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. 2022 May 4:16:885849.
doi: 10.3389/fnbeh.2022.885849. eCollection 2022.

A Preliminary Quantitative Electron Microscopic Analysis Reveals Reduced Number of Mitochondria in the Infralimbic Cortex of Rats Exposed to Chronic Mild Stress

Dávid Csabai  1   2 , Abigél Sebők-Tornai  1 , Ove Wiborg  3 , Boldizsár Czéh  1   2
Affiliations

A Preliminary Quantitative Electron Microscopic Analysis Reveals Reduced Number of Mitochondria in the Infralimbic Cortex of Rats Exposed to Chronic Mild Stress

Dávid Csabai et al. Front Behav Neurosci. .

Abstract

Exposure to severe, uncontrollable and long-lasting stress is a strong risk factor for the development of numerous mental and somatic disorders. Animal studies document that chronic stress can alter neuronal morphology and functioning in limbic brain structures such as the prefrontal cortex. Mitochondria are intracellular powerhouses generating chemical energy for biochemical reactions of the cell. Recent findings document that chronic stress can lead to changes in mitochondrial function and metabolism. Here, we studied putative mitochondrial damage in response to chronic stress in neurons of the medial prefrontal cortex. We performed a systematic quantitative ultrastructural analysis to examine the consequences of 9-weeks of chronic mild stress on mitochondria number and morphology in the infralimbic cortex of adult male rats. In this preliminary study, we analyzed 4,250 electron microscopic images and 67000 mitochondria were counted and examined in the brains of 4 control and 4 stressed rats. We found significantly reduced number of mitochondria in the infralimbic cortex of the stressed animals, but we could not detect any significant alteration in mitochondrial morphology. These data support the concept that prolonged stress can lead to mitochondrial loss. This in turn may result in impaired energy production. Reduced cellular energy may sensitize the neurons to additional injuries and may eventually trigger the development of psychopathologies.

Keywords: animal model for depression; chronic mild stress (CMS); electron microscope; infralimbic cortex; medial prefrontal cortex (mPFC); mitochondria; quantitative analysis; ultrastructure.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Illustrating the protocol for the systematic quantitative analysis of mitochondrial morphology and number in the infralimbic cortex of rats. (A) Two pieces of 80 μm thick coronal sections from the prefrontal cortex were selected between Bregma coordinates of 2.2 - 2.3 mm. Afterward, tissue fragments containing the infralimbic cortices were cut out and re-embedded in durcupan resin to produce tissue blocks for further ultra-sectioning. (B,C) First, semi-thin sections were cut and stained with toluidine blue dye to determine the exact area for further ultra-sectioning. (C) Afterward, serial 60 nm ultrathin sections were sectioned and we collected every 5th sections on single slot copper grids. The red lines on the image indicate the ultrathin sections. From each animal, we examined 10 ultrathin sections with the electron microscope. (D) In every ultrathin section, in each cortical layer, we made at least 10 non-overlapping photomicrographs using a sampling line with a random starting point. We analyzed 90 photomicrographs in each cortical layer of each animal. Mitochondria were counted and their cross-sectional profiles were outlined in these 40000× magnified images. (E) For the quantification we used an unbiased counting frame (3.87 × 3.87 μm). Mitochondria profiles touching the exclusion (red) lines were not counted.
FIGURE 2
FIGURE 2
Representative high-resolution electron microscopic images showing the neuropil in the infralimbic cortex in control (A,B) and stressed (C,D) rats. (A) An image with 30000 × magnification, where numerous mitochondria are visible both in dendritic (brighter areas) and in axonal (darker areas) structures. Scale bar: 1 μm. (A) Representative images with mitochondria with higher magnification (40000×). The outer and inner mitochondrial membranes, matrix and cristae are clearly visible. Scale bar: 500 nm. (C) An image with 30 000× magnification displaying the neuropil in a stressed rat. Asterisks identify the mitochondria as it was marked with the Neurolucida software when we counted them. Scale bar: 1 μm. (D) An image with 40000 × magnification showing several mitochondria with their cross-sectional profiles outlined for the morphological analysis. Scale bar: 500 nm.
FIGURE 3
FIGURE 3
Results of the sucrose consumption test. The sucrose intake of the rats was determined every week in order to behaviorally phenotype the animals and to evaluate their response to the chronic mild stress exposure. The original CMS study involved 335 rats. 260 rats were subjected to the CMS protocol, whereas the remaining 75 rats were kept undisturbed in separate rooms and used as controls. About 40% of the stressed rats (n = 98) were vulnerable to stress and significantly reduced their sucrose intake by > 30%. These animals were then labeled as “anhedonic” rats. Out of these 98 anhedonic rats 4 animals were selected for the present quantitative electron microscopic analysis based on their marked reduction in sucrose intake. About 20% of the stressed rats (n = 46) were unaffected by the stress exposure and even increased their sucrose intake, similarly to the control animals. These stress resistant rats were labeled as “stress-resilient”. The remaining stressed animals (n = 116) with intermediate sucrose consumption phenotype were excluded from the experiment. Baseline sucrose consumption was defined as the mean sucrose consumption during three sucrose tests conducted before the stress protocol started. Statistics: one-way ANOVA, followed by Dunnets’s multiple comparison tests where results are compared of the baseline sucrose consumption of the corresponding group. *P < 0.05 versus baseline value of the same group.
FIGURE 4
FIGURE 4
(A) Chronic mild stress significantly reduced the total number of mitochondria in the infralimbic cortex (t test, *P = 0.04). Total mitochondria numbers in the infralimbic cortex were calculated by multiplying mitochondria densities with the volume of the infralimbic cortex. (B) Mitochondria densities were only marginally reduced in the different cortical layers of the stressed animals, but these changes were not statistically significant (stress effect: P = 0.07). (C) Mitochondria numbers in the different cortical layers were compared with two-way ANOVA (stress × cortical layer) which revealed a significant difference between cortical layers (P < 0.0001) and also a significant stress effect (P < 0.0001). Sidak’s post hoc test found significant difference between control and stressed animals only in the first cortical layer (*P = 0.004). Mitochondria numbers within the cortical layers were calculated by multiplying mitochondria densities with the different cortical layer volumes.
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