Early Life Social Stress Does Not Alter the Number of Dopaminergic Neurons nor the Total Area of the VTA and SN
Since it has been demonstrated that dopaminergic neurons undergo a wave of physiological death/apoptosis at PD14 , and that social stress either in the first two postnatal weeks or in adulthood can affect the number of DA neurons [31, 53], we tested whether our model of social stress (SS) applied during the third postnatal week could alter the number of DA neurons and the total area of the VTA (PBP and PN) and SN (pars compacta) in male and female PD22 pups (Fig. 1A–C, F). DA neurons were identified by means of tyrosine hydroxylase (TH) immunostaining (Fig. 1C). Multivariate two-way ANOVA (MANOVA) of stereological measures (number of TH + neurons, total area of the TH + region, and number of cells/area) in the VTA failed to reveal any significant group (CTR vs SS; λ = 0.99, F3,17 = 0.07, P = 0.98), sex (M vs F; λ = 0.95, F3,17 = 0.31, P = 0.82), or group*sex (λ = 0.93, F3,17 = 0.44, P = 0.73) effect (Fig. 1H’-H’’’; males: N = 6 animals/group, females: N = 5–6/group). Similar results were obtained for the SN (Fig. 1I’-I’’’, MANOVA, group effect: λ = 0.92, F3,13 = 0.36, P = 0.78; sex effect: λ = 0.68, F3,13 = 2.03, P = 0.16; group*sex effect: λ = 0.87, F3,13 = 0.64, P = 0.60; males: N = 4–6 animals/group, females: N = 4–5/group). These results indicate that male and female CTR and SS mice display similar DA neurons number and VTA/SN total area.
SS Alters the Morphology of Dopaminergic Neurons in the VTA but Not SN of Male Mice Only
Chronic adult social stress can reduce the size of DA neurons , and soma size and morphology of DA neurons have been associated with altered functionality of these cells and aberrant behaviors [12, 54]. Therefore, we characterized morphological profiles of the soma of DA neurons in SS and CTR mice by evaluating cell body area and other shape indices, including the aspect ratio and roundness  (Fig. 1D, E, G). MANOVA of somatic shape indices in the VTA showed a significant main effect of the group (λ = 0.93, F3,114 = 2.90; P = 0.036), but no significant effect of sex (λ = 0.97, F3,114 = 1.30; P = 0.29) or group*sex interaction (λ = 0.97, F3,114 = 1.30; P = 0.27; males: N = 6 animals/group, 6 hemisections/animal; females: N = 4 animals/group; 6 hemisections/animal). Univariate results indicated a significant group effect only on aspect ratio (F1,116 = 8.84; P = 0.0036) and roundness (F1,116 = 6.88; P = 0.0099), but not on cell body area (Fig. 1J’-J’’’). Post hoc comparison revealed significant differences between male CTR and SS mice (aspect ratio: P = 0.0060; roundness: P = 0.0071), but not between female CTR and SS animals (Fig. 1J’’,J’’’). The difference in somatic shape between male CTR and SS VTA was further confirmed by nested ANOVA (cell(animal(group)): aspect ratio F1,564 = 16.78, P < 0.001; roundness F1,564 = 20.43, P < 0.001; N = 6 animals/group, 48 cells/animal), demonstrating that SS male DA neurons present an altered, elongated shape, with reduced short axis and augmented long axis, resulting in increased aspect ratio and decreased roundness compared to CTR (Fig. 1L, M).
This effect was restricted to the VTA, as no significant main or interaction effect was detected in the SN (Fig. 1K’-K’’’, MANOVA group effect: λ = 0.97, F3,126 = 1.10, P = 0.35; sex effect: λ = 0.96, F3,126 = 1.70, P = 0.16; group*sex effect: λ = 0.95, F3,126 = 2.30, P = 0.077; males: N = 6 animals/group, 6 hemisections/animal; females: N = 5 animals/group; 6 hemisections/animal).
