Ongoing effects of preterm birth on the dopaminergic and noradrenergic pathways in the frontal cortex and hippocampus of guinea pigs

Children born preterm have an increased likelihood of developing neurobehavioral disorders such as attention‐deficit hyperactivity disorder (ADHD) and anxiety. These disorders have a sex bias, with males having a higher incidence of ADHD, whereas anxiety disorder tends to be more prevalent in females. Both disorders are underpinned by imbalances to key neurotransmitter systems, with dopamine and noradrenaline in particular having major roles in attention regulation and stress modulation. Preterm birth disturbances to neurodevelopment may affect this neurotransmission in a sexually dimorphic manner. Time‐mated guinea pig dams were allocated to deliver by preterm induction of labor (gestational age 62 [GA62]) or spontaneously at term (GA69). The resultant offspring were randomized to endpoints as neonates (24 h after term‐equivalence age) or juveniles (corrected postnatal day 40, childhood equivalence). Relative mRNA expressions of key dopamine and noradrenaline pathway genes were examined in the frontal cortex and hippocampus and quantified with real‐time PCR. Myelin basic protein and neuronal nuclei immunostaining were performed to characterize the impact of preterm birth. Within the frontal cortex, there were persisting reductions in the expression of dopaminergic pathway components that occurred in preterm males only. Conversely, preterm‐born females had increased expression of key noradrenergic receptors and a reduction of the noradrenergic transporter within the hippocampus. This study demonstrated that preterm birth results in major changes in dopaminergic and noradrenergic receptor, transporter, and synthesis enzyme gene expression in a sex‐ and region‐based manner that may contribute to the sex differences in susceptibility to neurobehavioral disorders. These findings highlight the need for the development of sex‐based treatments for improving these conditions.


