2. Results
2.1. Differentially Expressed Genes (DEGs) in the VTA of Winners vs. Control Mice
A total of 13,750 expressed genes were identified. Among them, 44 genes were characterized as being differentially expressed in the VTA of aggressive mice (winners) compared to controls (
Supplementary Materials Table S1). Of these, 28 genes (63.6%) had a lower level of transcription in the winners compared to controls. A functional annotation of DEGs revealed 14 genes associated with a behavior and neurological phenotype (
Table 1). One of these genes (
Tph2) is known as associated with abnormal aggression-related behavior and increased aggression towards mice. Three genes from the list (
Slc17a7,
Slc6a4 and
Tph2) are associated with the terms abnormal fear/anxiety-related behavior and abnormal depression-related behavior. Increased expression levels of transcription of the
Tph2 and
Slc6a4 genes suggest an increase in the synthesis and transport of serotonin in the VTA of winning mice compared to controls.
A total of 9 out of 44 DEGs encode transcription factors (
Table 2), five of which (
Ercc2,
Lbx1,
Maf,
Nkx6-1,
Tcf7l2) are associated with the behavior/neurological phenotype and exhibit reduced expression levels in the VTA of winners. An analysis carried out in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database did not reveal metabolic pathways significantly enriched by DEGs.
2.2. DEGs in the VTA of Losers vs. Controls
A total of 13,656 expressed genes were identified, among which 188 genes were characterized as differentially expressed in the VTA of defeated mice (losers) compared to controls (
Supplementary Materials Table S2). Most DEGs, namely 112 genes (59.6%), showed a decreased level of transcription in defeated mice compared to controls. Altogether, 64 genes associated with the behavior/neurological phenotype were found among the DEGs (
Table 1). Six of these genes (
Camk2a,
Egr1,
En2,
Fev,
Gad2,
Tph2) are known to be associated with abnormal aggression-related behavior and an increased aggression towards mice. Sixteen genes from the list in
Table 1 are associated with abnormal fear/anxiety-related behavior, six of which (
Camk2a,
Gad2,
Slc6a2,
Slc6a3,
Slc6a4,
Tph2) are also known to be involved in abnormal depression-related behavior.
A total of 28 out of 188 DEGs encode transcription factors (
Table 2), most of which (16 genes) are expressed at reduced levels in the VTA of defeated mice. Among the DEGs encoding transcription factors, 16 genes are associated with the behavior/neurological phenotype.
The analysis of DEGs detected by comparing the level of gene expression in defeated mice to controls revealed several metabolic pathways (
Table 3). The expression characteristics of the DEGs linked with these metabolic pathways are presented in
Supplementary Materials Table S3. According to the changes in the level of transcription of genes related to the metabolic pathways in defeated animals compared with control animals, the activation of GABAergic synapses should be assumed, which includes the activation of GABA synthesis (
Gad1,
Gad2) and GABA receptors (
Gabra1,
Gabrb2,
Gabrg2); in addition, activation of the synthesis (
Th,
Ddc) and transport (
Slc6a3) of dopamine can be assumed. These genes are also implicated in drug (nicotine, amphetamine, cocaine) dependence.
2.3. DEGs with a Changed Expression Both in Winners and Losers (Common DEGs)
A comparison of the corresponding DEG lists revealed 23 common genes which significantly changed their expression in the VTA of both winners and losers compared to controls (
Table 4).
It can be seen that 22 of these genes either increased or decreased the level of transcription unidirectionally in both experimental groups, and only one gene (Nrgn, neurogranin) changed the level of transcription in winners and defeated animals in different directions with respect to the controls. In the VTA of winners, the Nrgn gene transcription level was decreased, and in the defeated mice the Nrgn gene transcription level was increased compared to control animals. Therefore, it can be concluded that, regardless of whether the animals win or are defeated, the level of transcription of many genes in the VTA of experimental mice changes unidirectionally, which is probably the result of the reaction of the animal organism to conditions of social stress. It can be assumed that genes whose expression level varies in different directions can make the most significant contribution to the formation of alternative (aggressive or depressive) behaviors. According to our results, Nrgn is one of such genes.
