Mitochondria Are Critical for BDNF-Mediated Synaptic and Vascular Plasticity of Hippocampus following Repeated Electroconvulsive Seizures

Abstract

Background: Electroconvulsive therapy is a fast-acting and efficient treatment of depression used in the clinic. The underlying mechanism of its therapeutic effect is still unclear. However, recovery of synaptic connections and synaptic remodeling is thought to play a critical role for the clinical efficacy obtained from a rapid antidepressant response. Here, we investigated the relationship between synaptic changes and concomitant nonneuronal changes in microvasculature and mitochondria and its relationship to brain-derived neurotrophic factor level changes after repeated electroconvulsive seizures, an animal model of electroconvulsive therapy.

Methods: Electroconvulsive seizures or sham treatment was given daily for 10 days to rats displaying a genetically driven phenotype modelling clinical depression: the Flinders Sensitive and Resistant Line rats. Stereological principles were employed to quantify numbers of synapses and mitochondria, and the length of microvessels in the hippocampus. The brain-derived neurotrophic factor protein levels were quantified with immunohistochemistry.

Results: In untreated controls, a lower number of synapses and mitochondria was accompanied by shorter microvessels of the hippocampus in "depressive" phenotype (Flinders Sensitive Line) compared with the "nondepressed" phenotype (Flinders Resistant Line). Electroconvulsive seizure administration significantly increased the number of synapses and mitochondria, and length of microvessels both in Flinders Sensitive Line-electroconvulsive seizures and Flinders Resistant Line-electroconvulsive seizures rats. In addition, the amount of brain-derived neurotrophic factor protein was significantly increased in Flinders Sensitive Line and Flinders Resistant Line rats after electroconvulsive seizures. Furthermore, there was a significant positive correlation between brain-derived neurotrophic factor level and mitochondria/synapses.

Conclusion: Our results indicate that rapid and efficient therapeutic effect of electroconvulsive seizures may be related to synaptic plasticity, accompanied by brain-derived neurotrophic factor protein level elevation and mitochondrial and vascular support.

Figure 1.

Estimation of synapses and mitochondria in consecutive serial sections, inserted survey with details (boxed areas). The synapses were identified primarily based on the presence of a postsynaptic density (PSD) with vesicles in close proximity to the presynaptic zone. Electron micrographs of consecutive ultrathin sections (a–d) showed nonperforated synapses (arrows), a perforated synapse (big arrows), and shaft synapse (arrow heads). The postsynaptic spine exhibited PSD discontinuities (black stars). The criteria for identifying mitochondria were the presence of distinctive cristae and a double membrane. Axon terminals were identified as presence of 3 or more synaptic vesicles. Dendrites were identified postsynaptic to a synapse or having an attached spine. Mitochondria are identified in each section plane, and a change between planes is deduced as being 1 of 2 significant possibilities: a new isolated part, a so-called “Island,” I, or a new connection between isolated mitochondria, a “Bridge”, B. Mitochonria (M); vesicles (V); branch dividing (white stars). Scale bar, 0.5 μm.

Figure 2.

(A) The length of microvessels was measured within a 3-dimensional sampling box. Green test lines were superimposed on the live image by newCAST software, and they represented the intersection between isotropic virtual planes intersect and the focal plane. Microvessel is defined as a vessel with a 1-celled wall and endothelial cells lining blood vessel walls and a diameter ≤10 µm. The sampling box area was 7200 µm² and the box height was 20 µm. When the microvessels are in focus and virtual planes intersect them, they are counted. The 4 box corner points are used to estimate the reference volume. One microvessel is intersecting a green line of virtual plane (small black arrow). Pyramidal cell (*), glial cell (black thick arrow) and endothelial cell (white thick arrow). (B–C) Effect of electroconvulsive seizures (ECS) on hippocampal vascular plasticity: length of microvessels. (**P < .01) (∆Flinders Resistant Line (FRL)-Sham rats; ▲FRL-ECS rats; ▽Flinders Sensitive Line (FSL)-Sham rats; ▼FSL-ECS rats). The length density (B) and the total length (C) of the microvessels in the CA1.SR were significantly higher in FRL-sham rats compared with FSL-sham. ECS treatment significantly increased both the length density (B) and the total length (C) of the microvessels in the CA1.SR in FSL rats.

Figure 3.

