PET/CT imaging of the late-gestation fetal brain in pregnant rats: A proof-of-concept study

Abstract

Preclinical PET studies offer the opportunity to elucidate molecular mechanisms underlying early neurodevelopment with minimal invasiveness. We demonstrated the feasibility of fetal brain PET in four pregnant rats (n = 42 fetuses). [¹⁸F]FDG uptake in rat fetuses was readily visualized by PET imaging. Additionally, in vivo fetal brain [¹⁸F]FDG concentration (standardized uptake value (SUV)) was significantly correlated with ex vivo SUV from matched post-mortem brains (R² = 0.90, p < 0.001). We further investigated the effect of the dopamine receptor antagonist haloperidol on cerebral glucose metabolism (CMR_glu) and [¹¹C]raclopride binding in maternal and fetal brains. Dopamine D2 receptor blockade by haloperidol resulted in significant decreases (p < 0.001, n = 33 vs 9 fetuses) in in vivo CMR_glu and ex vivo [¹⁸F]FDG SUV. Consistently, haloperidol pretreatment significantly decreased [¹¹C]raclopride SUV ratio (SUVR) by 17% (p < 0.001, n = 6 vs 6 fetuses) in the fetal whole-brain, using the maternal cerebellum as the reference region. In all, our results show that PET/CT imaging of the fetal rat brain can reliably quantify specific molecular targets in vivo, and future translational studies of neurodevelopment are feasible in this model.

Introduction

Identifying quantifiable biomarkers associated with environmental risk and resilience factors offers new opportunities for exploring neurotypical or neurodiverse development. There is a growing interest in molecular targets involved in disease origins, especially during the exquisitely sensitive period of embryonic development, ¹ as research shows that non-genetic influences during pregnancy impact long-term health. For example, malnutrition during pregnancy can influence cardiovascular disease incidence in offspring ² and prenatal infection may increase risk for psychiatric diseases later in life. ³ Several early life risk factors, such as obstetric complications and infections during gestation, are thought to underlie dysregulated synaptic pruning and abnormal neurodevelopment in schizophrenia. ⁴ These findings underscore the developmental origins of disease hypothesis and suggest that shared disruptions across neurodevelopment can be viewed with a transdiagnostic approach. ⁵

While environmental risk factors, including exposure to substances of abuse during pregnancy, affect neurodevelopment, the detailed mechanisms underlying their transmission and long-lasting effects remain unclear. Noninvasive imaging techniques enable studies without disturbing the physiology of the maternal-fetal dyad, allowing longitudinal tracking from in utero to after birth. In vivo positron emission tomography (PET) imaging offers additional advantages over ex vivo methods, namely capturing dynamic information about pharmacokinetics, receptor availability, and interactions with endogenous neurotransmitters, thus providing greater insight into developmental processes. PET imaging has been successfully used in pregnant non-human primate (NHP) models to study fetal exposure to drugs of abuse, ⁶ fetal brain function, ⁷ and synaptogenesis during gestation, ⁸ despite potentially limited clinical translation due to ionizing radiation. These preclinical studies demonstrate the potential utility of PET to investigate specific mechanisms underlying fetal brain development and probe in vivo biomarkers. ⁹ However, such NHP studies face barriers, including cost, limited access, ethical concerns, and extended timeline for longitudinal investigations.

In contrast to NHP PET studies, pregnant rodent models provide several advantages: shorter gestation period, larger litter size for higher throughput, and greater experimental flexibility, all while preserving the ability to conduct longitudinal measurements under intact maternal–fetal physiology. PET/CT has been used in rodents to assess whole-fetus glucose metabolism changes during chronic hypoxia. ¹⁰ Other studies have investigated placental–fetal transfer of substances beneficial or deleterious to fetal health, such as vitamin B₇ ¹¹ and environmental toxins ¹² in pregnant rodents. However, prior in utero rodent PET studies have not resolved signals at the level of fetal organs, which limits its utility in neurodevelopmental research.