SS Reduces Expression of the Dopamine Transporter and Tyrosine Hydroxylase Proteins in the Developing VTA but Not SN of Male Mice Only
We performed immunofluorescence staining and confocal imaging to assess distribution and expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins (Fig. 2A, B), which are canonical markers of dopaminergic neurons [29, 55] and have shown to be modulated by adult and early life social stress . Two-way ANOVA on quantitative measurements of DAT intensity showed significant group*sex interaction effect in the VTA (F1,116 = 5.07, P = 0.026) but not in the SN (F1,116 = 1.40, P = 0.24), and no main effect of group or sex in either region (Fig. 2C, E; N = 5 animals/group/sex, 6 sections/animal). Similar results were obtained for TH intensity (Fig. 2D, F, Two-way ANOVA, VTA group*sex effect: F1,75 = 4.29, P = 0.042; SN group*sex effect: F1,76 = 0.11, P = 0.75; N = 5 animals/group/sex, 4 sections/animal). Post hoc comparisons of VTA data revealed significant differences between male CTR and SS DAT (P = 0.039) and TH (P = 0.038) intensity, but not between female CTR and SS (Fig. 2C, D). This was further confirmed by nested ANOVA analysis of DAT and TH immunoreactivity in male CTR and SS VTA (section(animal(group)): DAT F1,50 = 12.48, P < 0.001; TH F1,30 = 18.26, P < 0.001), demonstrating that SS induced down-regulation of DAT and TH fluorescence intensity in male VTA compared to CTR.
SS Reduces DAT Currents in the Developing Male VTA
To assess whether the DAT immunoreactivity reduction induced by SS in the male VTA was associated with changes in DAT-dependent currents, we performed electrophysiological recordings on individual DA neurons at PD22. We analyzed DA-induced DAT-mediated depolarizing currents in the lateral VTA. Using whole-cell voltage clamp recordings of individual DA neurons, we measured the amplitude of the inward current that developed in response to bath application of DA + sulpiride, a selective DRD2 antagonist, in midbrain slices. DA neurons of SS mice (N = 10 cells) displayed significant lower amplitudes of DAT dependent currents compared with CTR (N = 12 cells) (t-test: t20 = − 3.41, P = 0.0028; N = 3–4 cells/animal, 3 animals/group; Fig. 2H, I).
Minocycline Treatment During SS Restores Morphology of Dopaminergic Neurons and Expression of DAT and TH in SS Male Mice
We have previously demonstrated that SS induces microglia activation in the VTA . Pharmacological inhibition of SS-induced microglia activation by systemic administration of minocycline prevented emergence of dopaminergic electrophysiological alterations at PD22 and cocaine conditioned place preference in adult stressed mice. Here, we employed minocycline to explore if and how microglia contributed to the observed morphological and molecular dopaminergic alterations caused by ELS exposure in the VTA of male mice (Fig. 3A).
First, to evaluate whether developmental minocycline treatment alone had an effect on dopaminergic markers and microglia cell number in CTRs, we compared TH intensity and ionized calcium-binding adapter molecule 1 (Iba1, microglia/macrophages marker) expression in CTR-saline and CTR-minocycline male VTA at PD22. TH intensity and Iba1 + cell count were comparable between minocycline- and saline-injected mice (TH: t-test: t31 = 1.42, P = 0.17; Iba1 + cell count: t-test: t31 = 1.58, P = 0.12; Supplementary file1 Fig. 1A-C), further supporting the specificity of minocycline action on microglia activation only in the SS condition .
Thus, we proceeded by comparing CTR, SS, CTR-minocycline, and SS-minocycline mice to evaluate group (stress) and treatment (minocycline) effects. MANOVA of somatic indices showed a significant effect of group (λ = 0.94; F2,115 = 3.90; P = 0.024), treatment (λ = 0.89; F2,115 = 7.10; P = 0.0011), and group*treatment interaction (λ = 0.89; F2,115 = 7.30; P < 0.001). Univariate results indicated a significant group*treatment effect only on roundness (F1,116 = 8.51; P = 0.0042), but not on the aspect ratio (F1,116 = 3.87; P = 0.052, data not shown), and no main effect of group or treatment on either index. Post hoc comparisons confirmed significant differences in the roundness only between CTR and SS untreated mice (P = 0.012), but no difference between the other groups, suggesting that roundness levels in SS-minocycline mice returned to CTR levels (Fig. 1B, F, CTR and SS: N = 6 animals/group, CTR-minocycline: N = 3 animals/group; SS-minocycline: N = 5 animals/group; all: 6 hemisections/animal).
Two-way ANOVA of DAT immunoreactivity showed a significant effect of group*treatment interaction (F1,97 = 19.95; P < 0.001), and no effect of the group or the treatment alone. Post hoc comparisons confirmed significant differences between CTR and SS untreated mice (P = 0.035), CTR-minocycline vs SS-minocycline (P = 0.0037), SS untreated vs SS-minocycline (P < 0.001), but no difference between CTR untreated and CTR-minocycline (P = 0.33), and in CTR untreated vs SS-minocycline (P = 0.13), indicating that minocycline treatment in SS mice restored immunoreactivity of DAT to CTR levels (Fig. 3C, G; CTR and SS: N = 5 animals/group, CTR-minocycline: N = 3 animals/group; SS-minocycline: N = 4 animals/group; 6 sections/animal).