Brain-derived neurotrophic factor (BDNF) expression levels were measured by immunohistochemistry in hippocampus. (A) BDNF expression in the subregions of hippocampus. (B) BDNF expression in serial sections of hippocampus in each animal. (B–D) Immunohistochemistry examined BDNF expression levels in each group. Mean optical density (MOD) was calculated with the following formula: OD=log10 (max pixel intensity/mean pixel intensity), where max pixel intensity =255. MOD in the electroconvulsive seizures (ECS)-treated group significantly increased compared with sham group in both Flinders Resistant Line (FRL) and Flinders Sensitive Line (FSL) rats in DG (B), CA2/3 (C), and CA1 (D) subregions of hippocampus.

Figure 4.

The volume of hippocampal CA1 stratum radium. The volume of hippocampal CA1-SR in the Flinders Resistant Line (FRL) sham rats is significantly larger than that of the Flinders Sensitive Line (FSL) sham group. After electroconvulsive seizure (ECS) treatment, the volume of hippocampal CA1-SR in the FSL-ECS group is significantly increased compared with the FSL sham group. (*P < .05) (∆FRL-Sham rats; ▲FRL-ECS rats; ▽FSL-Sham rats; ▼FSL-ECS rats).

Figure 5.

The number of synapses including subtypes of synapse in CA1 (*P < .05; **P < .01) (∆Flinders Resistant Line (FRL)-Sham rats; ▲FRL-electroconvulsive seizure (ECS) rats; ▽Flinders Sensitive Line (FSL)-Sham rats; ▼FSL-ECS rats). (A) The total number of synapses was significantly higher in FRL-Sham rats compared with FSL-Sham rats. ECS treatment significantly increased the total number of synapses in FSL-ECS rats compared with FSL-sham rats. (B) The number of nonperforated spine synapses was significantly higher in FRL-Sham rats compared with FSL-Sham rats. ECS treatment significantly increased the number of nonperforated spine synapses in FRL-ECS rats compared with FRL-sham rats and FSL-ECS rats compared with FSL-sham rats. (C) The number of perforated spine synapses was significantly higher in FRL-Sham rats compared with FSL-Sham rats. ECS treatment significantly increased the number of perforated spine synapses in FSL-ECS rats compared with FSL-sham rats. (D) Conversely, ECS treatment significantly decreased the number of shaft synapses in FRL-ECS rats compared with FRL-sham rats and FSL-ECS rats compared with FSL-sham rats.

Figure 6.

The number of mitochondria in the various structures (neuropil, axons, and dendrites) and the mean volume of mitochondria in CA1. (*P < .05; **P < .01; ***P < .001) (∆Flinders Resistant Line (FRL)-Sham rats; ▲FRL-electroconvulsive seizure (ECS) rats; ▽Flinders Sensitive Line (FSL)-Sham rats; ▼FSL-ECS rats). (A) The total number of mitochondria in neuropil was significantly smaller in the FSL-sham group compared with FRL-sham group. Following treatment, the FSL-ECS group showed a significant increase in total mitochondria number in neuropil compared with the FSL-sham group. (B) The number of mitochondria in axon terminal also displayed significantly smaller in the FSL-sham group compared with the FRL-sham group. Following treatment, the FSL-ECS group showed a significant increase in mitochondria number in axon terminal compared with the FSL-sham group. (C) The number of mitochondria in dendrites showed no significant differences in the FSL sham group compared with the FRL sham group. ECS treatment did not make any changes in the number of mitochondria in dendrites between the FSL sham group and FSL ECS group. (D) The mean volume of mitochondria in CA1 stratum radiatum was significantly greater in the FSL-sham group compared with FRL-sham group. Following treatment, the mean volume of mitochondria in FSL-ECS group showed a significant increase compared with the FSL-sham group.

Figure 7.

The correlations between synaptic plasticity and nonneuronal plasticity of hippocampus (*P < .05; **P < .01; ***P < .001). The levels of brain-derived neurotrophic factor (BDNF) expression correlated positively with synapse number (A) and mitochondrial number (D), but not the total length of microvessels (G). Total length of microvessels showed a significant positive correlation with synapse number (B) and mitochondrial number (E). In addition, the volume of hippocampal CA1-SR correlated positively with synapse number (C), mitochondrial number (F), and total length of microvessels (I). Furthermore, there was a significant positive correlation between the total mitochondria number density and total number of synapses (H).