INTRODUCTION
Neurodevelopment involves a series of finely orchestrated events that occur throughout gestation and early postnatal life.Disruptions to these processes can have major implications for the developing infant, including an increased risk of neurodevelopmental disorders (Soleimani et al., 2014).Preterm birth (birth defined as <37-week gestation) represents approximately 10% of all births worldwide, with ∼50% of preterm delivered children going on to develop longterm neurodevelopmental disabilities (Chawanpaiboon et al., 2019).Two of the neurodevelopmental disorders that are most frequently observed following moderate-late preterm birth (32-37-week gestation) include attention-deficit hyperactivity disorder (ADHD) and anxiety disorders (Fitzallen et al., 2021;Palumbi et al., 2018).These disorders tend to occur in a sex-dependent manner, with ADHD typically being diagnosed in males and anxiety being more commonly observed in females; however, the mechanisms behind this sex bias remain unclear (McLean et al., 2011;Ramtekkar et al., 2010).
There are several potential mechanisms that may contribute to the neurodevelopmental disorders linked to pretermassociated brain injury.Premature exposure to the stimulating ex utero environment, hypoxic-ischemic events, alongside the removal of the inhibitory placentally derived neurosteroid allopregnanolone, ultimately exposes the neonate to excitotoxic damage (Gopagondanahalli et al., 2016;Shaw, Berry, et al., 2019).The hippocampus and frontal cortex are two regions of the brain that develop in the latter half of gestation and are very susceptible to excitotoxic damage (Halliwell et al., 2009;Jacob et al., 2011).Likewise, this is a window of developmental susceptibility for oligodendrocyte (OL) lineage maturation and subsequent myelination, furthering cerebral susceptibility to excitotoxic damage (Shaw et al., 2021).Preterm birth has been shown to cause long-lasting changes to the OL population and subsequent myelination development, particularly within the frontal cortex and hippocampus (Ortinau & Neil, 2015;Shaw et al., 2016;Volpe et al., 2011).These regions are densely innervated by dopaminergic and noradrenergic pathways, of which these neuromodulators are particularly susceptible to injury caused by hypoxia and associated excitotoxic damage (Buller et al., 2008;Bywood & Johnson, 2001;Chen et al., 2017;Descarries et al., 1987;Devoto & Flore, 2006;Jenkins et al., 2016;Lindvall et al., 1978;Moreno-Castilla et al., 2017).Studies have indicated that these neurotransmitters also play roles in OL development and myelin production (Azizi, 2022;García-Ramírez et al., 2014).Additionally, it has been found that OLs express dopamine receptors D2 and D3, which have been proposed to modulate the maturation of OLs (Bongarzone et al., 1998;Howard et al., 1998).In a rat model of embryonic corti-cal cells, quetiapine, a D2 agonist, was shown to increase the synthesis of myelin (Xiao et al., 2008).
Dopamine and noradrenaline are both catecholamine neuromodulators that have multiple projections across the central nervous system and support a wide range of brain functions, including within the frontal cortex and hippocampus (Ranjbar-Slamloo & Fazlali, 2020).Dopamine plays a role in almost all aspects of cognitive functioning and is critical in attention and emotional control, the stress response, and the filtering of appropriate information to the brain (Weele et al., 2019).Noradrenaline, as a metabolite of dopamine, attenuates these functions through its shared biosynthetic pathway, with a large proportion of the dual effects of these neurotransmitters being observed in the frontal cortex and hippocampus (Ranjbar-Slamloo & Fazlali, 2020).Disruptions in dopaminergic and noradrenergic systems have been implicated in several neurobehavioral disorders.These neurotransmitters tend to work in an "inverted U shape" fashion, whereby programing leading to too little or too much can have negative effects on behavioral outcomes, increasing vulnerability to neurobehavioral disorders (Floresco, 2013;Holland et al., 2021).ADHD has been widely associated with dysfunction in several parts of the dopaminergic pathway.Positron emission tomography (PET) scanning revealed individuals with ADHD have decreased levels of the dopamine D1 receptor within the prefrontal cortex and anterior cingulate cortex that were associated with hyperactive behavior (Yokokura et al., 2021).Similar patterns have emerged with noradrenergic transmission, whereby adult ADHD patients are shown to have reduced levels of noradrenaline transporter (NET) levels as detected by PET-MRI scanning (Ulke et al., 2019).Dysfunction to these neurotransmitter pathways within associated regions such as the frontal cortex and hippocampus is also associated with anxiety conditions.Studies have shown reduced noradrenaline results in a drowsy state, whereas excessive noradrenergic activity is associated with symptoms of hyperarousal and anxiety (Bouras et al., 2023).The roles of the dopamine D1 and D2 receptors in anxiety were further elucidated in a rat model of anxious behavior, whereby the administration of D1 antagonists decreased conditioned fear responses, and administration of D2 antagonists attenuated fear reflexes (Pavlova et al., 2015).
Interestingly, some studies have investigated the effects of perinatal disruptions to these neurotransmitters.Prenatal stress in 2-month-old rats was found to cause decreased dopamine and noradrenaline levels in the bed nucleus of stria terminalis, an extension of the amygdala (Soares-Cunha et al., 2018).Similarly, in a study examining late prenatal stress in rats, the adult offspring were found to have increased levels of the dopamine D2-like receptors in key areas of the frontal cortex and hippocampus (Berger et al., 2002).The previous work has also extensively investigated the effects of prenatal drug exposure on these pathways.One study in particular showed that nicotine administered to pregnant rats caused reduced noradrenaline levels in offspring at 30-day postpartum (Seidler et al., 1992), highlighting the in utero programing of this system.However, it remains to be determined whether preterm birth results in similar dysfunction to these neurotransmitter systems in turn leading to the altered neurodevelopmental and behavioral outcomes associated with prematurity.Studies investigating the impact of preterm birth on the development of dopamine and noradrenaline pathways are lacking.Several postmortem studies have investigated the effects of hypoxia-ischemia (HI) in infants and concluded that a reduction in D2 receptor density was associated with an increase in the severity of brain injury (Kapucu et al., 1998;Meng et al., 1998;Tranquart et al., 2001).A recent study in which fetal HI was used to model ischemic brain injury, increasingly appreciated as an important comorbidity in preterm infants, concluded that HI resulted in a reduction of D1 and D2 receptor densities, as well as a reduction of dopamine transporter (DAT) expression within the caudate nucleus (Wong et al., 2020).Similarly, in a rat model of HI, postnatal day (PND) 3 pups were found to have reduced noradrenergic neurons and a reduction to tyrosine hydroxylase (TH)-producing neurons (Buller et al., 2008).However, there are currently limited studies on the long-term effects of preterm birth on either the dopamine or noradrenaline pathways in childhood or adolescence.
In this study, we hypothesize that preterm birth is associated with dysfunction to both the dopaminergic and noradrenaline pathways in both the frontal cortex and hippocampus, including their transporters and synthesis enzymes, and furthermore that this may occur in a sex-dependent manner.This study was performed using a guinea pig model, which is similar to human brain development but unlike other rodent species, key neurodevelopmental time points such as myelination and synaptogenesis begin before birth (Hirst et al., 2018;Kalusa et al., 2021;Morrison et al., 2018;Piorkowska et al., 2014;Shaw et al., 2022).Additionally, similar to a human pregnancy, the guinea pig placenta produces a continual supply of progesterone up until term (Hirst et al., 2018).As such, this model is clinically relevant and provides insight into the mechanisms underpinning preterm-associated brain injury and the increased risk of neurobehavioral disorders in children born preterm (Berry et al., 2015;Hirst et al., 2018).

