Titolo della tesi: MECHANISMS OF FEAR MEMORY PROCESSES AND IMPLICATIONS FOR TRAUMA-RELATED DISORDERS
Exposure to stressors or threatening events can lead to the development of stress-related diseases, such as anxiety, depression and post-traumatic stress disorder (PTSD) (De Kloet et al., 2005; McEwen, 2003). Understanding the neurobiological underpinnings of PTSD can be of great help for the identification of innovative therapeutic strategies and to this aim, animal models represent essential tools to gain a deeper insight on the maladaptive responses to trauma. Several studies demonstrated that the medial prefrontal cortex (mPFC) plays a pivotal role in the modulation of the fear memory(Koenigs & Grafman, 2009). The infralimbic portion of the mPFC has been shown to negatively modulate aversive behaviours and promoting fear extinction by inhibiting the amygdala output (Giustino & Maren, 2015). Not only, it well known that beyond the mPFC, amygdala and hippocampus are two other essential brain areas involved in fear memory modulation. The amygdala is a key brain area of the fear memory circuitry, in fact large evidences show its critical role into the formation and storage of fear memories (LeDoux, 2000; Maren & Holmes, 2016) and its deep interconnection with the hippocampus, which is pivotal for establishing and modulating contextual fear memory (Fanselow, 2000; Maren, 2001; Maren & Holmes, 2016). Clinical studies in PTSD patients showed that fearful experiences enhance amygdala activation but conversely, they reduce prefrontal cortex activity (Shin et al., 2005). A meta-analysis of PTSD fMRI studies confirmed this pattern of mPFC hypoactivity and amygdala hyperactivity and has been interpreted as a lack of regulatory control over emotion (Etkin and Wager, 2007). Taken together, this evidence led to the hypothesis that PTSD patients might present a malfunctional top-down control, from the medial prefrontal cortex (mPFC) onto amygdalar and hippocampal domains, either via direct or indirect synaptic connections that orchestrate the over-consolidation, excessive retrieval, generalization, and impairment of extinction of fear memories.
To this aim, in the context of a collaborative ERA-NET NEURON research project in which our team is involved, we proposed to identify and investigate the neurobiological functions of PTSD-fear memory engrams in an animal model of PTSD. Specifically, the project aimed at identifying dysfunctional neuronal populations of fear-relevant brain regions, including the mPFC, amygdala and hippocampus, involved in aberrant alterations of fear memory processing observed in PTSD. For this purpose, Prof. Mazahir T. Hasan’s laboratory developed a novel genetic technique to permanently tag and selectively manipulate these fear memory neuronal ensembles which are activated by learning. This novel genetic system allows activity dependent tagging of infected neuronal populations and their subsequent selective optogenetic activation (or inhibition) through the viral-induced expression of activating (or inhibiting) light-sensitive rhodopsins. Hasan’s laboratory engineered this novel genetic system called “virus-delivered, Genetic Activity-dependent Tagging of Ensembles” (vGATE) (Dogbevia et al., 2016; Hasan et al., 2013). During the first year of my PhD program I spent 8 months in the Hasan’s laboratory working on the development of the above mentioned vGATE system. Specifically, I worked on the development of the following vector-viruses: i) rAAV-(tetO)7-FOS-rtTA virus which consists of a c-fos promoter linked to heptamerized tetracycline (tet) operators, (tetO)7, to drive the expression of a reverse tet transactivator (rtTA); ii) rAAV-Ptetbi-Cre/YC3.60 which presents a bidirectional tet promoter (Ptetbi) that allows to drive Cre gene expression on one side and a fluorescent protein variant, such as a GFP variant on the other opposite side; iii) a AAV virus is equipped with a constitutive promoter (elongation factor 1, EF1a) with a flip-excision (FLEX) elements to drive Cre-dependent expression of channelrhodopsin fused to mCherry (ChR2-mCherry). In this research project, the development of this genetic system was addressed to be applied in PTSD-like animal models developed in Prof. Campolongo’s laboratory: this PTSD-like rat model is able to chronically reproduce both cognitive (over-consolidation, excessive retrieval and impaired extinction of traumatic memory) etiological alterations of the disease and a peculiar set of emotional dysfunctions related to the social domain(DSM-V, criterium g) (Berardi et al., 2014). This PTSD-like model allows to discriminate between susceptible and resilient rats to develop PTSD-like symptomatology(Colucci et al., 2020). Specifically, the project aimed at manipulating, through the vGATE system, neuronal populations of the mPFC and amygdala,to identify mechanisms of resilience and vulnerability to develop PTSD-like behavioral alterations.
