Timing of vagus nerve stimulation during fear extinction determines efficacy in a rat model of PTSD.

Studies have indicated that vagus nerve stimulation (VNS) enhances extinction learning in rodent models. Here, we investigated if pairing VNS with the conditioned stimulus is required for the enhancing effects of VNS. Adult Sprague-Dawley rats were exposed to intense stress followed by fear conditioning training to produce resistant fear. Rats were then implanted with a cuff electrode around the left vagus. After recovery, rats underwent extinction training paired with VNS (0.5 s, 0.8 mA, 100 µs, and 30 Hz) or with Sham VNS (0 mA). VNS rats were randomized into the following subgroups: During VNS (delivered during presentations of the conditioned stimulus, CS), Between VNS (delivered between CS presentations), Continuous VNS (delivered during the entire extinction session), and Dispersed VNS (delivered at longer inter-stimulation intervals across the extinction session). Sham VNS rats failed to extinguish the conditioned fear response over 5 days of repeated exposure to the CS. Rats that received Between or Dispersed VNS showed modest improvement in conditioned fear at the retention test. During and Continuous VNS groups displayed the greatest reduction in conditioned fear. These findings indicate that delivering VNS paired precisely with CS presentations or continuously throughout extinction promotes the maximum enhancement in extinction learning.

Vagus Nerve Stimulation as a Treatment for Fear and Anxiety in Individuals with Autism Spectrum Disorder.

Anxiety disorders affect a large percentage of individuals who have an autism spectrum disorder (ASD). In children with ASD, excessive anxiety is also linked to gastrointestinal problems, self-injurious behaviors, and depressive symptoms. Exposure-based cognitive behavioral therapies are effective treatments for anxiety disorders in children with ASD, but high relapse rates indicate the need for additional treatment strategies. This perspective discusses evidence from preclinical research, which indicates that vagus nerve stimulation (VNS) paired with exposure to fear-provoking stimuli and situations could offer benefits as an adjuvant treatment for anxiety disorders that coexist with ASD. Vagus nerve stimulation is approved for use in the treatment of epilepsy, depression, and more recently as an adjuvant in rehabilitative training following stroke. In preclinical models, VNS shows promise in simultaneously enhancing consolidation of extinction memories and reducing anxiety. In this review, we will present potential mechanisms by which VNS could treat fear and anxiety in ASD. We also discuss potential uses of VNS to treat depression and epilepsy in the context of ASD, and noninvasive methods to stimulate the vagus nerve.

Vagus nerve stimulation enhances fear extinction as an inverted-U function of stimulation intensity.

Studies in rodents indicate that pairing vagus nerve stimulation (VNS) with extinction training enhances fear extinction. However, the role of stimulation parameters on the effects of VNS remains largely unknown. Identifying the optimal stimulation intensity is a critical step in clinical translation of neuromodulation-based therapies. Here, we sought to investigate the role of stimulation intensity in rats receiving VNS paired with extinction training in a rat model for Posttraumatic Stress Disorder (PTSD). Male Sprague-Dawley rats underwent single prolonged stress followed by a severe fear conditioning training and were implanted with a VNS device. After recovery, independent groups of rats were exposed to extinction training paired with sham (0 mA) or VNS at different intensities (0.4, 0.8, or 1.6 mA). VNS intensities of 0.4 mA or 0.8 mA decreased conditioned fear during extinction training compared to sham stimulation. Pairing extinction training with moderate VNS intensity of 0.8 mA produced significant reduction in conditioned fear during extinction retention when rats were tested a week after VNS-paired extinction. High intensity VNS at 1.6 mA failed to enhance extinction. These findings indicate that a narrow range of VNS intensities enhances extinction learning, and suggest that the 0.8 mA VNS intensity used in earlier rodent and human stroke studies may also be the optimal in using VNS as an adjuvant in exposure therapies for PTSD.

Vagus nerve stimulation promotes extinction generalization across sensory modalities.