Animals
Time-mated Dunkin Hartley female guinea pigs were obtained from the University of Otago Wellington Biomedical Research Unit.The animal study was conducted in accordance with approval given by the University of Otago, Wellington Animal Ethics Committee (AUP 18-174).Guinea pigs were housed indoors under a 12-h light/dark cycle and were supplied with a diet of commercial guinea pig pellets, hay, and fresh vegetables.A total of 53 dams were used for the study.Litter effects were minimized as one pup from each dam was used (littermates formed part of a larger collaborative study).Pregnant dams were randomly allocated to either preterm (GA62) or term (GA69) delivery.Dams allocated to term pregnancy received no intervention during pregnancy, with pups delivering spontaneously and with no additional respiratory or nutritional support.Preterm pups were born via preterm induction of labor as previously described (Berry et al., 2015;Shaw et al., 2015;Shaw, Dyson, et al., 2019).In brief, dams received subcutaneous betamethasone (1 mg/kg, Celestone Chronodose; Merck Sharp and Dohme) 48 and 24 h prior to preterm delivery to ensure lung maturation and surfactant production.Aglepristone (10 mg/kg, Provet) was administered subcutaneously 24 h prior to, and on the morning of delivery to inhibit progesterone-based continuance of pregnancy.Intramuscular oxytocin (3 IU/kg, Provet) was administered to stimulate uterine contractions 1 h after the second aglepristone dose and repeated until all pups and placentas were delivered.
Resuscitation and respiratory support of preterm pups occurred as previously described (Berry et al., 2015;Shaw et al., 2015;Shaw, Dyson, et al., 2019).Continuous positive airway pressure (CPAP) at 5 cm H 2 0 was supplied to all preterm pups for respiratory support for at least 3 min, using the "Neopuff" t-piece infant resuscitator (Fisher and Paykel).All preterm pups were also given an initial fractional inspired oxygen concentration of 30% that was titrated depending on the pup's well-being.Once stable, pups were housed with their mothers and siblings in a warm humidified incubator (Dräger 8000 IC; Drägerwerk AG & Co.), with ambient temperature 33˚C (titrated down to 28˚C by 24 h), and 60% humidity (titrated down to 35% by 12 h).
Preterm pups were fed 0.3-0.5 mL of Impact guinea pig colostrum replacement (Wombaroo Food Products) orally within the first hour after birth and then every 2 h until 24h old.Thereafter, pups were fed 0.5-2 mL of Impact guinea pig milk replacement (Wombaroo Food Products) every 2 h or as needed until independent feeding was established.No additional nutritional or environmental support was provided for the pups once they had reached term equivalent age (TEA): a chronological age of 7 days and a corrected PND 0.

Tissue collection
All pups were euthanized by exsanguination under isoflurane, with tissue and organ weights recorded.For the neonatal group, preterm pups were euthanized at 24 h after TEA and term pups within 24 h of birth.For the juvenile group, pups were euthanized at corrected PND40.Each brain was sectioned in the sagittal plane to separate the hemispheres.Each left hemisphere was fixed in 4% paraformaldehyde, whereas the right was further dissected into key regions of interest and frozen in liquid nitrogen.

Immunohistochemistry
To characterize the preterm-associated differences in brains from animals born preterm or at term, immunostaining for a mature myelin marker (myelin basic protein [MBP]) and total neuron population (neuronal nuclei [NeuN]) was performed.
The caudal frontal cortex section (used to analyze MBP) was determined morphologically by the corpus callosum joining at the genu, at the most rostral point before the genu separated.The lateral ventricle was also present and used to further confirm the relative depth.The rostral frontal cortex section (used to analyze NeuN) was determined morphologically by the absence of the corpus callosum, and the presence of the isolated forceps minor.The lateral ventricles were not present at this point (Crombie et al., 2023(Crombie et al., , 2021;;Markowitsch & Pritzel, 2008;Shaw, Dyson, et al., 2019;Shaw et al., 2018;Tindal, 1965).In brief, dewaxing and rehydrating of tissue sections occurred by incubating in xylene and ethanol before antigen retrieval in citrate buffer (pH 6.0) at 90-95˚C for 25 min.Endogenous peroxidases were blocked by incubation in phosphate-buffered saline (PBS) containing 3% hydrogen peroxide for 20 min, and nonspecific staining was blocked by incubation in a goat serum block for 1 h (2% goat serum, 0.4% BSA, 0.3% Triton-X in PBS).Slides were incubated in primary antibodies overnight at 1:1000 dilution (MBP M9394, Sigma; NeuN MAB377, Millipore), before incubation in secondary antibodies at 1:300 dilution for 1 h at room temperature (biotinylated anti-rat IgG B7139; biotinylated anti-mouse IgG B6649).Slides then had a 1-h tertiary incubation in streptavidin-biotin-horseradish peroxidase complex (ab7403, Abcam) at 1:400 dilution for 1 h.Immunostaining was revealed by incubation in 3,3′-diaminobenzidine tetrahydrochloride solution (Metal Enhanced DAB Substrate Kit; Thermo Fisher Scientific).The Aperio imaging system was used to image stained sections at 20× magnification (Leica Biosystems).NeuN were quantified by cell count (cells/μm 2 ) in four regions of the prefrontal cortex, including the cingulate, prelimbic, infralimbic, and motor cortices.For mature myelinating OL protein, relative area coverage (%) of positive staining was quantified in three regions, including the cingulate and motor cortices, as well as the forceps minor of the caudal frontal cortex.Three serial consecutive sections from each sample were used to image each of the regions and were quantified with the HALO 3.1 cytonuclear stain and area quantification analysis software (Indica Labs).Appropriately sized boxes were used to quantify protein expression within each region over three consecutive sections, encompassing as much of the region as possible without overlapping adjacent regions.The total area of MBP quantification consisted of 1.5, 4.0, and 1.0 mm 2 for the cingulate cortex, motor cortex, and forceps minor, respectively, in the neonate per section and 2.0, 6.0, and 1.2 mm 2 , respectively, in the juvenile brains.
The area of NeuN quantification consisted of 2.0, 1.0, 0.6, and 3.0 mm 2 for cingulate, prelimbic, infralimbic, and motor cortices, respectively, in the neonate and 3.0, 1.5, 1.0, and 6.0 mm 2 , respectively, per section in the juvenile brains.The average of the three consecutive sections was calculated per region.

