Early Adolescence Prefrontal Cortex Alterations in Female Rats Lacking Dopamine Transporter

Monoamine dysfunctions in the prefrontal cortex (PFC) can contribute to diverse neuropsychiatric disorders, including ADHD, bipolar disorder, PTSD and depression. Disrupted dopamine (DA) homeostasis, and more specifically dopamine transporter (DAT) alterations, have been reported in a variety of psychiatric and neurodegenerative disorders. Recent studies using female adult rats heterozygous (DAT+/−) and homozygous (DAT−/−) for DAT gene, showed the utility of those rats in the study of PTSD and ADHD. Currently, a gap in the knowledge of these disorders affecting adolescent females still represents a major limit for the development of appropriate treatments. The present work focuses on the characterization of the PFC function under conditions of heterozygous and homozygous ablation of DAT during early adolescence based on the known implication of DAT and PFC DA in psychopathology during adolescence. We report herein that genetic ablation of DAT in the early adolescent PFC of female rats leads to changes in neuronal and glial cell homeostasis. In brief, we observed a concurrent hyperactive phenotype, accompanied by PFC alterations in glutamatergic neurotransmission, signs of neurodegeneration and glial activation in DAT-ablated rats. The present study provides further understanding of underlying neuroinflammatory pathological processes that occur in DAT-ablated female rats, what can provide novel investigational approaches in human diseases.


Introduction
Genetically determined changes in dopaminergic (DA) activity might contribute to the occurrence of neuropsychiatric diseases such as PTSD [1,2] and ADHD [3]. Sex-specific studies have demonstrated that the prevalence of negative outcomes associated with childhood ADHD [4] and PTSD [5] are higher in women, as well as the incidence of PTSD [6]. However, little research has focused on sex-specific studies of these pathologies at both clinical [4,6] and preclinical level [7,8]. Recent studies focusing on adult dopamine transporter deletion in rats, in the heterozygous and homozygote genotypes, highlighted that female adult rats from both genotypes display model validity for PTSD and ADHD [9]. On the other hand, male adult DAT−/− rats also display model validity for ADHD [10,11]. Furthermore, Adinolfi and coworkers have shown that adolescent DAT−/− rats display

Animals
Wistar Han heterozygous (DAT+/−) and homozygous (DAT−/−) rats were generated as previously described [29]. Wildtype (DAT+/+), DAT+/− and DAT−/− female rats were weaned at post-natal day (PND) 21 and housed in groups of 2-3, with food and water available ad libitum. The colony was maintained under standard conditions (12 h light/dark cycle 6 a.m. on-6 p.m. off, 21 ± 1 • C, 40-70% humidity). The breeding scheme was performed as reported from our group, mating mature DAT+/− female rats of fertile age (>2 months old) with mature DAT+/− male rats [9,30]. We acknowledge the possible effect that the breeding strategy could have on the study [30], out of our scope of work. All of the experiments were conducted in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the University of Miami IACUC (Protocol #17-016, Approval Date 15 June 2017). The replacement, reduction and refinement (3R) principle was applied, and the total number of animals used was 45.

Locomotor Activity
Locomotor activity was evaluated using Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH, USA) under illuminated conditions. The plexiglass apparatus (40.4 × 40.5 × 30.3 cm chamber) was equipped with four open field monitors, each made of 16 light beams placed on the horizontal x and y axes. The hardware detected beam breaks, while the software determined the location of the animal in the locomotor box apparatus. The horizontal activity was expressed in beam cross number in the x axes, in counts. Vertical activity represented the number of beam breaks on the y axes, in counts, which originated from rearing activity of the rat. Stereotyped behavior (stereotypies count), which measures the beam break patterns occurring in time intervals lower than 1 s, was also provided in counts.
The animals were individually tested for 85 min at the age of PND35. The number of rats per group was as follows: DAT+/+ n = 14, DAT+/− n = 18, DAT−/− n = 13.