A crucial aspect of PTSD is the susceptibility/resilience to develop the disorder. Even if humans experiencing a traumatic event, all show an acute response to trauma, only a subset of them (susceptible) ultimately develops the PTSD, while the others (resilient) fully recover after a first acute response (Colucci et al., 2020). Susceptible individuals may switch from a normal response to trauma to a maladaptive expression of memory specificities characterized by over-consolidation, memory generalization and impaired extinction (Desmedt et al., 2015; Finsterwald et al., 2015). Thus, understanding the neurobiological mechanisms sustaining such pathological expression in susceptible subjects and the possibility of having predictive power on the susceptibility to the development of PTSD prior to the pathological phase, would allow the design of targeted prophylactic, and therapeutic approaches in a clinical setting. As mentioned above, the majority of animal models of PTSD do not take into account the susceptibility factor and fail to reproduce a chronic cognitive and emotional symptomatology, thus lacking translational value (Kar, 2011). Among those spare studies considering the individual variability in response to trauma, only the anxiety-like symptoms following an acute stress response are used to discern between susceptible and resilient phenotypes to develop PTSD-like alterations (Musazzi et al., 2018; Ritov & Boltyansky, 2016). In an effort to improve preclinical research for the investigation of the neurobiological substrates underlying the individual variability towards PTSD development, our laboratory has identified a predictive variable (i.e. exploratory activity in a novel environment after trauma exposure) which allowed for the screening of rats in three different phenotypes: normal responders, susceptible and resilient to develop chronic PTSD-like cognitive and emotional alterations (Colucci et al., 2020). Based on these findings and on the evidence that PTSD-like phenotypic features are highly heritable over generations (Nievergelt et al., 2019), I was also involved in a research project aimed at the generation of PTSD-like susceptible and resilient lines of rats. Therefore, behavioral screening and subsequent mating procedures between subjects with the same phenotypic profile were carried on, up to the eleventh generation. The behavioral characterization conducted on the eleventh generation of the two rat lines showed that the susceptible and resilient PTSD-like phenotypes were inherited, such that susceptible rats, when subjected to a PTSD paradigm showed increased fear recall, poor fear extinction and reduced sociability as compared with resilient rats. The availability of these two rat populations represents an important tool to gain more knowledge on the neurobiology of PTSD and for the identification of tailored prophylactic and therapeutic strategies for trauma-related disorders.
Depending on the inter-individual variability of the population, the exposure to stressful events may result into maladaptive changes and lead to the development of PTSD. Several clinical data report that women are more susceptible to develop PTSD compared to men, and the neural basis of this discrepancy are still poorly understood (Breslau, 2002; Mancini et al., 2021; Seedat & Stein, 2000). Several studies demonstrated sex-differences in the use of illicit drugs, such as the psychostimulants, both in the drug-dependence and the biological and behavioral responses to them (Becker et al., 2001; Roth et al., 2004). Psychostimulants such as amphetamines are well known to interfere with memory processes (Baumann et al., 2019; Fleckenstein et al., 2007; LaLumiere et al., 2005; Roozendaal et al., 2008). Many studies have demonstrated that amphetamine can enhance memory consolidation in rodents, (Colucci et al., 2019; Doty & Howard, 1966; Kelemen & Bovet, 1961; Krivanek & McGaugh, 1969; McGaugh, 1973; McGaugh & Roozendaal, 2009) but other studies reported that psychostimulants have effects also on memory generalization (Ballard et al., 2014; Easton & Bauer, 1997). There is evidence that susceptible individuals to develop PTSD have comorbidity to psychostimulant drug addiction, such as cocaine (Schwendt et al., 2018). Based on this evidence, we investigated how psychostimulants affect fear memory dynamics in both male and female rats. Chapter 1 describes a study examining the effects of a sub-chronic treatment with the psychostimulants amphetamine or 3,4-Methylenedioxypyrovalerone (MDPV) on traumatic memory in a contextual fear conditioning/generalization paradigm in adult male and female rats. Our laboratory had previously shown that both drugs, if acutely administered soon after an inhibitory avoidance training session, induced generalization of fear memory in male rats (Colucci et al., 2019). Based on these findings and to better mimic the clinical setting, we investigated the effects of a subchronic MDPV and amphetamine treatment on fear generalization and examined the possibility of sex-divergent effects of the drugs in male and female Sprague Dawley rats.