Real-time PCR
Frozen frontal cortex and hippocampal tissue were prepared for PCR as previously described (Shaw et al., 2015(Shaw et al., , 2018;;Shaw, Dyson, et al., 2019).Briefly, frozen tissue was homogenized in RLT Plus Buffer (Qiagen), using a Precellys 24 dual-tissue homogenizer (Bertin Technologies).RNA extraction was then performed using the Qiagen RNeasy Plus Mini Kit (Qiagen) by following manufacturers' instructions.Synthesis of cDNA was performed with the Superscript III Reverse Transcription kit (Invitrogen) using a GeneAmp 9700 PCR Machine (Applied Biosystems, Life Technologies Pty Ltd).Primers for the genes of interest (Table 1) were designed using the predicted Cavia porcellus sequence (National Centre for Biotechnology Information [NCBI], Bethesda, MD, USA) and optimized to ensure maximum efficiency and specific amplification.Real-time PCR (RT-PCR) was then performed using the QuantStudio 6 Flex RT-PCR system (Applied Biosystems) for primer pairs shown in Table 1, with beta actin (ACTB) used as a housekeeper.Samples were run in duplicate along with a negative control sample (reverse transcribed with MilliQ).PCR products were detected with SYBR Green (Applied Biosystems) and analyzed using the Sequence Detection Software v2.01 (Applied Biosystems).Relative fold changes were calculated using the comparative Ct method (2 −ΔΔCt ).A calibrator of pooled brain samples was used to ensure consistency among plates.

Statistical analyses
Data were analyzed using Prism v7.0 (GraphPad Software Inc.) and presented as mean ± SEM for each group with significance considered p < .05.The data were first analyzed by a two-way ANOVA, before post hoc tests with Tukey  2. In this study, preterm males had a survival rate of 50%, and preterm females had a survival rate of 65%.All term pups had a survival rate of 100%.
At the juvenile age, preterm-born males and females weighed significantly less than term-born animals (p < .0001,.0004).There were no significant changes observed in overall body weight for neonates (PND1).
Interestingly, juvenile preterm-born females had an increased brain-body weight ratio compared to termdelivered counterparts, indicating a potential brain-sparing mechanism (p = .03).Compared to term-born sex-matched animals, male neonates and female juveniles born preterm had an increased liver-body ratio (p = .02,.04),and both male and female neonates born preterm had a higher kidney-body ratio (p < .0001and <.0001).This increase in kidney-body ratio persisted for female juveniles born preterm (p = .01).There were no other significant changes to organ-body weight ratios between preterm and term-delivered animals at either neonatal or juvenile age.

Relative mRNA expression of dopamine receptor subtypes in the frontal cortex
Preterm-born males had a significant reduction in DRD1 expression at both the neonatal and juvenile age, compared to term-born animals (p = .02and .01,respectively; Figure 1a).Conversely, preterm-born female neonates had a significant increase in expression of DRD1 compared to those born at term (p = .008;Figure 1a).
In male juveniles born preterm, there was a significant reduction in D2-like receptors DRD2, DRD3, and DRD4 compared to term controls (p = .01,.004,and .01,respectively; Figure 1b,d,e).Conversely, female neonates born preterm had a significant increase in expression of DRD2 and DRD3 compared to term-born controls (p = .006and .02,respectively; Figure 1b,c).
Lastly, there was a reduction of DRD5 in male juveniles born preterm compared to those born at term (p = .01;Figure 1e).Main effects of delivery and age as well as interactions are provided in Table S1.

Relative mRNA expression of noradrenergic receptors in the frontal cortex
Compared to term-born controls, male neonates and juveniles showed an increased expression of ADRA1 (p = .002and .01,respectively; Figure 2a), whereas female neonates born preterm showed an increased expression of ADBR1 (p = .01;Figure 2b).There were no significant differences identified in ADRA2 and ADBR2 expressions for either sex (data supplied in Figure S1).Main effects of delivery and age as well as interactions are provided in Table S2.