To identify more genes that changed the transcription levels in the VTA of the winners and losers in opposite directions, a comparative analysis of gene expression in these two experimental groups was performed.
2.4. DEGs in the VTA of Winners vs. Losers
When comparing the level of gene expression in the VTA of winners and losers, a total of 207 DEGs (
Supplementary Materials Table S4) were identified, of which 89 genes (43.0%) had a decreased transcription level in defeated mice.
The significantly enriched metabolic pathways and expression characteristics of the related DEGs are shown in
Table 5.
The changes in gene expression levels suggest an activation of the dopaminergic metabolic pathway in the VTA of defeated mice, differences in calcium signaling, and activation of several genes related to the axon guidance metabolic pathway, representing a key stage in the formation of neuronal networks, as well as a decrease in the expression of several extracellular matrix receptor interaction genes.
An analysis of DEG expressions in winners vs. losers, revealed six genes with an expression that level changed in opposite directions relative to controls (
Table 6). Four of them encode transcription factors. Several genes (
Ercc2,
Nrgn,
Otx2, and
Tcf7l2) are annotated as being related to the behavior/neurological phenotype, and
Ercc2 as involved in abnormal emotional/affective behaviors. Among these DEGs (listed in
Table 6), there may be the genes whose expression patterns determine behavioral differences between winners and losers.
2.5. DEGs that Can Maximize the Differences between the Groups of Winners and Losers
The genes with the most significant contribution to the formation of intergroup differences were sought using a partial-least squares discriminant analysis (PLS-DA). The location of the mice from the experimental groups (winners and losers) in the constructed PLS-DA axes is shown in
Figure 1A. Next, a correlation analysis between the obtained coordinates of the mice and the level of gene expression in the winners and losers was performed. A histogram showing the distribution of expressed genes according to the obtained values of correlation coefficients is given in
Figure 1B. Genes characterized by maximum correlation coefficients are believed to contribute most significantly to intergroup differences.
For further analysis in the KEGG database, 506 genes characterized by correlation coefficients greater than 0.90 or −0.90 (the list of these genes is presented in
Supplementary Materials Table S5) were selected. Their analysis revealed the significance of several metabolic pathways (
Table 7), among which melanogenesis and calcium signaling pathways are noted as the most significantly enriched. Among the DEGs assigned to the identified metabolic pathways, the most common gene is
Camk2a, encoding calcium/calmodulin-dependent protein kinase II alpha.
Camk2a is one of the 63 DEGs characterized by correlation coefficients greater than 0.90 or −0.90. The detailed description of these DEGs is given in
Supplementary Materials Table S6. The list of these genes includes 23 genes associated with the behavior/neurological phenotype, six of which (
Ercc2,
Bcl11b,
Pitx2,
Otx2,
En1,
Foxp2) encode transcription factors.
Supplementary Materials Table S6 presents a total of 17 genes encoding transcription factors.
The list of genes presented in
Supplementary Materials Table S6 includes four genes (
Six3,
Ercc2,
Otx2, and
Nrgn) whose expression levels in the winners and defeated mice changed in opposite directions. DEGs bidirectionally changing the level of transcription in winners and defeated mice and associated with the behavior/neurological phenotype (
Ercc2,
Otx2,
Nrgn) can be considered as the key genes in the formation of alternative behaviors in the conditions of social stress. It should be noted that the most highly expressed of these genes are the
Ercc2 and
Nrgn genes.
According to the Gene Ontology, the Ercc2 gene is associated with biological processes such as neurogenesis (gliogenesis), the regulation of transcription from RNA polymerase II promoter (regulation of gene expression), programmed cell death, stem cell differentiation, response to stimulus, response to hypoxia, immune system development, and aging, and the Nrgn gene is associated with biological processes such as nervous system development, cell–cell signaling, trans-synaptic signaling, behavior, cognition, the regulation of signaling, learning or memory, response to a stimulus, signal transduction, regulation of synapse structure or activity, modulation of synaptic transmission, regulation of synaptic plasticity, associative learning, and intracellular signal transduction.