To address this, we applied PET/CT imaging in late-gestation pregnant rats to quantify radiotracer uptake in the fetal brain in vivo. We used [¹⁸F]FDG and [¹¹C]raclopride, both of which are well-characterized radiotracers in adult brain imaging, for novel in utero applications. First, we compared fetal brain [¹⁸F]FDG uptake in vivo with matched post-mortem samples ex vivo. Additionally, we estimated in vivo cerebral metabolic rate of glucose (CMR_glu) in the fetal brain and its ratio to maternal brain metabolism. In a subset of two pregnant rats, we further explored baseline dopamine receptor availability in fetal brains estimated by [¹¹C]raclopride, using the standardized uptake value ratio (SUVR) and the maternal cerebellum, which served as the reference region. Finally, we administered a typical antipsychotic, haloperidol, to one subject prior to [¹¹C]raclopride and [¹⁸F]FDG PET scans to assess its pharmacological effects on the fetal brain. Because haloperidol is known to reduce [¹¹C]raclopride binding and [¹⁸F]FDG metabolic rate in adults, we hypothesized that similar reductions would be detected in the fetal brain.

Materials and methods

General

All animal housing and experiments were conducted in strict accordance with the institutional Guidelines for Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. Four pregnant female Sprague–Dawley rats (weight = 341 ± 9 g, litter size = 10–14) were imaged on gestational day (GD) 21 after a minimum 48 h acclimation period. GD1 was defined by first observation of the vaginal plug. Animals were housed individually with standard bedding and with standard rodent food and water ad libitum under a 12 h light/dark cycle.

PET/CT scans were performed on a MultiScan™ Large Field of view Extreme Resolution (LFER) 150 imaging system (Mediso USA, Arlington, TX, USA). ¹³ Vital signs (heart rate and oxygen saturation (spO₂)) were dynamically recorded using a PhysioSuite (Kent Scientific, Torrington, CT, USA) equipped with a MouseSTAT Pulse Oximeter and body temperature was maintained by external heat source. PET and CT image processing was performed in PMOD (v3.3; PMOD Technologies, Zurich, Switzerland) and data figures were generated in MATLAB (v2023b; MathWorks, Natick, MA, USA). This study followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines.

In vivo PET/CT imaging

Pregnant rats were imaged on gestational day 21 after an overnight fast (~13 h). A summary of PET/CT scans is available in Supplemental Table 1. Inhaled anesthesia was induced and maintained by isoflurane (1.5%–3%) in 100% O₂ carrier gas (0.2 L/min). Intravenous access was established through the tail vein for radiotracer injections. Animals were positioned head-first prone in the scanner bore and both whole-body and uterus CT scans were performed. The whole body CT was acquired for CT-based scatter and attenuation correction of PET data using a circular trajectory, X-ray tube energy of 60 kVp, 360 projections, and 200 ms exposure time. CT image reconstruction was performed using filtered back-projection and Shepp-Logan filter to a resolution of 200 µm³. Additionally, a uterus CT was acquired using a semicircular trajectory, 40 kVp X-ray tube power, 3000 projections with 300 ms exposure time, and no binning. The resolution was increased to 106 µm³ and this enabled fetal anatomy to be delineated by ossified bony features (Supplemental Figure 1).

PET data acquisition (90 min) began simultaneously with intravenous [¹⁸F]FDG (18.2 ± 2.9 MBq) or [¹¹C]raclopride (23.5 ± 1.0 MBq, <0.14 µg) administration. Dynamic reconstruction of PET data was subsequently performed for 29 frames (8 × 15, 6 × 30, 4 × 60, 3 × 120, 3 × 300, 3 × 600, and 2 × 900 s) using a version of the ordered subset expectation maximization (OSEM) algorithm (Tera-Tomo 3D; Mediso USA, Arlington, TX, USA). Twelve iterations of 3 subsets were performed with normal regularization and correction for random events, attenuation, and scatter. The final PET image matrix was 231 × 231 × 249 consisting of 600 µm isotropic voxels.

