We mainly found that chronic fluoxetine treatment enhanced the subsequent effects of fear extinction in a PTSD rat model with in vivo PET imaging. The glucose metabolism in the hippocampus was significantly increased after the chronic fluoxetine treatment, suggesting that this region may be a particularly interesting target. To the best of our knowledge, this study is the first to evaluate glucose metabolic changes after chronic fluoxetine treatment for PTSD in a non-invasive way.
PTSD-like behaviours could be induced by a Pavlovian fear conditioning paradigm, while glucose metabolism was increased in the right amygdala and left primary visual cortex but decreased in the left primary somatosensory cortex. However, the chronic fluoxetine treatment and behavioural extinction training could alleviate the PTSD-like symptoms. Our study showed that freezing (%) was lower than before in all three groups. Fluoxetine could enhance the subsequent effects of fear extinction (Pedraza et al., 2019). In the EXT + FLX group, glucose metabolism was increased in the left hippocampus, left striatum, right insular cortex, left posterior parietal cortex and right secondary visual cortex but decreased in the cerebellar lobule after the extinction retrieval. The rats in the EXT group exhibited increased [18F]FDG uptake in the left striatum, left cochlear nucleus and right primary visual cortex but decreased uptake in the anterior cingulate cortex. Glucose metabolism was increased in the left hippocampus and right primary visual cortex but decreased in the bilateral primary somatosensory cortex, left primary/secondary motor cortex and cuneiform nucleus in the FLX group.
The right amygdala was activated after fear conditioning in our study. Fear conditioning has been extensively used as a model of PTSD (Johansen, Cain, Ostroff, & LeDoux, 2011; Parsons & Ressler, 2013). The amygdala is considered the core brain region responsible for fear memory acquisition and storage (Maren & Quirk, 2004). Functional magnetic resonance imaging (fMRI) has shown greater activation of the amygdala in humans exposed to fear-relevant visual stimuli (Garrett et al., 2012). In clinical PET imaging studies, the amygdala of PTSD patients reacted more strongly than that of the control group when exposed to odours related to prior fear memories (Vermetten, Schmahl, Southwick, & Bremner, 2007). Similar results were also observed in an animal study (Brydges et al., 2013). Our results are consistent with the previous literature.
More importantly, we found that glucose metabolism was increased in the hippocampus in the FLX group and EXT + FLX group but not in the EXT group. The hippocampal volume of PTSD patients is smaller than that of controls based on MRI analyses (Bromis, Calem, Reinders, Williams, & Kempton, 2018). In addition, the reduction in the hippocampal volume is associated with the persistence of symptoms (Chao, Yaffe, Samuelson, & Neylan, 2014) and a poor treatment response in patients with PTSD (Rubin et al., 2016). Multiple animal studies have revealed that chronic fluoxetine treatment leads to changes in the hippocampus (Castren & Hen, 2013; Patricio et al., 2015), including the regeneration and metabolism of neurons, synaptic plasticity, and the complexity of dendritic spines (Kobayashi et al., 2010; Maya Vetencourt et al., 2008). These changes may be the result of some growth factors upregulated by fluoxetine, such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2) and serotonin (5-HT). As a major emotion regulator, BDNF plays an important role in the pathology of various mental diseases, such as depression (Rogoz, Kaminska, Panczyszyn-Trzewik, & Sowa-Kucma, 2017). Increased BDNF expression levels have been observed in the hippocampus in rodents following chronic fluoxetine treatment (Mendez-David et al., 2015). Acute, unilateral BDNF infusion into the rat dentate gyrus induced the long-term potentiation of medial perforant path-evoked synaptic transmission and, concomitantly, enhanced bilateral hippocampal neurogenesis (Kuipers et al., 2016). Chronic treatment with fluoxetine also increased the expression of FGF2 in hippocampus (Bachis, Mallei, Cruz, Wellstein, & Mocchetti, 2008; Evans et al., 2004). In addition, the concentrations of FGF2 were negatively correlated with fear expression in both humans and rats (Graham, Dong, & Richardson, 2018; Graham, Zagic, & Richardson, 2017). Notably, the antidepressant effect of fluoxetine is partially due to an increase in the concentration of FGF2, which in turn promotes nerve regeneration and increases the survival rate of new cells in the hippocampus (Perez, Clinton, Turner, Watson, & Akil, 2009). In addition, the tissue levels of 5-HT were consistently decreased in fear circuit areas (Lin, Tung, & Liu, 2016), but it could be adjusted by treatment with fluoxetine. Fluoxetine increased the extracellular serotonin levels in the brain by blocking the reuptake of serotonin. A study showed that chronic fluoxetine treatment enhanced excitatory synaptic transmission in the hippocampus by slowly elevating serotonin accumulation in vivo (Van Dyke, Francis, Chen, Bailey, & Thompson, 2019). The reduced freezing behaviour was related to increased concentrations of 5-HT in the hippocampus. After chronic treatment with fluoxetine, glucose metabolism in the hippocampus was significantly increased than before, suggesting that the hippocampus is a particular target for PTSD therapy.
