The mammalian target of rapamycin (mTOR ) is a phosphoinositide kinase family serine-threonine protein kinase that modulates cell growth, proliferation, and synaptic plasticity via the regulation of protein synthesis [35, 36]. Several reports indicate that components of the mTOR pathway are engaged following learning [35, 37–39], and rapamycin, a selective inhibitor of mTOR activity, when administered around training, blocks LTM formation in a number of behavioral paradigms [35, 37, 40–43]. It is activated by a number of growth factors including brain-derived neurotrophic factor (BDNF) and regulates protein synthesis mainly through the phosphorylation of two downstream targets, p70S6K and eukaryotic initiation factor 4E-binding proteins, which are critically involved in initiation, the rate-limiting step of translation [35, 36]. The behavioral effects of intra-hippocampal and amygdalar microinjections of rapamycin (600 nM in ACSF) 1 h after PSS exposure were assessed. Behavior was assessed 7 days later in the EPM and ASR tests and freezing response to a trauma reminder on day 8. Rapamycin postexposure was ineffective in preventing PTSD-like behavioral responses. Neither prevalence rates of individuals with extreme behavioral response patterns nor subsequent freezing responses to a trauma cue differed between the treatment group and controls.

Protein kinase M zeta ( PKMζ) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long-term potentiation (LTP) and memory storage in the brain [44, 45]. PKMζ can maintain LTP because the kinase is autonomously active in neural tissue [45] and, thus, able to persistently enhance synaptic strength [46]. It has been shown that long-term memory can be “erased” by infusion of ZIP into the insular cortex (IC) [47]. The authors reported that in the IC, the activity of PKMζ is specifically involved in the storage of memories but not in their acquisition. Therefore, one may speculate that inhibiting PKMζ activity after traumatic experience may interfere with the process through which fearful memories become pathological and may lead to PTSD. We therefore assessed the long-term effects of ZIP, a specific, membrane-permeant peptide that mimics the autoregulatory domain of PKMζ and thus acts as an inhibitor, microinjected 1 h after stress exposure, into four brain structures widely implicated in the neurobiology of memory processes as well as in anxiety states: the dorsal hippocampus (DH), basolateral amygdala (BLA), lateral ventricle (LV), and insular cortex (IC) (Fig. 11.2). Stress-induced behavioral responses were assessed in the EPM and the ASR tests on day 7 and trauma cue-triggered freezing response on day 8.

The results showed that inactivation of PKMζ with ZIP injected to the LV or DH within 1 h of exposure to stress effectively reduced PTSD-like behavioral disruption and trauma cue response 8 days later, resulting in statistically significant reductions (37.5% and 44.4%, respectively) in the prevalence rates of EBR (PTSD-like) individuals at 7 days, with concomitant increases in the prevalence of MBR individuals of 12.5% and 11.1%, respectively, as compared to scrambled (Scr)-ZIP controls, indicating a significant shift toward less extreme stress-induced behavioral disruption. The DH/LV ZIP-treated individuals demonstrated markedly less extreme trauma cue freezing responses (27.7% and 23.9% of time freezing, respectively) than the Scr-ZIP control groups (49.3% and 57% of time freezing). In contrast, immediate post-stressor ZIP microinfusion to the amygdala and IC was ineffective in attenuating stress-induced behavioral disruptions. We hypothesize that ZIP microinjected to the hippocampus and lateral ventricle disrupted traumatic memory consolidation processes.

After the initial demonstration that suppressing protein synthesis during consolidation interfered with the formation of the traumatic memory, we were particularly interested to examine if the effects of manipulation of the native stress-related neuromodulators associated with encoding and consolidation processes would also participate in processes.

The key processes of the primary consolidation of the initial trauma memory into long-term traces and trauma cue-triggered reactivation take place at times during which the stress response cascade is activated, i.e., the fast-acting autonomic (sympathetic) nervous system (ANS) and the slow hypothalamic-pituitary-adrenocortical [HPA] axis. Sympathetic nervous system responses include the release of the catecholamines, epinephrine, and norepinephrine from the adrenal medulla. Activation of the HPA axis leads, via intermediate steps, to the release of glucocorticoids (cortisol in humans and corticosterone in rodents) from the adrenal cortex [48]. Glucocorticoids can enter the brain and exert their actions principally via intracellular receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These key memory-related processes are thus likely to be affected by components, mediators, and end products of these physiologic systems, directly or indirectly [49]. The complex interactions among the various stress-related hormones in the emotionally aroused state experienced during trauma exposure presumably impact on the formation of the characteristically durable traumatic memories in PTSD.