Ex vivo [¹⁸F]FDG quantification

Immediately at the end of scan (90 min), dams and fetuses were euthanized and whole fetal brain tissue was harvested. The position and orientation of each fetus in the uterine horns was carefully annotated to match post-mortem brains with corresponding in vivo PET/CT images. Maternal and fetal brain radioactivity was quantified by cross-calibrated automated γ-counter (Wallac Wizard 1480; PerkinElmer, Waltham, MA, USA) and decay corrected F-18 counts/min were normalized to SUV.

Haloperidol administration

In one dam, 0.5 mg/kg haloperidol was administered intravenously (IV) as a single pretreatment before [¹¹C]raclopride (30 min pretreatment) and [¹⁸F]FDG (150 min pretreatment) PET scans.

PET image analysis and quantification

PET and CT images were analyzed similarly to previous reports in the fetal NHP brain, ⁸ with additional preprocessing due to multiple fetuses in pregnant rats. Maternal brain time-activity curves (TACs) were generated from a standard rat brain atlas and normalized to SUV. ¹⁴ For fetal PET/CT data, a rectangular bounding box was centered over each whole fetal–placental unit (FPU) and cropped to a new image volume. Spatial registration for fetal brains was performed by CT-to-CT co-registration to an in-house CT template created using an iterative method. ¹⁵ The CT-based transformation matrix was applied to the dynamic fetal brain PET data. Then, time-activity curves were generated using a postnatal day 0 (P0) rat brain magnetic resonance imaging (MRI) atlas ¹⁶ in the same space as the in-house CT template. All transformations were carefully inspected using fused images and contours as quality control. P0 brain atlas whole-brain VOI was overlaid onto dynamic PET images for individual fetal brain time-activity curve generation and subsequent kinetic modeling.

For [¹⁸F]FDG data, the final time frame (75–90 min) was reported as SUV at the end of scan in order to correspond to the ex vivo time point (90 min). CMR_glu was calculated as previously described for the fetal NHP brain. ⁷ Briefly, the input function for Patlak modeling was derived from the PET image by placing 2D sphere ROIs (1.5 mm diameter) over the inferior vena cava (and away from other organs) during the early time frames. ¹⁷ For overnight fasted pregnant rats, a blood glucose concentration of 4.8 mmol/L has been previously reported, ¹⁸ corresponding to 3.84 mmol/L is the fetal brain (80%). ¹⁹ Finally, the FDG lumped constant for rats (0.71 ²⁰ ) and equilibrium time t* = 40 min were used to generate maternal and fetal brain CMR_glu values in PMOD’s kinetic modeling tool. For in vivo [¹¹C]raclopride binding, standardized uptake value ratio (SUVR; 45–90 min) was quantified using the whole fetal brain and the maternal cerebellum as the reference region, as described. ²¹

All results are given as mean ± standard deviation. Statistical significance was assigned by p < 0.05. For group mean differences, Student’s t-tests were performed. Pearson’s correlation was used to perform linear regressions.

Results

In vivo [¹⁸F]FDG whole-brain SUV correlates with ex vivo SUV

[¹⁸F]FDG accumulation was readily visualized in maternal and fetal organs on summed PET/CT images (Figure 1). Specifically, high accumulation (“hot spots”) could be seen corresponding to the placenta, fetal heart, and fetal brain. In the fetal whole-brain, in vivo [¹⁸F]FDG SUV at the end of scan (82.5 min) was significantly correlated (R² = 0.90, p < 0.001, n = 42) with ex vivo SUV in post-mortem brains harvested at the end of scan (Figure 2).

Figure 1.

Time-averaged (0–90 min) [¹⁸F]FDG PET/CT images normalized by body weight and injected dose to SUV in a representative dam and fetal subject. Insert 1 (square): axial slice of uterus. Insert 2 (oval): sagittal slice of individual fetus and placenta. Arrows indicate reslicing to orientation.