Western Blotting
Rats were sacrificed by decapitation following locomotor activity recording. Half brain hemispheres were sectioned, snap-frozen and kept at −80 • C until analysis.

Tissue Preparation for Histological Assessments
Rats were sacrificed as indicated in the "Western blotting" section. The remaining halfbrain was post-fixed in paraformaldehyde 4% in PBS for 3 days at 4 • C, then transferred in sucrose 0.5 M in PBS 0.1 M for 7 days at 4 • C for cryoprotection, frozen on dry-ice surface and kept at −80 • C until analysis.
Fluoro-Jade C (FJC) staining was performed on the same PFC slice series following manufacturer's instructions (TR-100-FJT). Briefly, slides were dried at 60 • C on a slide warmer for 10 min and incubated in a mixture of 80% ethanol/Solution A (9:1) for 5 min. Slides were then rinsed 2 times in ethanol 70% and 2 times in double-distilled (dd) water. Slides were incubated in a mix of dd water/Solution B (9:1) for 10 min and rinsed in dd water. A mixture of FJC staining solution, DAPI and dd water (1:1:8) was added to the slides and incubated in the dark for 10 min; after 2 rinses in dd water, slides were dried at 60 • C on a slide warmer for 10 min and then cleared in xylene. Slides were mounted in DPX mounting medium.

Image Analyses
In the case of NeuN + cell (neuronal) count, data were analyzed using FIJI software [32]. Briefly, 3-4 images acquired at 10× were obtained for 4 animals per genotype (pixel conversion: 1 pixel ≡ 1.024 µm). After conversion to 8-bit, images were set to a threshold lower than 8% and cell bodies counted using the Analyze > analyze particles function. Experimenters were blinded to the genotype conditions.
Morphometric analyses of Iba1 + microglia were performed as previously reported [33], with minor modifications. In summary, data were analyzed using FIJI software [32], and 3 image stacks acquired at 63× were obtained for 3 animals per genotype (pixel conversion: 1 pixel ≡ 0.1652 µm). Cell tracing was performed using the "Simple Neurite Tracer" plugin and Sholl analyses executed as previously reported [34]. Experimenters were blinded to the genotype conditions.

ELISA
PFC protein extracts prepared for Western blotting were used for ELISA analysis. Thirty µg of protein extract per sample was used. Concentrations of interferon gamma (IFNγ) were measured using commercially available enzyme-linked immunosorbent assays following manufacturer indications (Pierce biotechnologies, Rockford, IL 61105, USA, Thermo Scientific #EM10015). Each sample was analyzed in duplicate, and all the absorbance readings were performed at 450 and 550 nm. The final absorbance value was obtained subtracting the 450 nm readings from the 550nm values. Protein extract dilution was tested as per manufacturer instructions and chosen to be 30 µg to fall within the dynamic range of the calibration curve. DAT+/+ n = 5; DAT+/− n = 4; DAT−/− n = 5.

Statistical Analysis
GraphPad Prism 9 was used for all analyses, and the null hypothesis was rejected at the 0.05 level of significance. The locomotor behavior experiment results were analyzed with two-way ANOVA, followed by Tukey's multiple comparison post hoc test. One-way ANOVA with Tukey's post hoc test was used for analysis of Western blot and ELISA data. Nested one-way ANOVA followed by Tukey's multiple comparison post hoc test was used to analyze cell count data and morphometric analyses.

Locomotor Activity of Female Adolescent DAT+/− and DAT−/− Rats
At PND35, rats were tested for locomotor behavior and then sacrificed for biochemical analyses of the PFC ( Figure 1A).