Relative mRNA expression of dopamine and noradrenaline receptors in the hippocampus
Preterm birth affected the expression of dopamine receptors DRD3 and DRD5, as well as the noradrenaline receptor ADBR1 in the hippocampus (Figure 3).Compared to term controls, the expression of DRD3 was increased in male neonates born preterm (p = .014;Figure 3a).However, expression was reduced in male juveniles born preterm (p < .0001; Figure 3a).Preterm-born male and female neonates both had reduced expression of DRD5 relative to term controls (p = .003and .001,respectively; Figure 3b).Preterm-born female juveniles had significantly increased the expression of ADBR1 compared to term controls (p = .02;Figure 3c).There were no other significant differences identified for the other receptors examined: DRD1, DRD2, DRD4, ADRA1, ADRA2, and ADBR2 (see Figure S2).Main effects of delivery and age as well as interactions are provided in Table S3.Additionally, main effects of the supplementary figures are provided in Table S7.

Relative mRNA expression of dopamine synthesis enzyme and transporter, and noradrenaline transporter in the frontal cortex and hippocampus
Male neonates and juveniles born preterm had a significant reduction in expression of the rate-limiting dopamine synthesis enzyme TH in the frontal cortex, compared to term controls (p = .01and .01,respectively; Figure 4a).Male juveniles born preterm also had a significant reduction in the expression of dopamine reuptake transporter SLC6A3 within the frontal cortex (p = .02;Figure 4b).In males born preterm, both neonates (p = .0001;Figure 4c) and juveniles (p = .008;Figure 4c) showed significantly increased expression of the noradrenaline reuptake transporter NET (SLC6A2) within the frontal cortex.
Conversely, in the hippocampus, female juveniles born preterm showed significantly increased expression of TH (p = .02;Figure 4d) and SLC6A3 compared to term controls (p = .04;Figure 4e).Within the hippocampus, male juveniles born preterm had increased expression of SLC6A2 (p = .0003;Figure 4f), whereas preterm-born female neonates and juveniles had significantly reduced expression compared to term-born counterparts (p = .01and .0004,respectively; Figure 4c).Main effects of delivery and age as well as interactions are provided in Table S4.

EFFECTS OF PRETERM BIRTH ON MYELIN BASIC PROTEIN IN THE FRONTAL CORTEX
MBP immunostaining was quantified in the cingulate cortex and motor cortex of the frontal cortex by immunohistochemistry (Figure 5).Preterm birth resulted in a significant reduction of MBP immunostaining in the cingulate cortex of male neonates, which was maintained into juvenility compared to term controls (p = .01and .002,respectively; Figure 5a).Preterm-born juvenile females also had a reduction in MBP in the cingulate cortex compared to controls (p = .002;Figure 5a).
Similarly, in the motor cortex, male neonates and juveniles born preterm also had a reduced area coverage of MBP immunostaining compared to controls (p = .0001and .02,respectively; Figure 5b).There were no other significant differences identified in protein expression by immunostaining.However, MBP relative mRNA in frontal cortex tissue was also significantly reduced in male juveniles born preterm compared to controls (p = .001;data supplied in Figure 3).Main effects of delivery and age as well as interactions are provided in Table S5.

5
NeuN IN THE FRONTAL CORTEX

Neuronal nuclei in the frontal cortex
NeuN protein expression was quantified in the cingulate and infralimbic cortices of the frontal cortex (Figure 6).Male juveniles born preterm had reduced cell counts in the cingulate cortex (p = .01;Figure 6a), whereas female neonates born preterm had reduced cell counts compared to term controls (p = .001;Figure 6a).Preterm-born male juveniles also displayed a significant reduction in NeuN cell counts within the infralimbic cortex (p = .04;Figure 6b) compared to term controls.There were no other significant differences identified for protein expression or relative mRNA expression (see Figure S3).Main effects of delivery and age as well as interactions are provided in Table S6.