Social interactions are basic human needs (Williams, 2007). Nonetheless, subjects vulnerable to PTSD development, after traumatic experiences face a social breakdown; PTSD individuals often show social impairment and relationships with close relatives and partners are disrupted after the traumatic event (Nietlisbach & Maercker, 2009; Riggs et al., 1998). Notwithstanding, it is also stated that social support is beneficial to pace traumatic experiences (Brewin et al., 2000; Charuvastra & Cloitre, 2008; Ozer et al., 2003). In animals, it is plenty demonstrated that social interactions can ameliorate stress and fear responses (Ishii et al., 2016; Mikami et al., 2020) by a so-called “social buffering” phenomenon, defined as the ability to mitigate stress responses when a stressed-feared animal is mated with a non-stressed subject (Kiyokawa, 2017; Kiyokawa & Hennessy, 2018; Mikami et al., 2020). To date, there is not a gold standard therapeutical approach to treat PTSD symptomatology. The Cognitive Behavioral Therapy (CBT) represents the first line treatment for this disorder, even if it has not always been effective and it suffers from many dropouts (Abramowitz, 2013; Morena et al., 2018). Therefore, with the aim to examine the possible beneficial effects of a social support on PTSD-like symptomatology and assess a novel therapeutic approach to be eventually coupled with CBT and increase its success rate, in Chapter 2 we investigated the social interaction as a therapeutic strategy to treat memory and emotional long-term sequelae of trauma exposure in a chronic PTSD-like model in rats. Specifically, we examined the effects of social buffering on fear extinction and social anxiety in our PTSD-like animal model, recently developed in our laboratory (Berardi et al., 2014)
Stressful experiences during lifetime can play a role in etiology of several mental disorders, such as PTSD and other trauma-related disorders (Brukhnová et al., 2023). It is known that experiencing a stressful event activate hypothalamus–pituitary–adrenal axis (HPA axis), resulting in an increased glucocorticoids release (cortisol in humans, corticosterone in rodents) from the adrenal cortex which are responsible of complex effects on cognitive and memory functions (de Quervain et al., 2011; Finsterwald et al., 2015). Stress and fear memory share common neuronal mechanisms and exposure to high levels of stress, as it happens in PTSD patients, profoundly alters fear memory processing. Animal models represent invaluable tools to investigate the brain circuitry and neurobiological mechanisms involved in mechanisms of stress-induced alterations of fear memory processing. With this aim, in Chapter 3 we run a meta-analysis to investigate the effects of stress exposure or GC-based treatments on fear memory extinction in rodents, exposed to an Auditory or Contextual Pavlovian Fear Conditioning paradigm. The current literature is extremely wide and heterogeneous. The results of the various studies on stress responses are subjected to multiple variables that may depend on factors such as the species of animal models, genetic strain, sex, age, the type of fear conditioning paradigm applied, the nature and the intensity of the stressors or the possible administration of different GC drugs. Therefore, this study aims at generating a comprehensive meta-analysis by using homogenized variables across the considered datasets, and at standardising the multiple responses concerning the effects of stress on fear memory extinction in rodent models.
Chapter 4 summarizes and discusses the findings of this thesis and provides conclusions and future perspectives.