Glutamatergic and Synaptic Alterations in Female Adolescent DAT+/− and DAT−/− Rats
Cortical alterations of glutamate neurotransmission might contribute to the exhibition of hyperactive phenotype in rats [35]. Therefore, we investigated the expression levels of glutamate subunits in the PFC of DAT+/− and DAT−/− rats. While expression levels of the obligate NMDAR1 subunit were unchanged among the three genotypes ( Further, we sought to investigate if ablation of DAT had substantial effects on the main components of pre-and post-synaptic machinery. DAT−/− rats display a significant increase of protein levels of pre-synaptic protein VAMP2 ( Figure 2C; one-way ANOVA F (2, 13) = 6.051; p = 0.0139; Tukey's multiple comparison test p = 0.0186 DAT+/+ versus DAT−/−). In order to study possible post-synaptic alterations in the PFC, we analyzed the expression of PSD95 protein. Our data indicate that PSD95 levels were unchanged among the three genotypes ( Figure 2D; one-way ANOVA F (2, 14) = 0.1729; p = 0.8430].
These data indicate that either partial of total ablation of DAT affects glutamate receptor homeostasis in the developing PFC of DAT+/− and DAT−/−. Moreover, DAT−/− rats also show anomalies in the VAMP2 component of the pre-synaptic machinery.

Glutamatergic and Synaptic Alterations in Female Adolescent DAT+/− and DAT−/− Rats
Cortical alterations of glutamate neurotransmission might contribute to the exhibition of hyperactive phenotype in rats [35]. Therefore, we investigated the expression levels of glutamate subunits in the PFC of DAT+/− and DAT−/− rats. While expression levels of the obligate NMDAR1 subunit were unchanged among the three genotypes (

DAT−/− Rats Display Neuronal Cell Death in the Prefrontal Cortex
In order to assess whether absence of DAT could cause neuronal cell death as previously shown in the DAT−/− mouse model in the striatum [36], we initially sought to qualitatively investigate the occurrence of neuronal death by Fluoro-Jade C (FJC) staining [37]. As shown ( Figure 3A), elevated FJC staining positivity was observed in DAT−/− PFC, while a sparse signal was detected in DAT+/− and complete absence in DAT+/+ controls. Based on FJC qualitative data, we proceeded to investigate markers of apoptotic response such as cleaved caspase 3 [38]. Western blot analysis showed that levels of cleaved caspase 3 are higher in the PFC of DAT+/− rats (one-way ANOVA F (2, 13) = 5.898; p = 0.0150; Tukey's multiple comparison test p = 0.0545 DAT+/− versus DAT+/+ controls) albeit without reaching statistical significance ( Figure 3B). Nevertheless, DAT−/− rats display a 2-fold increase in the levels of cleaved caspase 3 (Tukey's multiple comparison test p = 0.0184 DAT−/− versus DAT+/+ controls; Figure 3B).
In order to further address the occurrence of neuronal cell death and concomitant elevation of cleaved caspase 3 expression, we performed PFC NeuN + cell body counting ( Figure 3C

DAT−/− Rats Display Neuronal Cell Death in the Prefrontal Cortex
In order to assess whether absence of DAT could cause neuronal cell death as previously shown in the DAT−/− mouse model in the striatum [36], we initially sought to qualitatively investigate the occurrence of neuronal death by Fluoro-Jade C (FJC) staining [37]. As shown ( Figure 3A), elevated FJC staining positivity was observed in DAT−/− PFC, while a sparse signal was detected in DAT+/− and complete absence in DAT+/+ controls. Based on FJC qualitative data, we proceeded to investigate markers of apoptotic response such as cleaved caspase 3 [38]. Western blot analysis showed that levels of cleaved caspase 3 are higher in the PFC of DAT+/− rats (one-way ANOVA F (2, 13) = 5.898; p = 0.0150; Tukey's multiple comparison test p = 0.0545 DAT+/− versus DAT+/+ controls) albeit without reaching statistical significance ( Figure 3B). Nevertheless, DAT−/− rats display a 2-fold increase in the levels of cleaved caspase 3 (Tukey's multiple comparison test p = 0.0184 DAT−/− versus DAT+/+ controls; Figure 3B).
In order to further address the occurrence of neuronal cell death and concomitant elevation of cleaved caspase 3 expression, we performed PFC NeuN + cell body counting ( Figure 3C