DISCUSSION
This study has demonstrated that preterm birth results in significant alterations in the gene expression of dopamine and noradrenaline receptors, synthesis enzymes, and transporters within the frontal cortex and hippocampus of the guinea pig brain.These changes were seen at TEA following preterm birth but, perhaps more importantly, frequently continued to the equivalent of late childhood in this species.Males appeared to have more alterations in dopamine receptor expression, whereas females showed more effects in NET expression.Children-born preterm have an increased risk of developing neurobehavioral disorders such as anxiety and ADHD; our results suggest that a disruption to the dopamine and noradrenaline systems may be a major contributor to both these altered neurodevelopmental states but may also explain the sexually dimorphic impact of preterm birth.We have also continued to demonstrate that males born preterm have reduced MBP and reduced neuronal cell counts compared to their term controls (Shaw et al., 2016;Shaw, Dyson, et al., 2019).Furthermore, the juvenile age preterm and term-born cohort of animals allowed us to uncover deficits that persisted up until the equivalence of late childhood age and highlight when neurodevelopmental trajectories may have been dysregulated.This is consistent with other clinical findings of long-term developmental damages in preterm infants, such as white matter abnormalities, and validates the use of the guinea pig as a model of preterm birth (van Beek et al., 2021;Vo Van et al., 2022).One of the key findings of this study was that preterm males have significantly reduced expression of all five of the dopamine receptor subunits in the frontal cortex.These deficits primarily occurred at the later juvenile time point, indicating that the changes following preterm birth appear to cause widespread long-term changes to the expression of these critical receptors.There is a significant interaction between developmental age and delivery with DRD3 and DRD5 expression in males (Table S1) within the frontal cortex, supporting that the trajectory of development of these receptors is altered.Indeed, the expression of most receptor subtypes appears to remain halted at the neonatal age following preterm birth.To date, there are no other studies looking at the dopamine system in the frontal cortex of guinea pigs.There are, however, studies on dopamine receptor expression in the frontal cortex of humans and rats that show that dopamine receptor expression changes across development.In human prefrontal cortex, it was found that D2 and D5 receptor expressions are highest early in development and decline with age, whereas D1 shows an opposite trajectory, with expression increasing into adulthood (Rothmond et al., 2012).Adolescence has been found in both rats and nonhuman primates to coincide with increases in dopaminergic receptor expression; however, there are limited studies on the direct changes to each receptor subtype in either species (Wahlstrom et al., 2010).Changes in receptor expression are both region-and age-dependent within species and, evidently, different across species.Unfortunately, the current available literature does not provide us with appropriate information to assess changes in the developmental trajectory in the guinea pig model beyond our neonatal to juvenile age observations.Dopamine receptors can be split into two main families: the D1-like family (receptors D1 and D5) and the D2-like family (receptors D2, D3, and D4) (Beaulieu & Gainetdinov, 2011).Neurons in the frontal cortex richly express both families, with the D1-like family being the most prevalent of the two (Puig & Miller, 2012).Attentional control is a key aspect of cognitive functioning that is controlled largely through modulation of the dopamine family within the frontal cortex (Bahmani et al., 2019).Previous studies have shown that pharmacological blockade of the D1 and D2 receptors results in attentional impairment in an adult mouse model (Wulaer et al., 2021).These observations suggest the reduced receptor expression found in the males born preterm occurred at a time point at which symptoms of ADHD would likely start to occur.A rat model of ADHD also found that hypofunction of the D1 receptors in the anterior cingulate cor-tex was associated with hyperactive behavior (Satoh et al., 2018).
The reduced expression of all dopamine receptor subtypes in this critical brain region in the preterm-born male pups could therefore cause deficits in attention and focus, which are the primary symptoms of ADHD.Similar changes may possibly underlie a mechanism behind the development of ADHD in children that are born preterm (Skogli et al., 2013).Preterm-born children have a markedly greater risk of the development of ADHD with male children at heightened risk.As we only saw reduced expression in males, this further highlights a potential sex bias that may explain these clinical rates of ADHD that are more prevalent in males (Skogli et al., 2013).We have previously shown that preterm-born male guinea pigs exhibit a hyperactive phenotype indicative of ADHD-like behavior in the juvenile age period (Shaw et al., 2016).
The observation of the reduced expression of the dopamine synthesis enzymes TH and DAT (SLC6A3) in pretermborn male guinea pigs may further contribute to disrupted dopaminergic and noradrenergic signaling.Additionally, the significant effect of age in the preterm male cohort for the expression of SLC6A3 in the frontal cortex reveals an altered developmental trajectory of this synthesis enzyme following preterm birth.TH is the rate limiting enzyme in the synthesis of dopamine and, subsequently, noradrenaline (Nagatsu, 2007).The observed reduction in the expression of this enzyme could indicate that this combination, with the reduced expression of dopamine receptors, could markedly reduce F I G U R E 5 Continued overall levels and action of dopamine and noradrenaline within the frontal cortex.Interestingly, we also saw a reduction of the catecholamine transporter DAT (SLC6A3).DAT removes dopamine and other catecholamines from the synaptic cleft and returns them to the presynaptic neuron (Mackie et al., 2018).Previous imaging studies have reported differing results regarding the levels of DAT in patients with ADHD.One study used PET scanning on adult ADHD patients and found that these patients showed increased DAT binding within the caudate (Spencer et al., 2007); conversely, another study using MRI-based imaging found that adult ADHD patients had reduced DAT binding within the thalamus and midbrain (Hesse et al., 2009).These findings suggest that there may be considerable regional differences in the expression of dopaminergic enzymes in ADHD patients.As we also saw a reduction in the expression of the dopamine synthesis enzyme, this additional reduction in transporter expression may be the result of a lack of dopamine available to be recycled.Marked differences were also observed in enzymes controlling noradrenergic action with preterm birth having opposing effects on the expression of the NET in males and females (SLC6A2).NET functions in a similar manner to DAT, by moving noradrenaline into the presynaptic neuron (Nagatsu, 2007).The finding that preterm-born males had significantly increased expression of NET continuing into the juvenile period in both the frontal cortex and hippocampus suggests a reduction in the overall action of noradrenaline in these regions.As noradrenaline aids dopamine in attentive functions, this reduction could further predispose preterm males to the development of, or susceptibility for, ADHD (Ulke et al., 2019).Additionally, males exhibited a significant interaction between delivery and age for hippocampal SLC6A2 expression (Table S4), suggesting that the developmental trajectory of this transporter was upregulated by preterm birth.