Traumatic experiences involve complex sensory information, and individuals with trauma-related psychological disorders, such as posttraumatic stress disorder (PTSD), can exhibit abnormal fear to numerous different stimuli that remind them of the trauma. Vagus nerve stimulation (VNS) enhances extinction of auditory fear conditioning in rat models for PTSD. We recently found that VNS-paired extinction can also promote extinction generalization across different auditory cues. Here we tested whether VNS can enhance extinction of olfactory fear and promote extinction generalization across auditory and olfactory sensory modalities. Male Sprague Dawley rats were implanted with a stimulating cuff on the cervical vagus nerve. Rats then received two days of fear conditioning where olfactory (amyl acetate odor) and auditory (9 kHz tones) stimuli were concomitantly paired with footshock. Twenty-four hours later, rats were given three days of sham or VNS-paired extinction (5 stimulations, 30-sec trains at 0.4 mA) overlapping with presentation of either the olfactory or the auditory stimulus. Two days later, rats were given an extinction retention test where avoidance of the olfactory stimulus or freezing to the auditory stimulus were measured. VNS-paired with exposure to the olfactory stimulus during extinction reduced avoidance of the odor in the retention test. VNS-paired with exposure to the auditory stimulus during extinction also decreased avoidance of the olfactory cue, and VNS paired with exposure to the olfactory stimulus during extinction reduced freezing when the auditory stimulus was presented in the retention test. These results indicate that VNS enhances extinction of olfactory fear and promotes extinction generalization across different sensory modalities. Extinction generalization induced by VNS may therefore improve outcomes of exposure-based therapies.

Daily Optogenetic Stimulation of the Left Infralimbic Cortex Reverses Extinction Impairments in Male Rats Exposed to Single Prolonged Stress.

Post-traumatic stress disorder (PTSD) is associated with decreased activity in the prefrontal cortex. PTSD-like pathophysiology and behaviors have been observed in rodents exposed to a single prolonged stress (SPS) procedure. When animals are left alone for 7 days after SPS treatment, they show increased anxiety-like behavior and impaired extinction of conditioned fear, and reduced activity in the prefrontal cortex. Here, we tested the hypothesis that daily optogenetic stimulation of the infralimbic region (IL) of the medial prefrontal cortex (mPFC) during the 7 days after SPS would reverse SPS effects on anxiety and fear extinction. Male Sprague-Dawley rats underwent SPS and then received daily optogenetic stimulation (20 Hz, 2 s trains, every 10 s for 15 min/day) of glutamatergic neurons of the left or right IL for seven days. After this incubation period, rats were tested in the elevated plus-maze (EPM). Twenty-four hours after the EPM test, rats underwent auditory fear conditioning (AFC), extinction training and a retention test. SPS increased anxiety-like behavior in the EPM task and produced a profound impairment in extinction of AFC. Optogenetic stimulation of the left IL, but not right, during the 7-day incubation period reversed the extinction impairment. Optogenetic stimulation did not reverse the increased anxiety-like behavior, suggesting that the extinction effects are not due to a treatment-induced reduction in anxiety. Results indicate that increased activity of the left IL after traumatic experiences can prevent development of extinction impairments. These findings suggest that non-invasive brain stimulation may be a useful tool for preventing maladaptive responses to trauma.

The peripheral effect of direct current stimulation on brain circuits involving memory.

An ongoing debate surrounding transcranial direct current stimulation (tDCS) of the scalp is whether it modulates brain activity both directly and in a regionally constrained manner enough to positively affect symptoms in patients with neurological disorders. One alternative explanation is that direct current stimulation affects neural circuits mainly indirectly, i.e., via peripheral nerves. Here, we report that noninvasive direct current stimulation indirectly affects neural circuits via peripheral nerves. In a series of studies, we show that direct current stimulation can cause activation of the greater occipital nerve (ON-tDCS) and augments memory via the ascending fibers of the occipital nerve to the locus coeruleus, promoting noradrenaline release. This noradrenergic pathway plays a key role in driving hippocampal activity by modifying functional connectivity supporting the consolidation of a memory event.

Efficient parameters of vagus nerve stimulation to enhance extinction learning in an extinction-resistant rat model of PTSD.

