**3.1 Microglia and PTSD**

Fear memory of PTSD has several characteristics. First, fear extinction is impaired. Fear extinction refers to the diminished fear of a particular traumatic stimulus by learning that it is safe to be exposed to that stimulus. In case measuring the degree of fear extinction in animal experiments, after loading the foot electric shock stimulus in a chamber, researchers repeatedly expose the animal to the chamber or the sound that was heard when the stimulus was applied, without the stimulus, and observe whether the freezing behavior weakens or if it can be maintained once weakened. Lai et al. loaded single prolonged stress (restraint, forced swim, and ether anesthesia) to rats. Then, the authors gave a foot shock stimulus to rats in a specific chamber 7 days later to measure the degree of fear extinction to the chamber at a later date and examined changes in the inflammatory response and microglial cell number. SPS impaired fear extinction and increased the microglial cell number and the expression of HMGB1 and TLR4 in the amygdala. Intra-BLA administration of HMGB1 inhibitor or TLR4 antagonist normalized these behavioral and molecular changes [19]. A separate SPS study reported that the degree of fear extinction was inversely correlated with the number of IL-10 genes, which is anti-inflammatory, expressed by microglia in the prefrontal cortex [20]. Another recent study showed

#### *Stress, Microglial Activation, and Mental Disorders DOI: http://dx.doi.org/10.5772/intechopen.103784*

that microglial synaptic phagocytosis in the hippocampal dentate gyrus was enhanced by foot electric shock stimulation, which impaired fear extinction [21].

The second characteristic of PTSD fear memory is an excessive fear generalization. Fear generalization refers to showing fear response to stimuli that are similar to those that remind us of past traumatic experiences. Excessive fear generalization leads to perceiving an inherently neutral stimulus as dangerous and causes unnecessary anxiety and fear. In case measuring the degree of fear generalization in animal experiments, after loading the foot electric shock stimulus in a chamber, researchers put animals in a chamber that is different in color, shape, and odor from those used for electric shock stimulation, and measure the freezing behavior. Nguyen et al. focused on the interaction of neurons and microglia in the hippocampus via the IL-33 signal and investigated the effect of inhibition of that signal on fear generalization. The authors showed that when IL-33 released from neurons acted on the IL-33 receptor (IL1RL1) in microglia, it promoted extracellular matrix phagocytosis by microglia and increased synaptogenesis in the hippocampus, and that fear generalization was enhanced in IL-33 or IL1RL1 conditional knockout mice [22].

If the fear of a particular stimulus diminishes without going through the process of fear extinction, it is defined as forgetting of fear memory. Being not to be able to forget is the third feature of PTSD fear memory [23]. Memories are considered to be stored in engrams, a specific neuronal population. Wang et al. generated mice that express CD55 only in hippocampal engram cells.CD55 is supposed to suppress microglial phagocytosis by inhibiting complement pathways. In these mice, microglial phagocytosis for components of engram cells was reduced and fear forgetting was impaired [24].

The fourth feature of PTSD fear memory is that while the sensory information of the trauma is clearly preserved, the contextual information of when/where/why/how it happened is not well integrated. A protocol of animal experiments was proposed to investigate such characteristics. In the protocol, in an environment where the sound of a specific frequency is regularly generated, an electric foot shock stimulus is given to an animal in a chamber, and after a certain period of time, freezing time is measured for the chamber and the sound, respectively under a condition without a shock stimulus [25]. If the freezing response to sound is enhanced while the response to the chamber is not enhanced, it is considered to capture the fourth feature described above. Although the association between this feature and microglia has not been directly investigated, it was reported that glucocorticoid variability in the hippocampus and norepinephrine variability in the amygdala were associated with this behavioral change. Changes in microglial cytokine release by these stress hormones may be associated with the fourth feature of PTSD fear memory.

Since 2020, imaging studies and postmortem brain studies in PTSD patients have been reported. Bhatt et al. showed that expression of TSPO in the insula and ventromedial prefrontal cortex was reduced in patients with PTSD and suggested that less microglia that release neurotrophic factors might be behind it. In addition, they have genetically analyzed postmortem brain samples from female PTSD patients and found reduced expression of the microglial-related genes TNFRSF14 and TSPOAP1 in addition to TSPO in the prefrontal cortex [26]. Conversely, Deri et al. reported in a similar PET study that the severity of PTSD symptoms was positively correlated with TSPO expression in the hippocampus and prefrontal cortex [27]. We summarize animal, PET, and postmortem studies in **Table 1**.