Pro-Inflammatory Phenotype in the Prefrontal Cortex of DAT−/− Rats
We sought to investigate whether absence of DAT could also potentially impact glia in DAT+/− and DAT−/− rats. To this aim, we measured the levels of ALDH1L1, a selective pan-astrocyte marker [39,40]. DAT−/− rats showed a 1.5-fold increase in the levels of ALDH1L1 (one-way ANOVA F (2, 14) = 11.79; p = 0.0010; Tukey's multiple comparison test p = 0.0007 DAT−/− versus DAT+/+ controls), while DAT+/− rats showed a trend toward elevation that did not reach statistical significance (Tukey's multiple comparison test p = 0.0586 DAT+/− versus DAT+/+ controls; Figure 4A). Moreover, we analyzed the levels of myelin basic protein (MBP), which is responsible for axon myelination and the expression of which is tightly regulated by mature oligodendrocytes [41]. We did not observe any

Pro-Inflammatory Phenotype in the Prefrontal Cortex of DAT−/− Rats
We sought to investigate whether absence of DAT could also potentially impact glia in DAT+/− and DAT−/− rats. To this aim, we measured the levels of ALDH1L1, a selective pan-astrocyte marker [39,40]. DAT−/− rats showed a 1.5-fold increase in the levels of ALDH1L1 (one-way ANOVA F (2, 14) = 11.79; p = 0.0010; Tukey's multiple comparison test p = 0.0007 DAT−/− versus DAT+/+ controls), while DAT+/− rats showed a trend toward elevation that did not reach statistical significance (Tukey's multiple comparison test p = 0.0586 DAT+/− versus DAT+/+ controls; Figure 4A). Moreover, we analyzed the levels of myelin basic protein (MBP), which is responsible for axon myelination and the expression of which is tightly regulated by mature oligodendrocytes [41]. We did not In order to further investigate the occurrence of inflammatory signal transduction, we measured the levels of expression of mitogen-activated protein kinase (MAPK) P38, which plays a pivotal role in the regulation of inflammatory mediators and cytokines [42]. Our data show that phosphorylation levels of phospho-p38 MAPK (Thr180/Tyr182) were significantly increased in the PFC of DAT−/− rats (one-way ANOVA F (2, 14) = 8.332; p = 0.0041; Tukey's multiple comparison test p = 0.0007 DAT−/− versus DAT+/+ controls). We then analyzed the levels of interferon gamma (IFNγ), a pro-inflammatory cytokine involved in glial activation [43], in the PFC of DAT+/− and DAT−/− rats. We observed a significant increase of IFNγ in female adolescent DAT−/− rats (one-way ANOVA F (2, 11) = 5.621; p = 0.0208; Tukey's multiple comparison test p=0.0311 DAT−/− versus DAT+/+ controls; Figure 4C).
Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 16 0.0163). Taken together, these data reveal that DAT ablation causes glial alterations highlighting a pro-inflammatory state in the PFC of female adolescent DAT−/− rats.
The functional state of DAT has been proven to affect neuronal plasticity and control of cognitive function in the PFC [56,57], where pharmacological inhibition has shown to induce pro-cognitive effects [58]. DA function in the PFC controls several processes including working memory, attention, flexibility in behavior and action planning [59]. The DAT−/− rat model displays aberrant PFC signaling [29,60] that contributes to the alterations of cognitive, social and sexual behavior [10,29,60] in adult rats, with occurrence of behavioral abnormalities starting during adolescence [12]. More recently, investigations on PFC during postnatal development revealed that DA homeostasis is tightly regulated by astroglial cells at this stage [13], thus highlighting the importance that glial cells play in regulating DA signaling during development. Indeed, several studies have confirmed that DA can exert either anti-inflammatory or pro-inflammatory effects in human and in vivo models through dopamine receptor activation on astrocytes and microglia [61][62][63]. In this framework, we sought to investigate the effects of DAT ablation in the prelimbic PFC, responsible for cognitive processing [64], that could lead to glial pro-inflammatory process that might contribute to the underlying pathophysiology of female DAT+/− and DAT−/− rats during early adolescence.
Our data indicate that adolescent DAT−/− female rats display an overt hyperactive phenotype in the locomotor chamber, characterized by increased sustained locomotion as well as stereotypic behavior. Our data confirm previous findings observed in this animal