B: fetal brain; H: fetal heart; P: placenta; S: fetal spine; SUV: standardized uptake value.

Figure 2.

[¹⁸F]FDG in vivo SUV in fetal brains is highly correlated with ex vivo SUV determined from post-mortem brains at the end of scan (y = 0.56x + 0.27, R² = 0.90, n = 42). Markers (circle, square, diamond, star) correspond to fetuses from the same pregnant dam and colors correspond to baseline (gray) or haloperidol pretreatment (red) [¹⁸F]FDG scans.

SUV: standardized uptake value.

[¹⁸F]FDG utilization is lower in the fetal brain

In vivo [¹⁸F]FDG accumulation at 82.5 min was lower (32%–53%) in the fetal whole-brain compared to the maternal whole-brain at 90 min in all four pregnant dams (Table 1). Consistently, ex vivo fetal brain [¹⁸F]FDG SUV was relatively lower (16%–41%) compared to maternal SUV at the end of the scan (Table 1). On average, the in vivo CMR_glu of the fetal brains was only 52% ± 10% (8.76/18.24 µmol/min/100 g) of the maternal whole-brain value (Figure 3).

Table 1.

Maternal and fetal brain [¹⁸F]FDG SUV: ex vivo versus in vivo.

Animal		Ex vivo SUV (g/mL)	In vivo SUV (g/mL)
1	Maternal		2.42
	Fetal (n = 8)	2.41 ± 0.08	1.65 ± 0.07
	F:M ratio		0.68
2	Maternal	3.36	3.08
	Fetal (n = 13)	2.83 ± 0.31	1.87 ± 0.12
	F:M ratio	0.84	0.61
3	Maternal	3.70	3.49
	Fetal (n = 12)	2.45 ± 0.08	1.65 ± 0.07
	F:M ratio	0.66	0.47
4 a	Maternal	2.90	2.49
	Fetal (n = 9)	1.72 ± 0.11	1.19 ± 0.06
	F:M ratio	0.59	0.48

F:M ratio: fetal-to-maternal SUV ratio; SUV: standardized uptake value.

Fetal data mean ± SD.

^aAnimal 4 pretreated with haloperidol (0.5 mg/kg, IV) prior to experiment.

Figure 3.

Mean CMR_glu (µmol/min/100 g) in fetal brains is lower relative to the maternal rate in vivo. Markers (circle, square, diamond, star) correspond to maternal–fetal pairs and bars indicate mean CMR_glu across maternal (n = 4) and fetal (n = 42) brains.

CMR_glu: cerebral metabolic rate of glucose.

Baseline [¹¹C]raclopride SUVR is reproducible

Mean [¹¹C]raclopride SUVR in the fetal brain was not significantly different between baseline scans (p = 0.20) and showed moderate variability. On average, fetal whole-brain SUVR was 1.13 ± 0.11 versus 1.20 ± 0.08 (n = 9 vs 6, Supplemental Figure 3(a)) and intersubject variability (coefficient of variation (CV)) was 8.99% (n = 15).

Haloperidol decreased [¹⁸F]FDG and [¹¹C]raclopride quantification in fetal brains

Haloperidol administration decreased [¹⁸F]FDG uptake in both the maternal and fetal brains in one pregnant rat (Figure 4). Specifically, haloperidol pretreatment produced 31% lower SUV (1.19/1.74 g/mL) measured in vivo and, similarly, 34% lower SUV (1.72/2.59 g/mL; n = 9 vs 33) measured ex vivo in fetal brains (Figure 5). These differences were significantly lower than the baseline SUV (p < 0.001). After calculation of CMR_glu, haloperidol significantly decreased in FDG utilization by 65% in fetal brains (3.8/10.8 µmol/min/100 g, n = 9 vs 33; p < 0.001) and 52% in the maternal brain (10.1/21.0 µmol/min/100 g, n = 1 vs 3), as shown in Figure 5.

Figure 4.