Two-way ANOVA of TH immunoreactivity revealed significant main effect of group (F1,63 = 6.12, P = 0.016) and group*treatment interaction (F1,63 = 36.55, P < 0.001), but no effect of treatment alone. Post hoc comparisons showed significant differences between CTR and SS untreated mice (P = 0.035), CTR-minocycline vs SS-minocycline (P < 0.001), SS untreated vs SS-minocycline (P < 0.001), and CTR untreated vs SS-minocycline (P = 0.0084), indicating that minocycline treatment in SS mice (but not CTR mice) increased immunoreactivity of TH to levels above CTR (Fig. 3D, H; CTR and SS: N = 5 animals/group, CTR-minocycline: N = 3 animals/group; SS-minocycline: N = 4 animals/group; 4 sections/animal).
SS Alters Transcriptomic Patterning in the Developing Male VTA
To elucidate potential mechanisms involved in the effects of SS on the developing male VTA, we investigated molecular pathways globally modulated by SS by means of RNA-sequencing at the end of the stress procedure (Fig. 4A). Transcriptome analysis was performed by comparing 4 CTR and 4 SS male VTAs at PD 22. SS significantly (padj < 0.05) modulated 81 genes (Supplementary file2): 60 genes resulted downregulated in SS mice, while 21 were upregulated, as depicted in the heatmap (Fig. 4B) and volcano plot (Fig. 4C). Among the downregulated genes, 10 genes are crucial for dopaminergic functionality (Fig. 4D), with 9/10 taking part to the dopaminergic neurogenesis pathway (wikipathways.org/index.php/Pathway:WP1498), encompassing all the phases of neuronal development: the regionalization (engrailed homeobox 1 and 2, En1 and En2), specification (aldehyde dehydrogenase 1 family member A1), differentiation (En1; En2; paired like homeodomain 3, Pitx3) and maturation (tyrosine hydroxylase, Th; dopa decarboxylase, Ddc; dopamine transporter; vesicular monoamine transporter member 2; Pitx3; RET receptor tyrosine kinase). Moreover, some of these genes encode for proteins that are critical for dopamine metabolism, such as the TH and the DDC, and for dopamine activity, such as the DAT and the dopamine receptor D2, DRD2. Other highly significant downregulated genes include the GTP cyclohydrolase 1 gene (Gch1), responsible for the production of a TH cofactor, nicotinic cholinergic receptors (cholinergic receptor nicotinic alpha 4 subunit; cholinergic receptor nicotinic alpha 6 subunit; neuronal acetylcholine (nicotine) receptor subunit beta-3), genes associated with synaptic vesicle exocytosis such as complexin 1 and 2 genes, neuron-derived neurotrophic factor, and the peptide guanylate cyclase 2c (Gucy2c). Among the few upregulated genes, the most interesting targets were the corticotropin releasing hormone binding protein (Crhbp), the thyrotropin releasing hormone (Trh), and the neuropeptide neurokinin 2/alpha (tachykinin 2, Tac2) (Fig. 4C).
Ingenuity pathway analysis (IPA) software was used to determine the functional annotation and to predict the biological pathways affected by SS (P < 0.05). We focused on pathways associated with dopaminergic function, developmental processes, and neuro-psychological disorders. Significant diseases or functions annotations have been grouped on the basis of IPA categories in (Fig. 5A–C): Cell-to-cell signaling and interaction (A), nervous system development (B), and neurological disease, organismal injury and abnormalities, developmental, and psychological disorders (C). Based on the pathways predicted, SS broadly affects dopamine functionality, from the metabolism to the release and trafficking, and impairs neurotransmission (Fig. 5A). Moreover, SS influences developmental trajectories, affecting neuronal formation, differentiation, and survival (Fig. 5B), possibly increasing susceptibility to neurological, psychological, and neurodevelopmental disorders (Fig. 5C). TOP predicted altered ingenuity canonical pathways (Fig. 5D) include dopamine and serotonin signaling, catecholamines biosynthesis, GPCR-mediated integration of enteroendocrine signaling exemplified by an L cell, tryptophan degradation X, and tetrahydrobiopterin biosynthesis I. Finally, gene ontology classification revealed alteration in hormonal-related biological processes, exocytosis, and synaptic vesicles trafficking (Fig. 5E).