A list of 63 DEGs presented in
Supplementary Materials Table S6 was used in STRING database utilities to identify possible associations between the encoded proteins. According to the results presented in
Figure 2, the network includes 31 genes (shown in red) encoding proteins associated with nervous system development, 24 of which are associated with the generation of neurons (shown in blue).
Among them, several nodes were identified. One of the main nodes is the protein encoded by the Camk2a gene. An analysis revealed the interaction of Nrgn with Camk2a, which in its turn is linked to a group of proteins associated with a number of ionic (calcium and sodium) channels. In addition, Nrgn may be associated with glycine transporters (Slc6a5). In addition to Camk2a, proteins encoded by the Six3 and Otx2 genes can also be considered nodal ones. Since the expression of these genes, like the expression of the Nrgn gene, is changed in opposite directions in winners and defeated mice compared to controls, they can be considered as potential candidates for further studies.
Thus, we identified four genes (Nrgn, Ercc2, Otx2, and Six3) that change the level of transcription in opposite directions in winners and losers, thereby contributing to the formation of intergroup differences, including, possibly, the formation of alternative forms of behavior.
2.6. Correlation Between the Expression of Nrgn, Ercc2, Otx2, and Six3 and the Genes Involved in the Synthesis and Transport of Dopamine in the VTA Was Determined by a Cluster Analysis Using Expression Data for the Genes Differentially Expressed in the VTA of Winners and Losers
The results are presented in
Supplementary Materials File S7. As can be seen, these four genes and
Th,
Ddc, and
Slc6a3 genes encoding tyrosine hydroxylase, DOPA decarboxylase, and dopamine transporters, respectively, are in the same cluster. Therefore, it can be assumed that genes with oppositely changed expressions in winners and losers may be coregulated or functionally associated with the synthesis and transport of dopamine in the VTA.
According to our data, two genes encoding dopamine receptors, Drd2 and Drd5, are expressed in the VTA of mice. Based on the criteria of the Cufflinks/Cuffdiff packages used in our work, the Drd2 gene was not characterized as a DEG in all the comparisons; however, its transcription level increased 1.55 times in the VTA of defeated mice as compared to controls (p < 0.009) and only 1.02 times in the VTA of winners (p < 0.91) as compared to controls, which remained practically unchanged. Therefore, it can be assumed that a change in the level of Drd2 transcription may play a functionally important role in the VTA of defeated mice. The expression level of the Drd5 gene remained stable in all three experimental groups.
To understand whether changes in the level of expression of the genes characterized above as key ones can be related to alternative dopaminergic signaling cascades in winners and losers, the correlation between their level of expression and expression levels of the
Drd2,
Th,
Ddc, and
Slc6a3 genes was calculated in both experimental groups (
Table 8). The results showed a strong correlation between the expression of
Nrgn and the above four genes in defeated animals. In contrast to the group of winners, a statistically significant correlation between the expression of
Nrgn and
Ercc2 and dopaminergic genes was obtained in the VTA of defeated animals. Three nodal genes (
Camk2a,
Otx2, and
Six3) were characterized by high correlation coefficients with the dopaminergic genes in the VTA of mice from both experimental groups.
3. Discussion
First of all, when discussing the results of this study, it is important to comment on the alterations in the expression levels of the genes involved in the synthesis or transport of neurotransmitters. The analysis showed that the
Tph2 and
Slc6a4 genes responsible for the synthesis and transport of serotonin, respectively, in the VTA are expressed at higher levels in both winners and losers compared to the controls. The role of serotonin in both aggression and anxiety/depression has been extensively studied and reviewed by many authors [
18,
19,
20]. Although serotonin is believed to be a potent inhibitor of aggressive behavior, there are numerous data in favor and against this hypothesis (reviewed in [
21]). The complexity of the mechanisms regulating serotonin-mediated behavior is noted in many studies [
19,
22]. It is reported that the pharmacological effects of drugs depend on age, environment or experimental conditions [
18,
21]. Therefore, when discussing the results of this study, one should bear in mind that they are mostly relevant to the development of mixed anxiety–depressive disorder and pathological aggressive behavior in mice within the framework of the used experimental model.
