Photoperiodic Effects on Monoamine Signaling & Gene Expression Throughout Development in the Serotonin & Dopamine Systems

Photoperiod or the duration of daylight has been implicated as a risk factor in the development of mood disorders. The dopamine and serotonin systems are impacted by photoperiod and are consistently associated with affective disorders. Hence, we evaluated, at multiple stages of postnatal development, the expression of key dopaminergic (TH) and serotonergic (Tph2, SERT, and Pet-1) genes, and midbrain monoamine content in mice raised under control Equinox (LD 12:12), Short winter-like (LD 8:16), or Long summerlike (LD 16:8) photoperiods. Focusing in early adulthood, we evaluated the midbrain levels of these serotonergic genes, and also assayed these gene levels in the dorsal raphe nucleus (DRN) with RNAScope. Mice that developed under Short photoperiods demonstrated elevated midbrain TH expression levels, specifically during perinatal development compared to mice raised under Long photoperiods, and significantly decreased serotonin and dopamine content throughout the course of development. In adulthood, Long photoperiod mice demonstrated decreased midbrain Tph2 and SERT expression levels and reduced Tph2 levels in the DRN compared Short photoperiod mice. Thus, evaluating gene x environment interactions in the dopaminergic and serotonergic systems during multiple stages of development may lead to novel insights into the underlying mechanisms in the development of affective disorders.


Introduction
Globally it is estimated that over 300 million individuals suffer from depression and 17 million Americans have reported experiencing at least one depressive episode 1,2 .
Throughout adulthood the prevalence of depression is approximately 7% 2 . However, it is highest during early adulthood between the ages of 18-25 (13%) 2 and in adolescence, between the ages of 12-17, the prevalence can reach similar rates peaking at 13% 3 .
Interestingly, the prevalence of childhood depression is only approximately 2%, yet there is evidence that this and the overall incidence of mood disorders is beginning to steadily increase over the last 10 years 4 . Thus, childhood, adolescence, and early adulthood may represent vulnerable periods in which individuals may be more susceptible to the risk of developing depression 5-7 . Exposures to environmental factors during sensitive periods of development have been associated with elevated risk for neurodevelopmental disorders later in adulthood 8 . To this point, studies have demonstrated significant development x environment effects resulting in increased prevalence for psychiatric disorders 9 . An important environmental factor, the duration of daylight or photoperiod, has been implicated in affective disorders in adolescence and adulthood [10][11][12] , and has been shown to have profound and lasting effects on mood-related behaviors [12][13][14][15][16][17] . Photoperiod has been linked to increased risks for psychiatric disorders 11,[18][19][20][21] , can modulate monoamine turnover of serotonin and dopamine 22,23 , and associations between photoperiod and genes relevant to the serotonin and dopamine systems have revealed significant gene x environment effects observed during the winter and fall months, when the duration of daylight is lowest during the year 24 .
Therefore, photoperiod appears to be a consistent environmental factor that may play a critical role in the development of affective disorders.
Rodent studies in the dopaminergic and serotonergic systems have shown that photoperiodic exposure significantly affects neuronal firing rate 15,32 , monoamine signaling [33][34][35] , and mood-related behaviors 17,36,37 , which can be sex-dependent 34,38 . Specifically, it has been shown that developmental photoperiod can program various aspects of the serotonin system 15,34 . Mice that developed under Long summer-like photoperiods demonstrate increased dorsal raphe (DRN) serotonin (5-HT) neuronal firing rate, elevated midbrain serotonin content, and reduced anxiety and depressive-like behaviors later in life in a melatonin receptor 1 (MT1R) dependent manner 15 . Our lab has recently shown that prenatal photoperiodic exposure results in enduring changes to DRN 5-HT neuronal activity, and there are critical temporal windows within perinatal development that are impacted by photoperiod resulting in enduring changes to monoamine signaling and affective behaviors during adolescence and early adulthood 34 .
The midbrain contains both dopamine and serotonin rich nuclei, is a critical brain region responsible for mood regulation, and disruptions in these circuits have been linked to depression 8,39,40 . Midbrain dopaminergic neurons have specifically been shown to regulate aspects of depressive-like behavior and the activity of these neurons may be important for resilience against depression 41,42 . Tyrosine hydroxylase (TH) is a key gene in the dopamine system as it is the rate-limiting enzyme for dopamine (DA) synthesis, alterations in this gene have been observed in patients with depression, and have led to investigations focused on targeting TH as a novel therapeutic treatment for mood disorders [43][44][45] . Importantly, it has been shown that photoperiod can modulate human gene expression levels of TH and the dopamine transporter (DAT) in the midbrain 29 .
The main center for serotonin synthesis and neuronal development is a structure within the midbrain, known as the dorsal raphe nucleus (DRN) 46 . Studies have demonstrated developmental x environment changes specifically in the serotonergic system as it pertains to mood related disorders [47][48][49][50][51] . Prior developmental work has shown that manipulations to serotonin (5-HT) receptors, the serotonin transporter, and environmental factors such as stress can dramatically alter serotonin synthesis, circuit formation along with anxiety and depressive-like behaviors that can persist throughout adulthood [52][53][54][55][56][57][58][59][60][61] . Tryptophan hydroxylase 2 (Tph2) is the rate limiting enzyme for serotonin synthesis, the serotonin transporter (SERT) is responsible for reuptake of serotonin back into presynaptic vesicles from the synaptic cleft, and the ETS transcription factor Pet-1 is the main regulator for serotonin neuronal development and differentiation 62 . These three key 5-HT genes are highly expressed in serotonergic neurons and thus largely expressed in the midbrain and specifically the DRN 62 . Critically, developmental roles for these genes have been shown such that modulation of Tph2, SERT and/or Pet-1 expression during prenatal or perinatal development can also result in vast molecular, circuit level, and behavioral changes relevant to mood disorders [63][64][65][66][67] .
In this study we evaluated the effects of photoperiod on midbrain dopamine (TH) and serotonin (Tph2, SERT and Pet-1) gene expression, midbrain monoamine content, and expression levels of these 5-HT genes specifically in the DRN. These assays were performed in the mouse during multiple sensitive periods of postnatal development, representing childhood, adolescence, and early adulthood 68 , which have been implicated in the development of mood disorders 7 , in order to investigate the role of photoperiod on the dopamine and serotonin systems during the course of development.