In this study, consistent with previous investigations of neurodevelopmental outcomes following preterm birth (Kozhemiako et al., 2020), female preterm-born pups responded in a strikingly different manner compared to their male preterm counterparts.We found that females born F I G U R E 6 Continued preterm showed markedly elevated dopamine receptor expression, with increased DRD1, DRD2, and DRD3 frontal cortex expressions during the neonatal period.Importantly, this change was resolved by the juvenile period, indicating potential corrective mechanisms that may mitigate the long-term impact of preterm-associated brain injury in females.Previous studies have reported that increased levels of testosterone in the adolescent period in males may suppress this mechanism, resulting in a greater susceptibility to dopamine dysfunction (Sinclair et al., 2014).Preterm-born females in this study typically showed greater alterations in the hippocampus, in contrast to the preterm-born males.This finding is clinically relevant, as differences in the hippocampus and amygdala have been shown to contribute to anxiety disorders that have a higher prevalence in females (McLean et al., 2011).Indeed, these current findings showed increased dopamine DRD3 receptor expression in preterm females in the juvenile period.Studies using a mouse model of preadolescent anxiety have shown consistent results with adult female mice having increased DRD3 expression in response to stressors.
As such, it has been suggested that this receptor subtype may be involved in the modulation of the anxiety response (Seo & Kuzhikandathil, 2015).In contrast to the males, preterm juvenile females were shown to have reduced NET (SLC6A2) within the hippocampus.This finding suggests a reduction in the expression of the transporter could result in an excessive level of noradrenaline possibly accumulating in this region.The amygdala receives direct input from the hippocampus in response to stress, whereby noradrenaline is a critical neurotransmitter involved in this regulatory response (Martin et al., 2009).Increased levels of noradrenaline, particularly within these limbic regions, are associated with fear responses and activation of anxiety (Montoya et al., 2016).Consistent with this notion, this study also observed that females born preterm have reduced expression of the noradrenaline beta-1 receptor ADBR1, which is also associated with fear responses (Nesse et al., 1984;Pavlova & Rysakova, 2019).Thus, these changes in preterm-born females may contribute toward their predisposition to developing anxiety-related disorders.We have previously shown that female guinea pigs exposed to prenatal stress develop an anxious phenotype, indicating that altered neurodevelopment may increase the likelihood of such behaviors (Crombie et al., 2021).Lastly, we showed that preterm birth led to persisting alterations to MBP and marked changes in the neuronal populations (Dean et al., 2014;Schmidbauer et al., 2019).Myelination occurs in a developmental lineage, whereby cells develop from early OL precursor cells (OPCs) to mature myelinating OLs (van Tilborg et al., 2018).During these early stages, OPCs undergo rapid proliferation in order to maintain an appropriate level of cells prior to the differentiation into mature OLs, which is an irreversible step.These early OPCs are highly susceptible to injuries such as hypoxia and ischemia, and therefore preterm birth has been proposed to cause an arrest in OPC development and failure to mature into myelinating OLs, which could explain the reduced MBP observed in this study (Back et al., 1998;Shaw et al., 2021).
Similarly, the cortical maturation of neurons is also susceptible to preterm birth.During later gestation, the cerebral cortex is undergoing dramatic changes.Cortical neurons are in the process of rapid axonal development and are particularly vulnerable to insults (Volpe, 2019).In one clinical study, by term equivalent age, preterm-born infants were found to have reduced neurite density in parts of the cortex (Dimitrova et al., 2021).This is proposed to occur for several reasons, including reduced dendritic and spine formation as well as dysmaturation of subplate neurons that are vital for the guidance of other migratory neurons.Additionally, abnormal pruning may contribute to deficits observed (Nosarti et al., 2008).In this study, preterm birth reduced NeuN, a marker of overall neurons, which could be explained by these developmental effects.It is worth noting that within the cingulate cortex in males, and within the infralimbic cortex in both sexes, there was a significant interaction between age and delivery (Table S6).NeuN expression was downregulated at juvenile age following preterm birth.This further suggests that preterm birth may derail the neurodevelopmental trajectory of neuronal development.
Preterm-born infants are characterized by lower birth weights and reduced fat mass (Casirati et al., 2022).In this study, preterm-born juvenile males and females show a reduced body weight compared to terms, indicating this reduced weight persisted through development.This also replicates what we have previously seen in our preterm model, with preterm-born males still exhibiting reduced body weight at PND28 (Shaw et al., 2018).Preterm birth is associated with reduced renal maturity at birth due to the late gestation development of the kidneys.Kidney:Body weight ratio was significantly higher between preterm and term for males and females at neonatal age; however, this difference was reduced by juvenile age in males and was of reduced magnitude in the females.Increased kidney volume follows the expected phenotype for this preterm model as the preterm neonates were 7 days ex utero at time of collection, allowing for renal hypertrophy due to hyperfiltration through the immature, low-nephron, preterm kidneys (Grillo et al., 2021).
One limitation of the study is that the dopaminergic and noradrenergic pathways were only examined at the mRNA level.Previous studies have concluded that for dopamine in particular, mRNA expression can reliably translate into similar protein levels (Araki et al., 2007).Despite this, it would be worthwhile, considering replicating these findings with protein work, to confirm that these changes are occurring at a functional level.However, in the guinea pig, we are currently limited by antibody specificity.Moving forward, it would be useful to quantify direct neurotransmitter levels as well and correlate this to the mRNA data observed.Another limitation is the administration of betamethasone, oxytocin, and aglepristone to the dams delivering preterm but not term.Betamethasone and oxytocin were given at clinically relevant times and doses and are necessary for the survival and delivery of the preterm pup.However, it is necessary to acknowledge that these compounds may impact results, as they were only administered to preterm dams and not term.Another limitation is that one area from each cortical region was analyzed, in triplicate, for immunohistochemistry analysis.Protein analysis of MBP and NeuN was only performed in the frontal cortex.Further investigation of these markers in the hippocampus would enhance this study.Future research is warranted in potentially delivering pups by survival C-section surgery, which would eliminate these confounding factors.
In conclusion, this study has shown that preterm birth changes both dopamine and noradrenaline receptor, transporter, and synthesis enzyme gene expression in both the frontal cortex and hippocampus, with males showing more alterations to the dopamine pathway and females to the noradrenaline pathway.This may explain why we see sex differences in neurobehavioral disorder development following preterm birth.The current work will shine a light on potential therapeutic targets and avenues that may be considered.Further studies are required to explore this, with possibility given to therapeutics that can modulate these neurotransmitter systems, and hopefully prevent these damages from occurring.Neurosteroid replacement therapies as zuranolone and ganaxolone may be a possibility, given the interplay between dopamine and noradrenaline and the GABAergic system (Bullock et al., 2021).Further investigation in this area is warranted for the prevention of neurobehavioral disorders given the serious long-term outcomes of these disorders.Open access publishing facilitated by The University of Newcastle, as part of the Wiley -The University of Newcastle agreement via the Council of Australian University Librarians.