In the current study, in the experimental groups of mice with alternative types of behavior, unidirectional changes in the expression of serotonergic genes indicate that the synthesis and transport of serotonin are activated as a reaction of the serotonergic system to experimentally induced social stress experienced by animals and probably are not associated with the formation of alternative forms of behavior. This view is in good agreement with the opinion of other authors who believe that changes in serotonin levels associated with abnormal behavior are conditioned by different factors [
19]. It should also be noted here that the defeated animals are likely to experience more severe stress as indicated by a significantly higher increase in the transcription of serotonergic genes in the VTA of defeated mice than in the winners compared to controls (
Table 4).
As already noted in the introduction, most VTA neurons are dopaminergic. Statistically significant increases in the transcription levels of several genes (
Ddc,
Slc6a3,
Th) controlling dopamine synthesis (
Th,
Ddc) and transport (
Slc6a3) were detected in the VTA of defeated mice as compared to controls. In winning mice, the respective changes were less significant. Compared to controls, in the VTA of winning animals the increase in the level of transcription of the
Ddc gene was characterized by a
p value = 0.0028, whereas for
Slc6a3 and
Th it was statistically insignificant according to the annotations of the Cuffdiff program. At the same time, we previously studied the expression of these genes in larger groups of animals by real-time PCR and showed that in the VTA of the winning mice the level of transcription of the
Slc6a3 and
Th genes was significantly increased [
23]. Therefore, it can be assumed that in the VTA of both winning (aggressive) and defeated mice, dopamine synthesis increases, however, as in the case of serotonin, the effect of stress on dopamine synthesis and transport in defeated mice is stronger than that in the winners.
GABAergic neurons also play an important role in VTA functioning [
14]. Several genes (
Gabra1,
Gabrb2,
Gabrg2,
Gad1,
Gad2) related to the functioning of GABAergic synapses demonstrated a significantly increased transcription in defeated mice compared to controls (
Table 4 and
Supplementary Materials Table S2). According to our RNA-Seq data, the level of transcription of all these five genes also increased in the group of aggressive animals, but the differences compared to the control did not reach statistically significant values. Accordingly, we can assume both an increase in the synthesis of GABA and an increase in the expression of its receptors (
Gabra1,
Gabrb2,
Gabrg2) in both experimental groups of mice, but in aggressive animals, changes in the function of GABAergic synapses were less pronounced than in the defeated ones. The possible functional consequences of significant changes in these genes in defeated animals were discussed in [
9]. Here we want to emphasize that the unidirectional activation of a number of genes that control the functioning of GABAergic neurons occurs in the VTA of mice of both experimental groups and that this activation is more pronounced in defeated animals. The fact that defeated mice are more stressed than winners also follows from almost four times the number of DEGs that were found in losers vs. controls than in winners vs. controls comparisons. These results are in very good agreement with our observation that both winners and defeated mice are characterized by increased anxiety [
6,
24,
25,
26].
It should be noted that in the current study the social stress experienced by animals caused a change in the expression level of multiple genes that encode transcription factors in both experimental groups of mice, suggesting a wide range of functional changes in the VTA and, with a high probability, in the areas of the brain onto which the VTA neurons project. As can be seen from
Table 4, which represents common DEGs, three DEGs encoding transcription factors (
Lbx1,
Maf,
Nkx6-1) reduced the level of transcription in both winners and defeated mice, therefore suggesting that these genes may play a key role in regulating the overall response to stress.
Almost all genes listed in
Table 4 have their transcription levels changed in the same direction in both winners and defeated mice, and only one (
Nrgn) has it changed in the opposite direction—its transcription level decreased in winners and increased in defeated mice. The
Nrgn gene encodes neurogranin (Ng), which is enriched at dendritic spines and can enhance synaptic strength by targeting calmodulin (CaM) [
27]. The bidirectional changes in the level of
Nrgn transcription found in our experiment are in good agreement with the fact that neurogranin is associated with both long-term potentiation and long-term depression [
28]. Thus, it can be assumed that this particular gene can be involved in the formation of alternative behaviors in the experimental groups of mice.