The functional state of DAT has been proven to affect neuronal plasticity and control of cognitive function in the PFC [56,57], where pharmacological inhibition has shown to induce pro-cognitive effects [58]. DA function in the PFC controls several processes including working memory, attention, flexibility in behavior and action planning [59]. The DAT−/− rat model displays aberrant PFC signaling [29,60] that contributes to the alterations of cognitive, social and sexual behavior [10,29,60] in adult rats, with occurrence of behavioral abnormalities starting during adolescence [12]. More recently, investigations on PFC during postnatal development revealed that DA homeostasis is tightly regulated by astroglial cells at this stage [13], thus highlighting the importance that glial cells play in regulating DA signaling during development. Indeed, several studies have confirmed that DA can exert either anti-inflammatory or pro-inflammatory effects in human and in vivo models through dopamine receptor activation on astrocytes and microglia [61][62][63]. In this framework, we sought to investigate the effects of DAT ablation in the prelimbic PFC, responsible for cognitive processing [64], that could lead to glial pro-inflammatory process that might contribute to the underlying pathophysiology of female DAT+/− and DAT−/− rats during early adolescence.
Our data indicate that adolescent DAT−/− female rats display an overt hyperactive phenotype in the locomotor chamber, characterized by increased sustained locomotion as well as stereotypic behavior. Our data confirm previous findings observed in this animal model [9,10,29] and demonstrate occurrence of sustained hyperactive phenotype in adolescent female rats.
Our present data indicate that neither heterozygous nor homozygous deletion of DAT affects the expression levels of the obligate NMDA receptor subunit R1 (NMDAR1), while a reduction of NMDA receptor 2B (NMDAR2B) glutamate subunit is observed in both genotypes. NMDAR2B is the main component of neuronal extra-synaptic NMDARs [66] that guarantees the maintenance of sustained activity and proper postnatal development of the PFC [67]. Our data suggest that reduction of NMDAR2B in the PFC of female DAT−/− rats during adolescence could contribute to aberrant cortical development, which includes pre-synaptic alterations observed in the increased levels of VAMP2. Further studies investigating the mechanisms responsible for such impacts on glutamatergic signaling are needed. However, the hyperdopaminergic state of DAT−/+ as well as DAT−/− could be underlying such impact on the glutamatergic pathway components.
In neuropsychiatric diseases, extrasynaptic NMDARs are involved in complex signaling mechanisms that promote cell death, ultimately by activation of P38 MAPKs [66]. The marked increase in P38 phosphorylation that we measured in the PFC of homozygote animals might therefore underlie the neuronal cell death observed in the PFC of adolescent female DAT−/− rats. A dual approach [37] was previously used to confirm neurodegeneration in DAT−/− PFC that included (i) Fluoro-Jade C (FJC) staining, where a positive result indicates the presence of degenerating neurons [37,68], and (ii) NeuN + cell bodies count, the reduction of which indicates neuronal loss. We further investigated neurodegeneration addressing the concurrent activation of apoptotic cleaved caspase 3. Ultimately, we detected no changes in the levels of glial cell type markers CD45 (microglia) and MBP (oligodendrocytes), and increased levels of ALDH1L1 astroglial marker. In conclusion, these data indicate that ablation of DAT is responsible of substantial changes that involve multiple pathways leading to neurodegeneration in the PFC of DAT−/− rats. Interestingly, a trend of increased cleaved caspase 3 and spare positivity to FJC staining was observed in female DAT+/− rats that, in combination with the reduced NMDAR2B protein levels, opens novel investigational scenarios oriented toward specifically addressing changes that could occur when a mutation is only present in one allele and DAT is only partially reduced. Furthermore, neurodegeneration is also observed in the striatum of a subpopulation of DAT−/− mice (~36%), leading to a fatal neurodegenerative phenotype reminiscent of DTDS symptomatology [36,47].