Haloperidol pretreatment (0.5 mg/kg, IV) reduced [¹⁸F]FDG uptake in the maternal (n = 1) and fetal (n = 9) rat brain. SUV PET images were averaged between 0 and 90 min and overlaid on a standard MRI brain atlas with size scales (top row) at midsagittal slice.

Figure 5.

Haloperidol (0.5 mg/kg, IV) decreases in vivo [¹⁸F]FDG metabolism rate (CMR_glu) and ex vivo uptake (SUV) in fetal brains. (a) CMR_glu is decreased in the maternal brain after haloperidol pretreatment (n = 1, red) compared to mean baseline CMR_glu (n = 3, gray). (b) In vivo CMRglu, (c) Ex vivo [¹⁸F]FDG SUV, and (d) in vivo [¹⁸F]FDG SUV are significantly decreased (p < 0.001) in fetal brains after haloperidol pretreatment (n = 9, red) compared to mean baseline level (n = 33, gray). Bars indicate group means and markers indicate individual data points. Marker shapes in b-d represent fetuses from the dam with the same marker shape in a.

Consistent with the pharmacological effect observed with [¹⁸F]FDG, haloperidol pretreatment decreased dopamine D2R/D3R availability in the maternal brain almost to the level of the cerebellum (Supplemental Figure 2). [¹¹C]Raclopride SUVR decreased from baseline in every fetal brain after haloperidol pretreatment (Supplemental Figure 3(b)). Specifically, mean SUVR decreased from 1.20 ± 0.08 to 1.00 ± 0.07 (17% decrease, n = 6 vs 6, p < 0.001) in the same fetuses after haloperidol pretreatment (Supplemental Figure 3(b)). Compared to mean baseline SUVR grouped from all fetuses (n = 15), haloperidol produced a 13% decrease (p = 0.005) in SUVR (Figure 6).

Figure 6.

Mean [¹¹C]raclopride SUVR is decreased after haloperidol pretreatment (0.5 mg/kg, IV) in fetal brains. Bars indicate mean baseline (gray) or haloperidol (blue) SUVR and markers (stars, circles) represent fetuses from the same pregnant dam.

Discussion

In this study, we report in vivo measurements of [¹⁸F]FDG uptake (SUV, CMR_glu) and [¹¹C]raclopride SUVR in fetal whole-brains of late-gestation pregnant rats using PET/CT. Specifically, we show that [¹⁸F]FDG distribution was clearly visualized in utero, and in vivo SUV significantly correlated with ex vivo brain uptake. We estimated that CMR_glu was 52% ± 10% lower than in the maternal brain. In two pregnant rats imaged with [¹¹C]raclopride, baseline fetal brain SUVRs were comparable. Following haloperidol pretreatment, we observed significant reductions in in vivo and ex vivo FDG accumulation, fetal CMR_glu, and [¹¹C]raclopride SUVR when compared to baseline conditions. These findings demonstrate that fetal glucose metabolism and D2R/D3R availability can be quantified noninvasively in vivo in the rat fetus brain by PET imaging.

To validate in vivo [¹⁸F]FDG SUV measurements, we quantified whole-brain SUV in dissected fetal brains ex vivo using a cross-calibrated γ-counter. Linear regression showed a significant positive correlation between in vivo and ex vivo SUV values across all four pregnant dams (Figure 2). This finding aligns with Sawatzke et al., who reported a strong correlation (R² = 0.86) between in vivo and ex vivo [^¹8F]FDG SUV in pregnant rat organs, whole-fetuses, and placentas. ¹⁸ The strong concordance supports the validity of fetal PET measurements in utero. Despite this correlation, we noticed in vivo [¹⁸F]FDG SUV values were significantly lower than ex vivo SUV (Table 1). However, both in vivo and ex vivo data consistently showed substantially lower [¹⁸F]FDG uptake in fetal versus maternal brains, and after haloperidol pretreatment. This discrepancy may be attributed to partial volume effects (PVEs) due to the relatively small fetal brain size (approximately three to four times the PSF). Yet, we expect this inherent limitation will not hinder our ability to infer differences between groups, as PVEs would similarly affect fetal brains across different experimental groups of similar sizes.