Vagus nerve stimulation (VNS) has shown promise as an adjuvant treatment for posttraumatic stress disorder (PTSD), as it enhances fear extinction and reduces anxiety symptoms in multiple rat models of this condition. Yet, identification of the optimal stimulation paradigm is needed to facilitate clinical translation of this potential therapy. Using an extinction-resistant rat model of PTSD, we tested whether varying VNS intensity and duration could maximize extinction learning while minimizing the total amount of stimulation. We confirmed that sham rats failed to extinguish after a week of extinction training. Delivery of the standard LONG VNS trains (30 s) at 0.4 mA enhanced extinction and reduced anxiety but did not prevent fear return. Increasing the intensity of LONG VNS trains to 0.8 mA prevented fear return and attenuated anxiety symptoms. Interestingly, delivering 1, 4 or 16 SHORT VNS bursts (0.5 s) at 0.8 mA during each cue presentation in extinction training also enhanced extinction. LONG VNS trains or multiple SHORT VNS bursts at 0.8 mA attenuated fear renewal and reinstatement, promoted extinction generalization and reduced generalized anxiety. Delivering 16 SHORT VNS bursts also facilitated extinction in fewer trials. This study provides the first evidence that brief bursts of VNS can enhance extinction training, reduce relapse and support symptom remission using much less VNS than previous protocols. These findings suggest that VNS parameters can be adjusted in order to minimize total charge delivery and maximize therapeutic effectiveness.

Vagus nerve stimulation produces immediate dose-dependent anxiolytic effect in rats.

BACKGROUND: Chronic vagus nerve stimulation (VNS) attenuates anxiety in rats and humans. However, it is unclear whether VNS can promote acute anxiolytic effects. Here we examined short-term anxiolytic effects of VNS using single or multiple trains in rats submitted to a battery of tests. METHODS: Three groups of rats were implanted with VNS cuffs and tested for anxiety using the elevated plus maze (EPM), novelty suppressed feeding (NSF), and acoustic startle response (ASR), after receiving either one or five VNS trains (0.4 mA/30 Hz/30­sec), or sham stimulation. RESULTS: Both single and multiple VNS trains reduced state anxiety as measured using the EPM, but only multiple trains reduced anxiety in the EPM and NSF. VNS did not decrease arousal as measured using the ASR. LIMITATIONS: The anxiolytic effects of VNS may be differently influenced by test order or prior-exposure to stress. VNS did not affect startle responses in naïve rats but the present findings do not determine whether VNS would affect startle responses that are potentiated by fear or anxiety. CONCLUSION: A single VNS train can produce an anxiolytic-like effect in the EPM minutes later, an effect that is not observed in the NSF. Delivering 5 VNS trains restores the immediate effects across tests of anxiety, indicating that more trains produce a more robust anxiolytic effect. The lack of effects on ASR suggests that VNS affects state anxiety but not baseline arousal in naïve rats. We suggest that the anxiolytic effect of VNS can increase tolerability and reduce dropout in exposure-based therapies.

Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.

Vagus nerve stimulation (VNS) enhances extinction of conditioned fear in rats. Previous findings support the hypothesis that VNS effects on extinction are due to enhanced consolidation of extinction memories through promotion of plasticity in extinction-related brain pathways however, alternative explanations are plausible. According to one hypothesis, VNS may produce a hedonic effect and enhance extinction through counter-conditioning. According to another hypothesis, VNS reduces anxiety during exposure and this weakens the association of conditioned stimuli with aversive conditioned responses. The present set of experiments (1) used conditioned place preference (CPP) to identify potential rewarding effects associated with VNS and (2) examined the peripheral effects of VNS on anxiety and extinction enhancement. Male Sprague-Dawley rats were surgically implanted with cuff electrodes around the vagus nerve and subjected to a CPP task in which VNS and sham stimulation were each paired with one of two distinct contexts over the course of 5 d. Following this procedure, rats did not show a place preference, suggesting that VNS is not rewarding or aversive. The role of the peripheral parasympathetic system in the anxiolytic effect of VNS on the elevated plus maze was examined by blocking peripheral muscarinic receptors with intraperitoneal administration of methyl scopolamine prior to VNS. Methyl scopolamine blocked the VNS-induced reduction in anxiety but did not interfere with VNS enhancement of extinction of conditioned fear, indicating that the anxiety-reducing effect of VNS is not necessary for the extinction enhancement.