C O N F L I C T O F I N T E R E S T S T A T E M E N T
The authors confirm that there are no financial ties to products, or conflicts of interest to disclose.

D A T A AVA I L A B I L I T Y S T A T E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Roisin A. Moloney: Conceptualization; formal analysis; investigation; methodology; project administration; writingoriginal draft preparation.Hannah K. Palliser: Funding acquisition; project administration; writing-review and editing.Rebecca M. Dyson: Methodology; writing-review and editing.Carlton L. Pavy: Writing-review and editing.Max Berry: Methodology; writing-review and editing.Jonathon J. Hirst: Funding acquisition; writing-review and editing.Julia C. Shaw: Study design; funding acquisition; investigation; methodology; supervision; writing-review and editing.A C K N O W L E D G M E N T S We would like to acknowledge Ryan Sixtus, Heather Barnes, Maureen Prowse and Taylor Wilson for their contributions to the animal work in this study.
Guinea pig-specific primers for used for real-time PCR.
T A B L E 1Note: Primer sequences for detection of genes of interest in the guinea pig frontal cortex and hippocampus.Primer sequences are displayed from 5′−3′ for forward and reverse primers.Abbreviations: DAT, dopamine transporter; NeuN, neuronal nuclei; NET, noradrenaline transporter.correctionsformultiplecomparisons were performed when the ANOVA was p < .05.Factors in the ANOVA included delivery (preterm or term) and postnatal age (neonate or juvenile).Data were split by sex.Main effects of delivery and age as well as interactions are provided in TablesS1-S6.At the time of tissue collection (at either the neonatal [PND1] or juvenile [PND40] age) animal weights and organbody ratios were recorded and are detailed in Table Physical characteristics.Means and standard error of the means for body (in grams) and organ weights (ratio to body or brain weight) recorded upon tissue collection at term-equivalence age for neonatal (corrected PND1) and juvenile (corrected PND40) pups.Dam numbers (n) per group are recorded.Comparisons are made within sex with difference to term at p T A B L E 2Note: Body and organ weights.