Fetal brain [¹⁸F]FDG SUV at 82.5 min post-injection reached 47%–68% of maternal brain SUV (Table 1), which is in agreement with reports in fetal NHP brain. ²² To explore whether SUV differences reflected true metabolic variation or differential FDG delivery across the blood-placenta barrier, we applied Patlak modeling to calculate CMR_glu. We found a 52% reduction in fetal brain CMR_glu relative to maternal levels (Figure 3), consistent with lower energetic demand in the anesthetized fetal brain during late gestation. While the biological basis underlying comparatively lower fetal brain energy expenditure than maternal brain is outside our scope, reproducing prior findings supports the feasibility of quantitative PET imaging in pregnant rat models. Indeed, Benveniste et al. demonstrated a 40%–50% lower CMR_glu in the fetal NHP brain, ⁷ while a 66% reduction in the metabolic rate of ¹⁴C-labeled glucose was observed in the fetal rat brain. ²³ Together, these results validate the pregnant rat model as a robust translational platform to study fetal brain metabolism in utero, highlighting opportunities to investigate fetal brain to drug or toxin exposure during pregnancy.

In contrast to [¹⁸F]FDG, [¹¹C]raclopride is a widely used radiotracer that binds to dopamine D2/D3 receptors, which are highly expressed in the striatum. ²⁴ In the fetal brain, significant levels of both dopamine receptors ²⁵ and the necessary enzymatic and transportation machinery for functional dopamine signaling are present. ²⁶ Therefore, we quantified baseline [¹¹C]raclopride binding in a subset of two pregnant rats. Across fetal whole-brains, SUVRs were not statistically different between scans (n = 9 vs 6), showing moderate intersubject variability (9%) comparable to baseline variability observed in adult rat brains (8%). ²⁷ This level of reproducibility supports the utility of the pregnant rat model for investigating neuroreceptor availability during fetal brain development. Because the fetal cerebellum is thin and flat, ¹⁶ we chose the maternal cerebellum as a reference region for [¹¹C]raclopride quantification, which has been validated in pregnant NHPs. Specifically, Bartlett et al. calculated the distribution volume ratio (DVR), a fully quantitative measure of specific binding, for [¹⁸F]fallypride in the fetal NHP brain using the plasma concentration, maternal cerebellum, or fetal cerebellum as the input function. Among these, they demonstrated consistent quantification between all three methods. ²⁸ We choose to report the SUVR relative to the maternal cerebellum as a measure of specific binding, which approximates DVR but has the key advantages of simple quantification without requiring full scan dynamic data. However, we used SUV from 45 to 90 min as a pseudo equilibrium period, which may introduce bias due to not achieving true steady state, yet we found that SUVR was highly correlated to DVR determined using Logan reference graphical analysis (Supplemental Figure 4).

Finally, to further assess the feasibility of in vivo PET quantification in the fetal rat brain, we administered a pharmacological dose of haloperidol to one pregnant rat before [¹¹C]raclopride and [¹⁸F]FDG PET/CT. As a dopamine D2R antagonist, haloperidol is known to have a long biological half-life and achieve robust displacement of [¹¹C]raclopride binding. ²⁹ Acute haloperidol administration is known to produce widespread decreases in cerebral [¹⁸F]FDG utilization in controls and subgroups of schizophrenic patients. ³⁰ We therefore hypothesized that haloperidol administration would reduce [¹¹C]raclopride binding and [¹⁸F]FDG CMR_glu, not only in maternal brains but also in fetal brains through the placenta-fetal brain barrier transfer of the drug. ³¹ Indeed, we found the fetal brain SUVR was significantly reduced to the level of the maternal reference region after haloperidol administration (Figure 6). In an exploratory analysis, we further found that [¹¹C]raclopride SUVR in the fetal heart, presumably with low density of D2Rs, did not decrease after haloperidol treatment, in contrast to the fetal brain and placenta with known D2R expression (Supplemental Figure 5). ³² While the maternal reference region may not fully capture differences like P-glycoprotein (P-gp) function differences at the blood-brain barrier versus the blood-placenta barrier, this is a less likely explanation because [¹¹C]raclopride does not have P-gp interactions. ³³ Additionally, haloperidol pretreatment also reduced fetal brain CMR_glu by 65% (n = 33 vs 9), consistent with its expected pharmacological effect measured indirectly through glucose metabolism changes. In summary, we first demonstrated [¹⁸F]FDG SUV in fetal brain, validating the static uptake of [¹⁸F]FDG with ex vivo measurement. We further estimated the PET parameters CMR_glu and [¹¹C]raclopride binding using maternal input functions for quantification in fetal brains, taking advantage of the dynamic information enabled by in vivo PET imaging. In all, this highlights the feasibility of fetal brain PET as a sensitive and specific measure of pharmacological effects and neuroreceptor availability changes using the pregnant rat.