Animals and Housing
Male and female C3Hf +/+ mice were used as these animals are melatonin-producing and lack the retinal degeneration alleles of the parent C3H strain 69 . For developmental midbrain quantitative RTPCR and monoamine content experiments mice were raised under either an Equinox (Eq) (12 hours of light 12 hours of darkness), Long (L) (16 hours of light and 8 hours of darkness), or Short (S) (8 hours of light and 16 hours of darkness) photoperiod (Figure 1 A-C). We have used these photoperiods commonly in our previous work 15,34,70 and these light-dark cycles (LD 16:8 and LD 8:16) mimic real-world photoperiods of summer-like (Long) and winter-like (Short) photoperiods at the high middle latitudes (e.g. equivalent to London, Paris, Berlin) and are experienced by significant portions of the human population. For all studies, mice were maintained continuously under these photoperiods from embryonic day 0 (E0) until they were assessed experimentally (Figure 1). Developmental midbrain quantitative RTPCR, measuring key dopamine (TH) and serotonin (i.e. Tph2, SERT, and Pet-1) genes, along with midbrain monoamine signaling assays were performed at P8, P18 (ranging from P17-P19), and P35 (ranging from P34-P37), representing perinatal development, early childhood, and adolescence in the mouse 68 (Figure 1). For early adulthood midbrain RTPCR 5-HT gene experiments, mice developed and were raised from E0 to maturity under either an Equinox, Long, or Short photoperiod (Figure 4 A-C) and RNAScope measurements were evaluated from the DRN in mice that developed under either a Long or Short photoperiod (Figure 4 B-C). Early adulthood quantitative RTPCR and RNAScope assays for serotonin related genes (i.e. Tph2, SERT, and Pet-1) were performed at postnatal day P50 (ranging from P50-P90), representing early adulthood in the mouse 71 (Figure 4). All tissues were isolated

Developmental Midbrain Quantitative RTPCR Analysis
Mouse midbrains (n = 5-6 per group per age) were collected and RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. All RNA samples were treated with TURBO DNase (Invitrogen) to remove contaminating genomic DNA following manufacturer's instructions. Reverse transcription was performed from isolated RNA using the QuantiTect Reverse Transcription Kit (Qiagen) containing a mixture of oligo-dT and random primers. Quantitative PCR (qPCR) reactions were set-up to include 1 µL of cDNA, 7 µL nuclease-free H20, 10 µL of TaqMan Universal PCR Master Mix (Applied Biosystems), 1 µL mouse b-actin primer-limited TaqMan Endogenous Control probe (ACTB VIC/MGB), and 1 µL target probe TaqMan probe (Mm00447557_m1 FAM-MGB). All samples were amplified for 36 cycles and relative TH, Tph2, SERT, and Pet-1 expression were calculated using the ΔΔCt method 72 . Each sample was assayed in triplicate.