Vagus nerve stimulation reverses the extinction impairments in a model of PTSD with prolonged and repeated trauma.

We have shown that vagus nerve stimulation (VNS) enhances extinction of conditioned fear and reduces anxiety in rat models of PTSD using moderate stress. However, it is still unclear if VNS can be effective in enhancing extinction of severe fear after prolonged and repeated trauma. Severe fear was induced in adult male rats by combining single prolonged stress (SPS) and protracted aversive conditioning (PAC). After SPS and PAC procedures, rats were implanted with stimulating cuff electrodes, exposed to five days of extinction training with or without VNS, and then tested for extinction retention, return of fear in a new context and reinstatement. The elevated plus maze, open field and startle were used to test anxiety. Sham rats showed no reduction of fear during extensive extinction training. VNS-paired with extinction training reduced freezing at the last extinction session by 70% compared to sham rats. VNS rats exhibited half as much fear as shams, as well as less fear renewal. Sham rats exhibited significantly more anxiety than naive controls, whereas VNS rats did not. These results demonstrate that VNS enhances extinction and reduces anxiety in a severe model of PTSD that combined SPS and a conditioning procedure that is 30 times more intense than the conditioning procedures in previous VNS studies. The broad utility of VNS in enhancing extinction learning in rats and the strong clinical safety record of VNS suggest that VNS holds promise as an adjuvant to exposure-based therapy in people with PTSD and other complex forms of this condition.

Vagus nerve stimulation as a tool for enhancing extinction in exposure-based therapies.

RATIONALE: Emotionally traumatic experiences can lead to maladaptive memories that are enduring and intrusive. The goal of exposure-based therapies is to extinguish conditioned fears through repeated, unreinforced exposures to reminders of traumatic events. The extinction of conditioned fear depends upon the consolidation of new memories made during exposure to reminders. An impairment in extinction recall, observed in certain patient populations, can interfere with progress in exposure-based therapies, and the drive to avoid thoughts and reminders of the trauma can undermine compliance and increase dropout rate. Effective adjuncts to exposure-based therapies should improve the consolidation and maintenance of the extinction memory or improve the tolerability of the therapy. Under stressful conditions, the vagus nerve responds to elevations in epinephrine and signals the brain to facilitate the storage of new memories while, as part of the parasympathetic nervous system, it slows the sympathetic response. OBJECTIVE: Here, we review studies relevant to fear extinction, describing the anatomical and functional characteristics of the vagus nerve and mechanisms of vagus nerve stimulation (VNS)-induced memory enhancement and plasticity. RESULTS: We propose that stimulation of the left cervical vagus nerve during exposure to conditioned cues signals the brain to store new memories just as epinephrine or emotional arousal would do, but bypasses the peripheral sympathetic "fight-or-flight" response. CONCLUSIONS: In support of this hypothesis, we have found that VNS accelerates extinction and prevents reinstatement of conditioned fear in rats. Finally, we propose future studies targeting the optimization of stimulation parameters and the search for biomarkers of VNS effectiveness that may improve exposure therapy outcomes.

The M-Maze task: An automated method for studying fear memory in rats exposed to protracted aversive conditioning.

BACKGROUND: Fear conditioning (FC) in rodents is the most used animal model to investigate the neurobiology of posttraumatic stress disorder (PTSD). Although research using FC has generated a better understanding of fear memories, studies often rely on mild or moderate FC training and behavioral analysis generally focuses on measuring freezing responses within few test sessions. NEW METHOD: We introduce the M-Maze task, a system that measures extinction of conditioned fear using suppression of operant behavior. The apparatus consists of an M-shaped maze where rats are trained to alternate nose poking at two pellet dispensers. Proximity sensors measure the animal's locomotion, as well as the latencies and number of operant behaviors. Here we also describe the protracted aversive conditioning (PAC), a rat model of severe fear that induces resistant extinction following a 4-day conditioning protocol that combines delay, unpredictable, and short- and long-trace conditioning. RESULTS: An intense one-day auditory FC protocol induced a sharp elevation in transit time and suppression of nose pokes by conditioned cues, but in contrast to what is found in PTSD patients, fear extinction was rapidly observed. On the other hand, PAC alone or in combination with exposure to single prolonged stress induced persistent extinction impairments in M-Maze tests, as well as enhanced anxiety, and social withdrawal. COMPARISON WITH OTHER EXISTING METHODS: The M-Maze task is fully automated and allows multiple animals to be tested simultaneously in long-term experiments. Moreover, PAC training can be an alternative approach to study extinction-resistant fear. CONCLUSIONS: The M-Maze task allows rapid and unbiased measurements of fear-induced suppression. We suggest that long-term assessment of extinction impairments would lead to a better understanding of the neurobiology of persistent fear and the screening for new therapies.