Animals were treated once with corticosterone at doses of 0.1, 3.0, 5.0, 15.0, or 25.0 mg/kg 1 h after stress exposure and compared with vehicle-treated and unexposed controls [50] (Fig. 11.3). Behavior was assessed at day 30 and freezing response to the trauma reminder on day 31 [50]. The results clearly showed that a single 25 mg/kg dose of corticosterone administered 1 h after stress exposure resulted in a statistically significant reduction of 22.0% in the prevalence rates of EBR (PTSD-like) individuals at 30 days, with a concomitant increase of 14.3% in the prevalence of MBR individuals, as compared to saline controls – i.e., a significant shift toward less extreme behavioral disruption ensuing from traumatic stress. Individuals in the high-dose corticosterone group responded markedly less to the trauma cue (24% of time freezing) than the saline-control group (80% of time freezing). This pattern of response suggests that the single high-dose corticosterone treatment conferred some degree of resilience to subsequent trauma-related stress exposure. Paradoxically, lower doses of corticosterone (0.1–5.0 mg/kg) were not only ineffective in attenuating stress-induced behavioral disruptions but in fact significantly increased the propensity of individuals to show extreme behavioral responses at day 30 and significantly increased freezing response to the trauma cue as compared to vehicle. The dose-response curve for corticosterone thus conformed to the inverted U-shaped curve previously reported [51]. The neurobiological mechanisms underlying the dose-dependent effects of corticosterone appear to be the result of differential activation of the mineralocorticoid and glucocorticoid receptor systems in different brain areas through a complex system of time-dependent modulators and/or modulation of gene expression through transcription or via transrepression [52–56].

The possibility that the high-dose corticosterone regimen affected behaviors via memory processes was subsequently borne out by a follow-up study employing a nonspatial object recognition memory task. Similar dose dependence was displayed. Low-dose corticosterone enhanced performance of the task, whereas high dose impaired it, presumably by interfering with memory consolidation, suggesting that one possible mechanism underlying the overall effect involved the disruption of consolidation of traumatic (fearful) memories [50].

A large body of evidence from animal studies indicates that adrenergic stress hormones released peripherally during emotionally arousing experiences modulate the storage of memory for the experience [57]. The ability of β-adrenoceptor blockade to reduce anxiety and fear has been quite firmly established by studies in human subjects and animals, although this is not true for every experimental paradigm involving anxiety and fear. In the clinical arena, Vaiva et al. [58] gave propranolol (40 mg) to 11 people admitted to the hospital following a motor vehicle accident or physical assault without any serious physical injuries. They were compared with eight patients who refused propranolol but agreed to participate in the study. Two months later, the propranolol group had fewer symptoms of PTSD (1/11) than controls (3/8). A pilot study by Pitman and colleagues [59] found similar results. The effect of two different doses of propranolol (10.0 and 15.0 mg/kg) administered immediately after PSS exposure was assessed at days 30 (EPM and ASR) and 31 (reminder response). Propranolol proved to be ineffective in attenuating stress-induced behavioral disruption. The propensity of treated individuals to develop extreme behavioral responses (PTSD-like) and the degree of vulnerability to a trauma cue 31 days after the index stress exposure were indistinguishable from exposed controls [60]. The physiological efficacy of the doses of propranolol was verified by collecting cardiovascular data telemetrically, and the results showed that the same treatment regimen effectively reduced post-stressor heart rate responses. Thus, propranolol effectively attenuated physiological functions, but failed to have behavioral effects in preventing posttraumatic responses, suggesting that propranolol was ineffective in preventing the development of posttraumatic responses in this animal model for PTSD [60]. In keeping with these results, a number of clinical studies published since then have reported similarly disappointing outcomes. In a double-blind, randomized, controlled trial of 14 days of propranolol administered within 48 h of injury to patients admitted to a surgical trauma center, no benefit over placebo on depressive or posttraumatic stress symptoms was found [61]. Sharp et al. [62] reported that propranolol does not reduce risk for acute stress disorder in pediatric burn patients. In a retrospective study performed on military burn soldiers injured in Operation Iraqi Freedom/Operation Enduring Freedom (OIF/OEF), propranolol did not decrease PTSD development. The prevalence of PTSD in patients receiving propranolol was the same as those not receiving propranolol [63].

Convergent evidence has accumulated that sleep serves as an off-line period in which newly encoded hippocampus-dependent memories are gradually adapted to preexisting knowledge networks [64–67]. Sleep following learning is known to enhance the consolidation of newly acquired memory traces [68–71] through an active reorganization of representations, whereas acute sleep deprivation (SD) may disrupt this process and impair retrieval functions [72]. Wagner et al. [71] reported that brief periods of sleep immediately following learning cause preservation of emotional memories over 4 years. We therefore hypothesized that interfering with memory consolidation processes by SD immediately after traumatic experience will reduce posttraumatic stress symptoms and incidence. Rats were deprived of sleep for 6 h throughout the first resting phase after predator scent stress exposure. Behaviors in the elevated plus maze and acoustic startle response tests were assessed 7 days later and served for classification into behavioral response groups. Freezing response to a trauma reminder was assessed on day 8. We found that 6 h of SD after PSS exposure resulted in a significant moderation of behavior patterns representing stress-induced anxiety, avoidance, and hyperarousal responses on the EPM and ASR tests. A resounding overall shift in the prevalence rates of animals fulfilling criteria for EBR, which were effectively reduced to nil, was mirrored by a concomitant increase in minimal behavioral responders (Fig. 11.4a). Freezing responses to the late (day 8) neutral trauma cue were markedly attenuated (16.4% of time freezing in the treatment group as compared with 75.8% for untreated controls) (Fig. 11.4b). As memory is required to bridge the time interval between stress exposure and trauma cue, and because the SD procedure intentionally spanned the time frame within which memory consolidation processes take place at the cellular level McGaugh [11], the reduction in freezing responses suggests that memory-related processes were affected. In other words, postexposure SD may affect traumatic memory consolidation and thereby effectively ameliorate long-term, stress-induced, PTSD-like behavioral disruptions.