*AMY, amygdala; PFC, prefrontal cortex; DG, dentate gyrus; HIP, hippocampus; cKO, conditional knockout; ECM, extracellular matrix; VMPFC, ventromedial prefrontal cortex.*

#### **Table 1.**

*List of papers of microglia and PTSD including animal studies, PET with TSPO, and a post mortem study.*

## **3.2 Microglia, depression, and suicide**

CUS, social defeat stress, repeated restraint stress, and social isolation stress are known as stress-induced animal models of depression. In these models, immobility time during forced swimming or when hung upside down is evaluated as an index of depressive symptoms [28]. While many studies report that microglia cause neuroinflammation in these model rodents [29–32], a few studies report reduced production of inflammatory mediators in microglia [5, 21]. However, depressive mood and suicidal ideation, which are important in the clinical setting of depression, are inherently subjective symptoms, and it is difficult to evaluate them from the behavior of model animals. In addition, it is not clear whether the phenomenon of suicide exists in animals other than humans, thus research on humans is indispensable for understanding the pathophysiology of depression and suicide.

Several PET studies using TSPO as a ligand have been conducted in depressed patients, and two systematic reviews have ever been reported. Gritti et al. examined nine original articles and reported that most studies suggested increased TSPO expression in the anterior cingulate gyrus, prefrontal cortex, hippocampal formation, and insula of depressed patients. In addition, the authors suggested treatment with antidepressants and cognitive-behavioral therapy might reduce TSPO expression [33]. Enache et al. performed a meta-analysis on six of the nine original articles above mentioned. The authors concluded that TSPO expression was increased in depressed patients in the anterior cingulate gyrus, prefrontal cortex, temporal lobe, insula, and hippocampus [34].

The results of postmortem brain studies in depressed patients examining microglial changes are mixed. One study showed an increase in the number of Iba-1 positive amoeboid-like microglia in the ventrolateral prefrontal cortex of depressed patients [35], while another study showed that the number of HLA-positive microglia in the amygdala did not change [36]. In the tryptophan-serotonin alternative pathway, the tryptophan-kynurenine pathway, microglia synthesize neurotoxic quinolinic acid. It has ever been reported that quinolinic acid expression is reduced in the hippocampus and ventrolateral prefrontal cortex of depressed patients [35, 37]. Several studies observed microglial changes in the brains of suicide victims. Steiner et al. found increases in HLA-DR-positive microglia in the dorsolateral prefrontal cortex, anterior cingulate gyrus, and mediodorsal thalamus, of suicide victims [38]. On the other hand, Brisch et al. found a decrease in HLA-DR-positive microglia in the dorsal raphe nuclei of non-suicidal depressed patients [39]. Schneider et al. observed an increase in CD68 highly positive microglia in the ventral prefrontal white matter of suicide victims [40]. In another study, the number of IBA-1-positive microglia did not change in the dorsal anterior cingulate gyrus of depressed suicide victims, but microglia in suicide victims had wider cell bodies than control groups [41]. In a recent study, Snijders et al. isolated and extracted microglia from the medial frontal gyrus, superior temporal gyrus, thalamus, and subventricular zone in the postmortem brain of depressed patients, and investigated gene and protein expression changes extensively. No inflammatory changes in microglia were detected in these regions, the expression levels of CX3CR1 and TMEM119 increased, and the expression levels of CD14 and CD163 decreased [42]. The authors hypothesize that these results reflect changes in microglial homeostatic function other than inflammation in depressed patients.

We are conducting reverse translational research to elucidate the dynamics of microglia in depression at the molecular level using the peripheral blood of patients. We performed a blood metabolome/lipidome analysis in patients with first-time depressive episodes who are not receiving medication and found that multiple metabolites in the tryptophan-kynurenine pathway, which are closely associated with microglial activation, correlate with the severity of depressive symptoms and the intensity of suicidal ideation [43]. In a separate study of peripheral blood samples from depressed patients, we evaluated nerve-derived exosomes in blood by the sandwich ELISA (enzyme-linked immune sorbent assay) method and found that IL-34 was increased in the patient group and that synaptophysin and TNF-α correlated with the severity of depression [44]. IL-34 is a cytokine essential for maintaining the function of microglia. We envision a process in which activated microglia damage synapses and lead to the formation of depressive symptoms. Additionally, we are developing our own technology to generate induced microglia-like (iMG) cells from human peripheral blood monocytes and obtained a US patent in 2018. Human iMG cells can be produced in 2 weeks by separating monocytes from the collected human peripheral blood and adding two types of cytokines, granulocyte colony-stimulating factor and IL-34. We analyzed gene profiling patterns of iMG cells from three patients


*ACC, anterior cingulate cortex; dACC, dorsal anterior cingulate cortex; PFC, prefrontal cortex; HIP, hippocampus; AMY, amygdala; DLPFC, dorsolateral prefrontal cortex; DRN, dorsal raphe nuclei, VLPFC, ventrolateral prefrontal cortex.*

**Table 2.**

*List of papers of microglia in depression and suicide including PET with TSPO, postmortem studies.*

with rapid cycling bipolar disorder during both manic and depressive states, respectively. We revealed that CD206 gene expression was upregulated in the depressive state compared to the manic state among all three patients [45]. We summarize PET, postmortem, and iMG studies in **Table 2**.
