19	0.003	0.167449
perirhinal ctx	1.34	0.15	<	1.52	0.24	0.009	0.163461
habenula nucleus	1.34	0.11	<	1.52	0.24	0.009	0.156466
central amygdaloid nucleus	1.32	0.11	<	1.49	0.18	0.002	0.155197
ventral tegmental area	1.45	0.23	<	1.64	0.30	0.045	0.153921
reticular nucleus	1.29	0.09	<	1.46	0.18	0.002	0.15359
caudate putamen (striatum)	1.23	0.09	<	1.39	0.18	0.002	0.153054
accumbens core	1.25	0.10	<	1.40	0.15	0.001	0.149244
posterior thalamic nucleus	1.24	0.11	<	1.39	0.17	0.004	0.146364
globus pallidus	1.22	0.10	<	1.37	0.17	0.004	0.144324
zona incerta	1.32	0.16	<	1.47	0.21	0.018	0.143221
lateral amygdaloid nucleus	1.40	0.14	<	1.56	0.20	0.008	0.141691
temporal ctx	1.30	0.13	<	1.45	0.21	0.016	0.141549
median raphe nucleus	1.33	0.16	<	1.48	0.21	0.019	0.139344
parabrachial nucleus	1.31	0.14	<	1.46	0.17	0.008	0.137927
medial geniculate	1.26	0.12	<	1.40	0.17	0.007	0.136556
basal amygdaloid nucleus	1.31	0.17	<	1.45	0.18	0.021	0.13487
pontine reticular nucleus oral	1.34	0.21	<	1.48	0.22	0.047	0.13411
pretectal nucleus	1.28	0.10	<	1.42	0.18	0.007	0.132915
reticular formation	1.33	0.14	<	1.47	0.19	0.015	0.132011
claustrum	1.23	0.11	<	1.36	0.17	0.009	0.131793
White Matter	1.37	0.11	<	1.51	0.20	0.011	0.131328
reuniens nucleus	1.28	0.13	<	1.42	0.18	0.015	0.130902
ventral thalamic nuclei	1.28	0.11	<	1.41	0.18	0.010	0.130631
subiculum	1.40	0.16	<	1.55	0.20	0.021	0.128979
dorsal raphe	1.35	0.14	<	1.49	0.20	0.020	0.127819
lateral septal nucleus	1.45	0.12	<	1.60	0.18	0.008	0.123361
auditory ctx	1.29	0.12	<	1.42	0.19	0.023	0.120441
periaqueductal gray	1.41	0.11	<	1.55	0.22	0.027	0.119783
lateral posterior thalamic nucleus	1.30	0.10	<	1.43	0.17	0.012	0.119406
CA3	1.39	0.13	<	1.53	0.21	0.028	0.118736
posterior hypothalamic area	1.32	0.17	<	1.44	0.18	0.039	0.118504
infralimbic ctx	1.48	0.16	<	1.62	0.18	0.018	0.117904
CA1	1.40	0.12	<	1.53	0.19	0.017	0.117489
bed nucleus stria terminalis	1.30	0.13	<	1.43	0.17	0.019	0.115845
orbital ctx	1.36	0.14	<	1.48	0.21	0.046	0.113322
solitary tract nucleus	1.30	0.13	<	1.42	0.20	0.044	0.113143
paraventricular nuclus	1.37	0.15	<	1.49	0.16	0.022	0.112125
medial septum	1.34	0.14	<	1.46	0.14	0.022	0.107011
lateral geniculate	1.31	0.11	<	1.42	0.18	0.034	0.106521
extended amydala	1.33	0.15	<	1.44	0.17	0.043	0.106481

Highlights.

• Voles exposed to oxytocin at birth had functional and anatomical brain changes.

• Male voles were particularly sensitive to oxytocin exposure at birth.

• Oxytocin males had increased functional connectivity throughout the brain.