Developmental Midbrain Monoamine Metabolism
Mouse midbrains (n = 11-14 per group and age) were dissected with clean razor blades from the inferior colliculus to the apex; -6.1mm to -4.1mm from Bregma. The tissue was placed in 1.5mL tubes and then frozen in liquid nitrogen and biogenic amine analysis was

Early Adulthood RNAScope and Confocal Microscope Imaging
Mouse (n = 6 per group) whole brain tissue was collected and submerged into isopentane Integrated density can be defined as the product of mean fluorescence and area. The draw tool in ImageJ was used in order to define each individual cell and ROI (i.e. the dorsal raphe nucleus).

Data Analysis
Prism 8 (Graphpad Software Inc., La Jolla, CA) was used for all statistical analyses.
Statistical significance was determined by either two-way ANOVA (for developmental midbrain RTPCR and monoamine signaling assays), one-way ANOVA (for early adulthood RTPCR 5-HT gene experiments), or paired t tests (for early adulthood DRN RNAScope experiments) with a p value less than 0.05 considered significant. Paired twotailed t tests were used for early adulthood RNAScope experiments because samples from each photoperiod were sectioned and stained together (i.e. one Long photoperiod animal and one Short photoperiod animal were sectioned and stained together on the same day).
All post hoc analysis with Holm-Sidak's multiple comparison tests were performed and standard error of the mean was used for all experiments unless otherwise specified.

Conditions in the Midbrain
We investigated the role of developmental photoperiod on the expression of key dopamine between Short and Long photoperiods (p < 0.0001). No significant multiple comparison effects were observed between photoperiods at either P18 or P35 for midbrain TH expression levels. Trend level differences at P18 were observed between Short and Equinox (p = 0.0793), however these comparisons did not reach statistical significance.
With quantitative RTPCR, we found that TH expression levels in the midbrain are significantly elevated during perinatal development (P8) and decrease throughout the course of development, normalizing by early adolescence (P35).
We also evaluated the effects of photoperiod throughout development on expression levels of key serotonergic genes (i.e. Tph2, SERT, and Pet-1). We found no significant main effects of photoperiod for these genes throughout development. When measuring Tph2 levels and utilizing a two-way ANOVA we found a non-significant main effect of

Short Winter-like Photoperiod Exposure Results in Decreased Midbrain Serotonin & Dopamine Content at Multiple Development Periods
We were also interested in determining the effects of photoperiod on midbrain monoamine content during sensitive periods of postnatal development. We evaluated mice that were raised and developed under either control Equinox, Long summer-like or Short winter-like photoperiods from E0 and measured monoamine concentrations at P8, P18, and P35  Figure 3). Holm's-Sidak's multiple comparison post hoc tests revealed a significant difference between Short and Long photoperiods at P8 (p = 0.0245). For midbrain epinephrine content, values across age and group were so low that they were undetectable. Overall, we observed that developmental Short photoperiod exposure results in decreased levels of midbrain serotonin and dopamine content along with their corresponding metabolites, with these differences manifesting at P18 and P35, representing early childhood and adolescence in the mouse, respectively 68 .

Midbrain Serotonergic Gene Expression in Early Adulthood is Significantly Reduced in Long Summer-like Photoperiod Mice
Based on the highest incidence of depression occurring during early adulthood 2 , we investigated the effects of photoperiod on expression levels of key 5-HT genes in the midbrain at P50, representing early adulthood in the mouse 71 . Quantitative RTPCR at P50 was used to evaluate the expression of serotonergic genes of interest (Tph2, SERT and Pet-1) for mice developed under control Equinox, Long summer-like, and Short winter-like photoperiod conditions from E0 (Figure 4). Using a one-way ANOVA, a significant main effect of photoperiod was found for Tph2 expression (p = 0.0118; F (2, 15) = 6.063) ( Figure 5A). In addition, a significant Holm-Sidak's multiple comparison post hoc test was observed between Equinox and Long conditions (p = 0.0123) and a trend level effect was found comparing Short and Long photoperiods (p = 0.0574). Also, using a one-way ANOVA, a significant main effect of photoperiod was found for SERT expression (p = 0.0014; F (2, 15) = 10.56) with significant Holm-Sidak's multiple comparison effects being observed between Equinox and Long photoperiod conditions (p = 0.0019), and between Short and Long photoperiods (p = 0.0058) ( Figure 5B). Lastly, no significant main effect of photoperiod was found for Pet-1 expression (p = 0.4921; F (2, 15) = 0.7437) ( Figure   5C). By utilizing quantitative RTCPR in the midbrain during early adulthood, it was observed that Tph2 and SERT expression levels are reduced under Long summer-like conditions compared to both Equinox control and Short winter-like photoperiodic conditions.