In addition to the behavioral tests, we launched a study to examine factors affecting neural/dendritic synaptic connectivity in response to interventions with sleep deprivation. We demonstrate that early post-stressor intervention with SD, which attenuates posttraumatic stress response, was associated with a dramatic increase in the number of dendrites of dentate granule cells, total dendritic length, and dendritic spine density, as compared to vehicle controls (Fig. 11.4 (C+D)). Although the precise molecular mechanisms underlying the factors that regulate the orientation, morphology, and elaboration of dendritic processes are largely unknown, there is now compelling evidence that outgrowth and morphogenesis of the dendritic arbor depends on the coordinated action of brain-derived neurotrophic factor (BDNF). Therefore, we also evaluated the BDNF expression. Hippocampal expression of BDNF demonstrated that postexposure SD also corrected the clear-cut stress-induced downregulation of hippocampal BDNF expression [73] displayed by exposed-untreated rats. In light of the involvement of neurotrophins, and particularly of BDNF, in neuronal plasticity [74, 75], axonal and dendritic branching and remodeling [76–79], and proliferation of pyramidal neurons and their proximal dendrite growth [80], such an upregulation would enhance synaptic plasticity and stabilization of synaptic connectivity, enabling adaptive behavioral responses, i.e., increased resilience. Overall, the results of this study suggest that prevention of sleep in the early aftermath of stress exposure may be beneficial in attenuating traumatic, stress-related sequelae. Postexposure SD impairs hippocampus-dependent traumatic memory formation and consolidation, a mechanism possibly pertinent to the development of PTSD.

To summarize, interference with consolidation processes appears to represent a valid avenue for attenuating subsequent PTSD symptoms by affecting the durability of long-term trauma memories. The findings that single high-dose corticosteroids might be effective in attenuating the neuromodulatory effects of the stress response and the lack of efficacy of β-blockade are of particular clinical relevance. In addition, intentional prevention of sleep in the early aftermath of stress exposure may well be beneficial in attenuating traumatic stress-related sequelae. These evidence shed light on the importance of posttraumatic “off-line” processing and consolidation, in a specific timing (i.e., natural sleep time), to long-term adaptive recovery from the traumatic event. Postexposure SD impairs hippocampus-dependent traumatic memory formation and consolidation and therefore may be a simple, yet effective, intervention for the secondary prevention of stress-induced psychopathologies.

Given that it is often not possible to administer a consolidation-blocking agent, the possibility of later affecting the traumatic memory by pharmacologically blocking reconsolidation is particularly clinically relevant [81]. The temporarily destabilized state of memories between their reactivation, often in response to triggering by trauma reminder or cues, and their subsequent reconsolidation presents an equally important target for study. The clinical impact of potential interventions at later stages hardly requires explanation. The benefit for patient populations could be significant, considering that the window of opportunity for preventive interventions, i.e. during consolidation, is quite narrow and that there are quite significant issues which arise from the lack of reliable predictive factors in the acute phase.

The next series of studies employed the same interventions as the consolidation studies and the same approach to data analysis. The reactivating stimulus was unused cat litter and was administered either 7, 10, or 14 days after the initial PSS exposure. The results show that the exposed groups displayed significant behavioral responses to the reminder cue comparable to the responses to the original traumatic event. Behavioral responses were assessed at days 30–31 in a manner identical to the consolidation study protocols.

A regimen of repeated corticosterone treatment paired with reactivating the traumatic memory over 15 consecutive days, with the same range of doses of corticosterone (3.0, 15.0, and 25.0 mg/kg) injected 1 h after memory reactivation, was next assessed (Fig. 11.5), beginning on day 14 postexposure. Behavioral responses were assessed in the EPM and the ASR tests at day 30. Exposure- and trauma cue-triggered freezing response was assessed 1 day later.

Low-dose corticosterone (3.0 mg/kg) resulted in a statistically significant reduction of 40.0% in the prevalence rates of EBR individuals at 30 days, with a concomitant increase of 16.7% in the prevalence of MBR individuals, as compared to saline controls. Moreover, these animals responded markedly less to the trauma cue (17.7% of time freezing) than the saline-control group (44.7% of time freezing). One plausible explanation for our data is that corticosterone may facilitate the extinction of a traumatic memory trace. Based on the finding that glucocorticoids impair the retrieval of emotional information, we thus suggest that by inhibiting memory retrieval, corticosterone may weaken the traumatic memory trace and thus reduce behavioral symptoms. We propose that repeated low-dose corticosterone administered following the reactivation of a traumatic memory could therefore potentially represent a novel treatment for PTSD. The results suggest that repeated paired low-dose corticosterone and trauma memory reactivation may be worthy of study in the clinical arena.