In physiological conditions, neurogranin forms a complex with CaM, and its CaM-binding affinity can be modulated by phosphorylation, oxidation and glutathiolation under the activation of protein kinase C or oxidant stress [
29]. It was shown that mice with an
Nrgn gene deletion displayed an apparently normal phenotype but were characterized by impairments in spatial and emotional learning and by changes in hippocampal short- and long-term plasticity (paired-pulse depression, synaptic fatigue, and long-term potentiation induction). These deficits were accompanied by a decreased basal level of the activated Ca(2+)/CaM-dependent kinase II (CaMKII) [
30,
31]. The behavioral testing revealed that neurogranin knockout mice were both hyperactive and socially withdrawn [
32]. Neurogranin overexpression in neurons enhances synaptic strength as well as long-term potentiation (LTP) and learning by promoting calcium-mediated signaling [
33]. Based on the above information, we can assume that the increase in
Nrgn transcription in the VTA of defeated mice and its decrease in the VTA of winners observed in the current study are adaptive reactions of the system working to achieve a homeostatic balance.
It was shown that the restorative effects of neurogranin on synaptic depression and LTP deficits are dependent on the interaction of neurogranin and CaM and CaM-dependent activation of CaMKII [
34]. The results of our experiment showed a coordinated increase in the expression of both
Nrgn and
Camk2a (encoding calcium/calmodulin-dependent protein kinase II alpha) genes in the VTA of defeated mice, while the level of
Camk2a transcription was unchanged in the VTA of the winners exhibiting a decreased level of
Nrgn transcription.
CaMKII is known as a mediator of Ca
2+-linked signaling. It phosphorylates a wide range of substrates playing a multifunctional role in the coordination and regulation of Ca
2+-mediated alterations in different intracellular events related to the neuronal functions: the synthesis and release of neurotransmitters, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, gene expression, etc. [
35,
36,
37].
Camk2a encodes a calcium/calmodulin-dependent protein kinase subunit that plays a key role in synaptic plasticity, AMPA receptor transmission, LTP, and long-term memory formation, and its dysfunction underlies neuropsychiatric disorders such as drug addiction, schizophrenia, depression, epilepsy, and multiple neurodevelopmental disorders, perhaps through maladaptations in glutamate signaling and neuroplasticity [
38,
39]. The data obtained in the current study are in a good agreement with the findings that calcium signaling is an important link in the regulation of processes associated with neurogenesis in various neurological disorders [
40].
In our work, when comparing the transcriptomes of the winning and defeated mice, the
Camk2a gene was assigned to the DEGs with maximum contributions to the intergroup differences (
Supplementary Materials Table S6).
Figure 2 clearly demonstrates that the protein encoded by the
Camk2a gene can be viewed as a nodal gene in the calcium modulating pathway, as well as in the regulation of sodium and potassium ion channels and a number of other processes responsible for functional differences in the VTA of winners and defeated animals.
The list of DEGs characterized as making a maximum contribution to intergroup differences contains three more genes (Ercc2, Otx2, Six3) that change the transcription level in winners and defeated mice in opposite directions. These three genes encode transcription factors.
The
Ercc2 gene is associated with the behavior/neurological phenotype. In the VTA, a decrease in the level of transcription of this gene was detected in the winners, and an increase in its transcription was detected in the defeated mice (
Table 6). Given that
Ercc2 is involved in repairing the damage caused by the redox process [
41], it can be assumed that oxidative stress processes in the VTA play a pivotal role in the formation of phenotypic features of defeated mice.
According to the results presented in
Figure 2, the proteins encoded by the transcription factor genes
Otx2 and
Six3, the expressions of which are changed in opposite directions in winners and defeated mice compared to controls, can also be considered as key (nodal) genes. The investigations of the mechanisms involved in neural functions have shown that the expression of several regulatory genes, including
Six3, cannot be initiated in the neural plate of Otx2−/− embryos [
42,
43], suggesting that the expressions of the
Otx2 and
Six3 genes are closely related.