Long Summer-like Photoperiod Conditions in the DRN
In addition, we wanted to investigate the role of developmental photoperiod on adult gene expression levels in the main hub for serotonin synthesis in the brain, the dorsal raphe and Pet-1 (Short -267.8 ± 11.17, Long -267.8 ± 10.03). By utilizing RNAScope methods targeting the DRN during early adulthood, we found Tph2 expression to be significantly reduced for Long compared to Short photoperiod mice using four different measures, and SERT, Pet-1 expression, and the total cell numbers were not significantly different between the two photoperiod conditions.

Discussion
In this study we evaluated the effects of photoperiod during key periods of postnatal development on gene expression levels and midbrain monoamine content in the dopaminergic and serotonergic systems. We found that gene expression levels of TH, the rate-limiting enzyme for dopamine synthesis, were elevated in Short winter-like photoperiod mice. This difference was specifically observed in P8 animals, representing perinatal development 68 , and this increase in TH expression gradually decreased, reaching trend level differences by P18, and eventually normalizing by P35. When evaluating serotonergic gene expression (i.e. Tph2, SERT, Pet-1), we observed that levels were not altered by photoperiod throughout development. In addition, we observed that midbrain serotonin (5-HT) and dopamine (DA) tissue content along with their corresponding metabolites (5-HIAA and DOPAC), were significantly reduced in mice developed under Short winter-like compared to Long summer-like photoperiods. This suggests that Short winter-like photoperiod exposure can decrease not only dopamine and serotonin content levels, but can reduce monoamine utilization, as observed via elevated metabolite concentrations. Interestingly, these differences manifested at either P18 and/or P35, representing early childhood and adolescence in the mouse 68 . Overall, when investigating through development, midbrain TH expression levels were elevated earlier in development, by P8, in Short winter-like mice, normalizing by P35 and midbrain monoamine content was significantly reduced in Short photoperiod animals by P18 and P35.
We also focused on early adulthood, as this is a consistent time period associated with mood disorders 7 , to assess the expression of key serotonergic genes in the midbrain.
Interestingly, at P50 we found that Tph2 and SERT expression levels were reduced for Long photoperiod animals compared to both Equinox and Short photoperiod conditions, while no differences were observed for Pet-1 expression. We then utilized RNAScope methods to specifically target the known key brain region for serotonin synthesis and neuron development (i.e. the DRN). We found that Tph2 expression was again significantly reduced for mice that developed under Long summer-like compared to Short winter-like photoperiods for integrated density of cell fluorescence, integrated density of ROI fluorescence, mean cell fluorescence, and ROI mean fluorescence. SERT and Pet-1 expression were comparable between photoperiods, however SERT expression levels were consistently reduced in Long photoperiod animals for all four fluorescence measures.
Lastly, there were no significant differences in total cell number observed between photoperiods or when evaluating these genes of interest. Importantly, this demonstrated that the current findings are not due to simply an overall increase in cell number, but rather consistently reduced gene expression levels in DRN cells for Long photoperiod animals.
Therefore, it appears that with two different measures and locations, quantitative RTPCR in the midbrain and RNAScope in the DRN, Tph2 and possibly SERT expression are reduced in animals raised under Long summer-like compared to Short winter-like photoperiods by early adulthood (P50), which could not be explained by potential differences in total cell numbers.
We recently demonstrated that Long developmental photoperiod can program DRN 5-HT neuronal firing rate prenatally in mice, whereas Long photoperiods can modulate monoamine content and the resulting affective behaviors during postnatal development 34 .
Therefore, we proposed a double hit hypothesis in which photoperiodic programming of the serotonin system may occur sequentially, impacting DRN 5-HT neurons prenatally, and then modulating monoamine signaling responsible for the underlying circuitry and resultant affective behaviors during specific periods of postnatal development 34 . While we have previously focused on the DRN, in the current study we aimed to determine these sensitive postnatal periods in the midbrain, which encompasses aspects of both the serotonergic and dopaminergic systems.
Studies have identified sensitive periods in which monoamine content, receptors, and the respective transporters of the dopamine and serotonin systems develop and the role these time windows may have in the development of affective disorders 74 . In the current study we observed similar effects, as found in early adulthood, for monoamine signaling of 5-HT, DA, and their corresponding metabolites, such that monoamine content was significantly reduced in mice developed under Short winter-like compared to Long summer-like photoperiods, however these effects arose by P18 or P35. These time periods mirror windows that are critical for the development of key aspects of the dopamine and serotonin systems 74 . In addition, this has intriguing implications as these periods represent childhood and early adolescence in the mouse 68 , respectively, and have been associated with mood disorders in humans 6 . Thus, we may have identified sensitive periods of postnatal development, which may be vulnerable to the effects of an environmental factor such as photoperiod, which could then impact the underlying circuitry responsible for the resultant affective behavior.