In our study, elevated [18F]FDG uptake was found in the left striatum after the extinction training in both the EXT and EXT + FLX groups. The striatum is the terminal field of dopamine (DA) which is related to the reward and motivational processes (Schultz, 2001). Evidence suggests that PTSD involves the reward circuitry in the brain (Enman, Arthur, Ward, Perrine, & Unterwald, 2015). The DA and dopamine transporter levels, dopamine receptor (DR) density and DA metabolites were decreased in the striatum in a rat model of PTSD (Lee, Shim, Lee, & Hahm, 2018). Emotional numbness is a manifestation of the alteration in moods observed in those with PTSD. An fMRI study revealed a significant negative correlation between emotional numbness and the activation of the right ventral striatum in PTSD patients (Felmingham et al., 2014). Moreover, the release of DA in the striatum is involved in putative dopamine signalling during fear extinction (Kobayashi et al., 2010). Behavioural extinction training of fear has neurobiological mechanisms similar to those of exposure therapy. We performed a CS-no-US association that consisted of repeated presentations of the CS without the US to the rats in the EXT group and EXT + FLX group, and the memory of extinction learning was tested on the following day (called extinction retrieval) (Milad, Rosenbaum, & Simon, 2014). The disappearance of an expected US could be considered a reward-like safety signal during extinction training. A rodent study suggested that extinction must be mediated by DA signalling via the D1R (El-Ghundi, O'Dowd, & George, 2001). DR-mediated signal transduction involves metabolic changes in neurons. Consequently, our findings highlight that the increase in glucose metabolism in the left striatum is strongly consistent with the above conclusions.
The glucose metabolism in the right insular cortex was increased in the EXT + FLX group. Only a few studies indicated that the insular cortex participates in the extinction of conditioned fear. The insular cortex is involved in the extinction of conditioned taste aversion (CTA), which is delayed by blocking protein translation (Hadamitzky et al., 2016). The synaptic plasticity of insular neurons could be enhanced by extinction training and BDNF. Acute BDNF infusions into the insular cortex were able to promote the extinction of CTA (Rodriguez-Serrano, Ramirez-Leon, Rodriguez-Duran, & Escobar, 2014). In addition, studies have demonstrated that chronic treatment with fluoxetine had a positive effect on the increase in BDNF. Thus, we speculate that the activation of the insular cortex was the result of the combination of extinction training and chronic fluoxetine treatment.
Interestingly, we observed decreased glucose metabolism in the anterior cingulate cortex only in the EXT group. The anterior cingulate cortex, which belongs to the prefrontal cortex, participates in the dysregulation of emotion control and cognitive function in PTSD (Wisdom et al., 2014; Woodward et al., 2015). Previous clinical studies have shown that the symptom severity and grey matter volume in PTSD patients were negatively correlated with the level of decrease in anterior cingulate cortex activity (Bromis et al., 2018). PTSD is the result of persistent energy metabolism disorders that ultimately lead to chronic low-grade inflammation (Naviaux, 2012). Gene clusters in inflammation pathways following fear conditioning are enriched in the anterior cingulate cortex. Studies have shown that chronic fluoxetine treatment after trauma could reduce PTSD-like symptoms by altering the expression of inflammatory genes in the anterior cingulate cortex (Kao et al., 2016). However,t only the EXT group showed metabolism changes in the anterior cingulate cortex in our study. Using c-Fos as a marker of neuronal activation, we confirmed that the expression of c-Fos in the anterior cingulate in the EXT group was decreased. Therefore, the effect of chronic fluoxetine treatment in the anterior cingulate cortex warrants further investigation.
As expected, we found increased glucose metabolism in brain areas involved in the audiovisual pathway, including the visual cortex, cochlear nucleus and posterior parietal cortex. When the rats were transferred from a dark chamber to the bright microPET scanning room, the light stimulation resulted in increased glucose metabolism in the visual cortex. The cochlear nucleus is not only the primary auditory information processing centre but also the only nucleus in the central auditory system (Schofield & Coomes, 2005). In addition, the posterior parietal cortex potentially integrates information from the visual, somatosensory and auditory cortices (Harvey, Coen, & Tank, 2012; Wilber et al., 2014). Thus, unsurprisingly, these brain areas showed increased glucose metabolism. We also observed decreased glucose metabolism in the primary somatosensory cortex, left primary/secondary motor cortex, cerebellar lobule and cuneiform nucleus. Environmental information obtained from whiskers could be sent the barrel field of the somatosensory cortex (Diamond, von Heimendahl, Knutsen, Kleinfeld, & Ahissar, 2008). In addition, the cerebellum receives facial sensory information and participates in cortical sensorimotor integration (Popa et al., 2013; Proville et al., 2014). Moreover, optogenetic stimulation of the dorsal periaqueductal grey increased glucose metabolism in the cuneiform nucleus in a rat model of panic disorder (He et al., 2019). Decreased glucose metabolism in the cuneiform nucleus was observed in our results, which was related to the decrease in freezing in the behavioural test.
Our study found that chronic fluoxetine enhanced the subsequent effects of fear extinction in a PTSD rat model. Our behavioural results were consistent with these findings, providing additional evidence for the evaluation of the treatment efficacy of fluoxetine combined with extinction training for PTSD. Extinction training combined with fluoxetine treatment were more effective in eradicating persistent fear memory (Karpova et al., 2011). Futhermore, [18F]FDG microPET imaging could not only visualize glucose metabolism in various brain areas across different behavioural stages but also help evaluating the effects of fluoxetine and extinction training; Thus,we recommend using this non-invasive in vivo imaging technique for future research concerning PTSD.