It is noteworthy that P38 MAPK module activation plays a pivotal role in inflammatory responses and cytokine release [69] and that increased inflammation in the central nervous system is a hallmark of psychiatric diseases in humans [18]. Dopamine is also an important regulator of cytokine secretion, regulating immune response [70] and neuropsychiatric disorders where aberrant DA neurotransmission is involved, such as PTSD and schizophrenia, displaying increased CNS inflammation [71]. Astrocytes are considered as initiators of and responders to inflammation in the CNS [72] and preside over the control of DA homeostasis in the developing PFC [13]. Based on our data and on previous literature, we sought to investigate glial markers indicative of pro-inflammatory signal transduction in the PFC of DAT+/− and DAT−/− adolescent rats. Homozygous ablation of DAT presented a characteristic profile of pro-inflammatory state [43] on the PFC indicated by increases in pan-astroglial marker (ALDH1L1), phosphorylation of P38 MAPK and an increase in the levels of pro-inflammatory cytokine IFNγ. Such an environment might likely affect microglia, which respond to inflammation processes in the brain and regulate neuronal activity as well as connectivity [73]. Indeed, the pro-inflammatory profile seen in the PFC of adolescent DAT−/− rats was accompanied by changes in microglial morphology consistent with neuroinflammation [74], characterized by increased arborization and complexity. Peculiarly, the absence of alteration of myelin basic protein MBP levels produced by mature oligodendrocytes suggests that the pro-inflammatory conditions present in the PFC of female DAT−/− rats might not affect this glial population.
Altogether, these data support the fact that the PFC of DAT−/− female adolescent rats presents a pro-inflammatory profile at early stages in life, concurrent with neurodegeneration. Importantly, these patterns indicative of increased inflammatory responses were not present in DAT+/− female adolescent rats. Our differential data obtained from DAT−/− and DAT+/− rats might, therefore, provide further insights in the PFC pathophysiology of neuropsychiatric diseases like ADHD and PTSD, respectively, the model validity of which was demonstrated in our previous work [9]. An increasing number of studies using DAT+/− and DAT−/− rats are underway, and based on our results and previous publications [10,12], partial mutation of DAT is sufficient to cause several alterations to a variety of underlying mechanisms controlling, among others, behavioral parameters relevant for the study of psychiatric disorders [9,10,12].
Prevalence of psychiatric and neurodegenerative disorders is determined by sex [75] and should be treated accordingly [76]. Psychiatric disorders such as ADHD [77] and PTSD present higher incidence and prevalence in women compared to men [78,79]. Although most psychiatric disorders cited across our study present higher prevalence in women, we acknowledge that our study focused only on female rats, and further studies are needed to expand our results also in male rats. Additionally, we studied female rats at PND35, the time point during development when hormones have been described to be almost ready to start the first proestrus cycle [80]. We acknowledge the need for future studies considering the role of hormones modulating glutamatergic synaptic response. The present investigation focused on early adolescent rats. We acknowledge the limitations of the time frame selected and the need to introduce additional time points during development. Future studies are needed to ensure better understanding of possible glial and neurodegenerative features during development and in adult rats.
Neuroinflammation is a common facet of a large variety of psychiatric and neurodegenerative disorders. In the present study we showed in early adolescent females that genetically engineered female rats lacking DAT present with a neurodegenerative phenotype in the PFC, accompanied by altered glutamatergic neurotransmission and glial activation at early stages of adolescence. To our knowledge, this is the first characterization of the neuroinflammatory phenotype of the PFC of female DAT KO rats during adolescence. Further studies are needed to deeply characterize neuroinflammation in our DAT+/− and DAT−/− rat model at different developmental stages, including adulthood and across sexes. Our study introduces a novel investigational perspective that may be pursued to shed light on the neuroinflammatory and neurodegenerative pathophysiology of disorders involving DAT and aberrant DA neurotransmission.