Long term effects of peripubertal stress on excitatory and inhibitory circuits in the prefrontal cortex of male and female mice

Abstract

The impact of stressful events is especially important during early life, because certain cortical regions, especially the prefrontal cortex (PFC), are still developing. Consequently, aversive experiences that occur during the peripubertal period can cause long-term alterations in neural connectivity, physiology and related behaviors. Although sex influences the stress response and women are more likely to develop stress-related psychiatric disorders, knowledge about the effects of stress on females is still limited. In order to analyze the long-term effects of peripubertal stress (PPS) on the excitatory and inhibitory circuitry of the adult PFC, and whether these effects are sex-dependent, we applied an unpredictable chronic PPS protocol based on psychogenic stressors. Using two strains of transgenic mice with specific fluorescent cell reporters, we studied male and diestrus females to know how PPS affects the structure and connectivity of parvalbumin expressing (PV+) interneurons and pyramidal neurons. We also studied the expression of molecules related to excitatory and inhibitory neurotransmission, as well as alterations in the expression of plasticity-related molecules. The structure of pyramidal neurons was differentially affected by PPS in male and female mice: while the former had a decreased dendritic spine density, the latter displayed an increase in this parameter. PPS affected the density of puncta expressing excitatory and inhibitory synaptic markers exclusively in the female mPFC. Similarly, only in female mice we observed an increased complexity of the dendritic tree of PV+ neurons. Regarding the perisomatic innervation on pyramidal and PV + neurons by basket cells, we found a significant increase in the density of puncta in stressed animals, with interesting differences between the sexes and the type of basket cell analyzed. Finally, the PPS protocol also altered the total number of somata expressing the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) when we analyzed both sexes together. These results highlight the strong programming effects of aversive experiences during early life for the establishment of cortical circuitry and the special impact of these stressful events on females.

Fig. 1

Timeline of the experiment.

Fig. 2

Behavioral effects of peripubertal stress in the Open Field Test at P84. Behavioral results of PV-tdT mice in the open field, 42 days (P84) after the PPS protocol or corresponding unstressed controls. A: Representative track-plot reports recorded during the OF test session to test the locomotion and anxiety-related behavior (A1-A4). B-D: Graphs showing changes in behavioral parameters related to locomotor activity (B1–B2, C1–C2, D1-D2) and anxiety-related (B3, C3, D3) in the OF test. Graphs in green include data from all experimental animals (not separated by sex, B; N: controls = 27, PPS = 17). Graphs in orange include data from males (C: N: controls = 16, PPS = 8). Graphs in purple include data from females (D; N: controls = 8, PPS = 9). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3

Analysis of the density of dendritic spines in pyramidal neurons of the infralimbic cortex. A & B: Representative 2D projections of distal segments of spiny apical dendrites of pyramidal neurons from control and stressed males (A) and females (B) at P84. C-E: Graphs showing the results of the analysis of dendritic spine density. Spine density was determined in four 50 μm-length segments located 0–50 μm (proximal), 50–100 μm (proximal-medial), 100–150 μm (medial-distal) and 150–200 μm (distal) from the soma and in the total length (total). The graph in green includes data from all experimental animals (not separated by sex) (C; N: controls = 13, PPS = 11). The graph in orange includes data from males (D; N: controls = 6, PPS = 6). The graph in purple includes data from females (E; N: controls = 6, PPS = 6). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: 5 μm.

Fig. 4

Analysis of the density of inhibitory and excitatory puncta in the infralimbic cortex. A & B: Representative single confocal planes of puncta expressing excitatory (blue, VGLUT1) and inhibitory (red, VGAT) synaptic markers in the neuropil of stressed and control mice in males (A) and females (B) at P84. C-D: Graphs representing the effects of PPS on the density of VGLUT1+ (C1, D1, E1) and VGAT + puncta (C2, D2, E2) in the IL subregion of the mPFC. The effects on the E/I ratio (density of VGLUT1+ puncta/density of VGAT + puncta) were also plotted (C3, D3, E3). Graphs in the first row (green) include data from all experimental animals (not separated by sex) (C; N: controls = 23, PPS = 13). Graphs in the second row (orange) include data from males (D; N: controls = 11, PPS = 6). Graphs in the third row (purple) include data from females (E; N: controls = 12, PPS = 7). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar 20 μm. VGLUT1: Vesicular Glutamate Transporter-1. VGAT: Vesicular GABA Transporter.

Fig. 5

Analysis of the number of somata and complexity of the dendritic arbor of parvalbumin expressing interneurons in the infralimbic cortex. A & B: Representative 3D reconstructions of the dendritic arbor of PV expressing interneurons in control and stressed animals, both in males (A) and females (B) at P84. C1- E1: Graphs representing the total number of TdTomato expressing somata in IL cortex. C2- E2: graphs showing the results of Sholl analysis of these PV-expressing interneurons, indicating the number of intersections per Sholl sphere (see material and methods for details). C3- E3: Graphs showing the results of Sholl analysis grouping the data in proximal (spheres in 0–60 μm), medial (spheres in 60–100 μm) and distal (spheres in 100–160 μm) regions. Graphs in green include data from all experimental animals (not separated by sex) (C; N: controls = 18, PPS = 15). Graphs in orange include data from males (D; N: controls = 10, PPS = 7). Graphs in purple include data from females (E; N: controls = 8, PPS = 8). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar 25 μm.

Fig. 6

Density of endocannabinoid receptor 1 and parvalbumin immunoreactive puncta in the perisomatic region of pyramidal neurons and parvalbumin expressing interneurons in the infralimbic cortex. A-D: Confocal images (single confocal planes) comparing the density of CB1R and PV expressing puncta (indicated by arrows) in pyramidal (A, B) and PV + cells (C, D) between control and stressed mice at P84. E-G: Histograms representing changes induced by stress on the density (number of puncta/μm of soma perimeter) of CB1R (E1-G1) and PV (E2-G2) expressing puncta in the perisomatic region of pyramidal neurons. Histograms in E3-G3 represent the density of CB1R positive puncta surrounding PV expressing cell somata. Graphs in green include data from all experimental animals (not separated by sex) (E; N: controls = 23, PPS = 13). Graphs in orange include the data from males (F; N: controls = 11, PPS = 6). Graphs in purple include the data from females (G; N: controls = 12, PPS = 7). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar 10 μm CB1R: endocannabinoid receptor 1. PV: parvalbumin.

Fig. 7

Analysis of plasticity-related molecules in the infralimbic cortex. A & B: Representative single confocal planes showing PV-immunoreactive somata surrounded by PNN in control and stressed males (A) and females (B) at P84. C & D: Representative single confocal planes showing PSA-NCAM-expressing somata in control and stressed males (C) and females (D). E-G: Graphs representing changes in the number of PNN (E1, F1, G1), PV + somata surrounded by PNN (E2, F2, G2), and the PSA-NCAM (E3, F3, G3). Graphs in the first line (green color) include data from all experimental animals (not separated by sex) (E; N: controls = 12, PPS = 12). Graphs in the second line (orange color) only include the data from male animals (F; N: controls = 6, PPS = 6). Graphs in the third line (purple color) only include the data from female animals (G; N: controls = 6, PPS = 6). Values represent mean ± S.E.M. Asterisks in graphs indicate statistically significant effects between groups (Control x Stress) after unpaired Student's t-test. Symbols: # 0.1 > p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: 30 μm. PV: parvalbumin. PNN: perineuronal nets. PSA-NCAM: polysialylated form of the neural cell adhesion molecule.

Fig. 8

Summary of the stress effects on prefrontocortical networks.