Recently the
Otx2 gene was characterized as an upstream mediator of increased susceptibility to social defeat stress in mice. Its transient knockdown in the VTA increased stress susceptibility, and its overexpression was associated with reverse effects [
44]. The role for the
OTX2 gene was confirmed in the study of stress-related depressive disorder pathophysiology in children [
45]. Otx2 is involved in the regulation of the number of VTA neurons with efficient dopamine uptake [
46] and is considered one of the genes with a key role in midbrain dopaminergic neuron development and the regulation of their survival and physiology [
47].
To date, the
Six3 gene has not been associated with the behavior/neurological phenotype, but its role in the control of the status of the neural progenitor cells has been reported. It was shown that the upregulation of
Six3 expression kept the progenitor cells of the embryonic telencephalon in an undifferentiated state. The authors suggested that
Six3 is involved in the control of the subtle equilibrium between the proliferation and differentiation of neural progenitor cells during mammalian neurogenesis [
48]. A conditional deletion of the
Six3 gene prevented the formation of most dopamine receptor DRD2-expressing medium spiny neurons being the principal projection neurons in the striatum [
49]. To date, the role of the
Six3 gene in the functioning of the VTA neurons has not been reported. However, based on the above-cited data on its role in the formation of the DRD2-expressing neurons of the striatum, it can be assumed that the
Six3 gene can also participate in the regulation of D2R-expressing neurons functioning in the VTA. Our results from the cluster and correlation analyses confirm the plausibility of this hypothesis. Accordingly, a further study of the effects of
Six3 gene expression in the VTA of the winners and defeated animals on dopaminergic signaling may be promising for understanding the formation of their behavioral characteristics.
The VTA is one of the major sites of dopamine synthesis. The VTA dopaminergic neurons are implicated in the regulation of emotional and motivational behaviors and are involved in the development of psychopathologies including depression [
50], aggression [
51], and addictions [
52,
53]. So, it was important to check if the prioritized genes could be implicated in VTA dopaminergic signaling. The results of the cluster analysis on expression data of the genes differentially expressed in the VTA of winners and defeated mice showed that the
Nrgn,
Ercc2,
Otx2,
Six3, and
Camk2a genes were assigned to the same cluster as the dopaminergic (
Th,
Ddc,
and Slc6a3) genes. According to the correlation analysis, the expression of
Otx2,
Six3, and
Camk2a genes is coregulated with the dopaminergic genes in both experimental groups. The expression of two other genes (
Nrgn and
Ercc2) correlated with the expression of dopaminergic genes only in the VTA of losers indicating a possible coregulation or functional relationship of these genes in the VTA of defeated mice. It should be emphasized that the most significant correlations were found between the expression of dopaminergic genes and the expression of the
Nrgn gene (see
Table 8).
The development of technologies generating large amounts of gene expression data called for the use of mathematical methods capable of grouping the genes based on the similarity of their expression patterns. Such methods include a correlation analysis [
54,
55], which is a potent tool to identify coregulated genes or groups of genes associated with particular experimental or environmental conditions [
56].
To date, the role of the
Nrgn gene in dopaminergic signaling remains unclear, however, there is indirect evidence for its involvement [
57]. Additionally, in favor of this hypothesis are the findings that neurogranin is enriched at dendritic spines [
27] whereas D2R is localized to the VTA dendrites [
58], which suggests their spatial colocalization.
According to our results,
Nrgn is involved in the network of 31 coregulated genes, encoding proteins associated with the nervous system development, including 24 genes encoding proteins associated with the generation of neurons. Several of them (Otx2, En1) are known as regulators of terminal differentiation, survival and the maintenance of midbrain dopaminergic neurons [
59]. We believe that other coregulated genes included in the network may also be essential for studying their role in VTA dopaminergic neuron maturation under the influence of social stress.
Of course, many genes with VTA expression levels significantly changed in one of experimental groups and not changed in the other group could be directly or indirectly related to the formation of behavioral characteristics in winners and defeated mice. However, we believe that the genes that change the level of transcription in opposite directions in winners and defeated mice deserve special research attention. As noted earlier, the Nrgn, Ercc2, Otx2, and Six3 genes that change their transcription levels in opposite directions in winners and defeated mice and make the maximum contribution to intergroup differences can be considered as key genes in the formation of alternative phenotypes associated with behavioral patterns in winning and defeated animals.