While previous studies have found intriguing effects of photoperiod in the dopaminergic system due to photoperiod [29][30][31] , no study has investigated photoperiodic effects due to developmental photoperiod, across multiple stages of development, or in mice that are melatonin competent, such as the C3Hf +/+ strain. In the current study we observed reduced levels of DA and DOPAC for Short winter-like photoperiod mice at P35, thus based on this data and prior work, we hypothesized that the expression of a key dopamine gene, TH, would be decreased for animals raised under Short photoperiodic conditions as well.
Interestingly, we found that midbrain TH expression was comparable across photoperiods at P35, but that mice developed under Short winter-like photoperiods demonstrated a significant increase in midbrain TH expression compared to Long photoperiod animals at P8. While transcriptional regulation of TH is important, there are multiple posttranslational mechanisms of TH regulation as well including phosphorylation, degradation, and protein binding 75 , which may explain these differences.
In addition, we observed age-dependent effects on midbrain Tph2, with levels highest at P8 and then declining, while SERT expression in the midbrain showed no main effects of age or photoperiod from P8 through P35. However, by P50, there were clear photoperioddependent effects on the midbrain expression of Tph2 and SERT although not on the 5-HT neuron-specific transcription factor Pet-1, with Tph2 and SERT being reduced in Long photoperiods. The tendency for Tph2 and SERT to be reduced in Long photoperiods was also observed in RNAScope assays targeting DRN 5-HT neurons specifically. Thus, these key serotonin signaling genes are consistently found to be down-regulated in Long photoperiods by P50, conditions when serotonin content has been found to be elevated in the dorsal raphe nucleus 15  In addition to these basic science findings, this study has potentially significant clinical implications. The dopaminergic and serotonergic systems have been implicated in various neurodevelopmental disorders such as major depressive disorder, anxiety, and autism spectrum disorder 39,40,[84][85][86][87][88][89] . With preclinical models it has been shown that modulation of serotonin and dopamine during key developmental time points can vastly alter neuronal firing, circuit formation, and the associated behaviors [90][91][92][93][94][95] . As we are beginning to identify the underlying mechanisms and the sensitive postnatal developmental periods impacted by the duration of daylight or photoperiod in the mouse model, this may be clinically relevant, as there is evidence to suggest that light therapy may be effective in treating children and adolescents with seasonal affective disorder 99 and major depression 100,101 .
Overall, it was found that mice developed under Short winter-like photoperiods demonstrate reduced midbrain serotonin, dopamine, 5-HIAA, and DOPAC content compared to Long summer-like photoperiod mice, with these differences manifesting by P18 and P35. This work follows similar results observed in multiple rodent species evaluated in early adulthood and suggests that these time periods, representing childhood and early adolescence in the mouse, may represent vulnerable periods in postnatal development. Interestingly, we observed that midbrain TH levels were significantly increased for Short photoperiod animals during perinatal development, at P8, and normalized by P35. In early adulthood, P50, we showed that animals raised under a Long photoperiod demonstrate decreased expression levels of Tph2 and SERT compared to mice that developed under either a Short or Equinox photoperiod. We observed similar photoperiodic effects using both quantitative RTPCR in the midbrain and RNAScope in the DRN, which were not driven by potential differences in cell number. Based on prior results, we hypothesize that there is an up-regulation of genes relevant to the dopaminergic (TH) system in Short photoperiod mice as observed during perinatal development, and a down-regulation of genes relevant to the serotonergic (Tph2 and SERT) system in early adulthood, (P50). Interestingly, these findings may indicate that depending on the sensitive period, gene expression is modulated differentially depending on the neurotransmitter system, potentially resulting in enduring changes to the underlying circuitry and related behaviors later in life. Thus, investigating the interactions between photoperiod, monoamine signaling, and gene expression levels in the dopaminergic and serotonergic systems during the course of development may provide novel insights into the etiology, underlying mechanisms, and potential therapeutic targets for mood disorders.