This study has several limitations. First, it is important to note that the translational potential may be restricted by ethical and safety concerns surrounding PET imaging in pregnant humans. That said, preclinical work in this field is a valuable way to learn about biological processes, such as fetal neurodevelopment, even if translation to humans is impractical. Additionally, anesthesia is a common confound in preclinical imaging studies and may influence metabolic or neurochemical measurements. Species differences in placental physiology and developmental timing may also present challenges. Notably, late gestation in rats aligns most closely with the second trimester of human development. ³⁴ We also note that CMR_glu calculations were based on literature values for blood glucose in pregnant rats and a standard lumped constant. However, these approximations do not affect the reported maternal–fetal differences in CMR_glu that we report. Due to the small size of the fetal rat brain relative to the PET system resolution, we focused on whole-brain analysis in this proof-of-concept study. While suitable for detecting global effects, this may limit sensitivity to detect regional changes, particularly for tracers like [¹¹C]raclopride, which targets discrete receptor populations. Therefore, caution should be exercised when choosing a radiotracer for investigating a specific target of interest. We did not apply partial volume correction (PVC), which may have contributed to underestimation of tracer uptake, and pursuing PVC in this model may increase the value of future experiments in this field. In the case of [¹¹C]raclopride, signal spill-out from high radioactivity in the intestines led to exclusion of some fetal scans depending on fetal head orientation. This limitation could be addressed in future work through voxel-wise PVC or intravenous CT contrast to improve fetal brain localization. ³⁵ Lastly, we did not perform formal power analysis or statistical tests for normality and acknowledge this as a limitation of our study. However, this preliminary data provides important foundational insights that can inform future research on disease mechanisms with clinical applications. Larger, independent samples in future studies will enable robust assessment of effect sizes, statistical power, and model assumptions, thereby enhancing the translational relevance of this approach.

In conclusion, the pregnant rat model is a feasible and informative platform for quantitative PET studies of fetal brain development. Many disorders first diagnosed in adolescence or adulthood are increasingly linked to in utero development, and adverse experiences in early life can lead to lasting changes in the brain’s response and resilience to stress. ³⁶ Studies in humans have largely focused on how early life experiences shape neurocircuits, for example, associating negative parenting behaviors with reduced activation in the mesolimbic pathway regulated by dopamine. ³⁷ However, how molecular-level adaptations in early life, especially involving neuroreceptors, shape future neurocircuitry is poorly understood and not easily investigated with clinical research paradigms. For this objective, our method can take advantage of the experimental flexibility and short gestational period of a rodent model to investigate longitudinal mechanisms in neurodevelopment. For example, dopamine D2Rs may play an important role in neurodevelopment changes underlying schizophrenia or externalizing disorders that originate from in utero infection or maternal substance abuse. ³⁸ Future studies examining changes in D2R availability in the fetal brain after maternal immune activation or chronic opioid exposure could add translatable insights into how the neurocircuitry changes observed in humans after adverse early life experiences arise.