Using the Single Prolonged Stress Model to Examine the Pathophysiology of PTSD.

The endurance of memories of emotionally arousing events serves the adaptive role of minimizing future exposure to danger and reinforcing rewarding behaviors. However, following a traumatic event, a subset of individuals suffers from persistent pathological symptoms such as those seen in posttraumatic stress disorder (PTSD). Despite the availability of pharmacological treatments and evidence-based cognitive behavioral therapy, a considerable number of PTSD patients do not respond to the treatment, or show partial remission and relapse of the symptoms. In controlled laboratory studies, PTSD patients show deficient ability to extinguish conditioned fear. Failure to extinguish learned fear could be responsible for the persistence of PTSD symptoms such as elevated anxiety, arousal, and avoidance. It may also explain the high non-response and dropout rates seen during treatment. Animal models are useful for understanding the pathophysiology of the disorder and the development of new treatments. This review examines studies in a rodent model of PTSD with the goal of identifying behavioral and physiological factors that predispose individuals to PTSD symptoms. Single prolonged stress (SPS) is a frequently used rat model of PTSD that involves exposure to several successive stressors. SPS rats show PTSD-like symptoms, including impaired extinction of conditioned fear. Since its development by the Liberzon lab in 1997, the SPS model has been referred to by more than 200 published papers. Here we consider the findings of these studies and unresolved questions that may be investigated using the model.

Acquisition and expression of fear memories are distinctly modulated along the dorsolateral periaqueductal gray axis of rats exposed to predator odor.

The dorsolateral region of the midbrain periaqueductal gray (dlPAG) modulates both innate and conditioned fear responses. However, the contribution of the rostrocaudal portions of the dlPAG to defense reactions and aversive memories remains unclear. Here, we sought to investigate the effects of N-methyl-d-aspartate (NMDA) receptor blockade within rostral or caudal dlPAG of rats exposed to innate and learned fear to cat odor. For this, adult male Wistar rats were microinjected with the NMDA antagonist D-2-amino-5-phosphono-pentanoate (AP5; 3 or 6nmol/0.2µl) into the rostral or caudal dlPAG before and after the exposure to the cat odor or to the context paired with the predator odor. The results demonstrated that cat odor exposure induced unconditioned defensive behaviors as well as contextual fear. AP5 microinjected in the rostral dlPAG reduced the defensive responses to cat odor and impaired the acquisition, but not consolidation of contextual fear. On the other hand, AP5 infused within the caudal dlPAG promoted long-lasting reduction of contextual fear expression. Altogether, our data suggest that NMDA receptors mediate a functional dichotomy in the rostrocaudal axis of dlPAG regulating unconditioned and conditioned defensive reactions to predatory cues.

Paradoxical mineralocorticoid receptor-mediated effect in fear memory encoding and expression of rats submitted to an olfactory fear conditioning task.

There is general agreement that the substantial modification in memory and motivational states exerted by corticosteroids after a traumatic experience is mediated in complementary manner by the mineralocorticoid (MR) and glucocorticoid (GR) receptors. Here we tested the hypothesis that pharmacological manipulation of MR activity would affect behavioral strategy and information storage in an olfactory fear conditioning (OFC) task. Male Wistar rats were submitted to the OFC with different training intensities. We observed that following high intensity OFC acquisition, a set of defensive coping strategies, which includes avoidance and risk assessment behaviors, was elicited when subjects were exposed to the conditioned stimulus (CS) 48 h later. In addition, following either OFC acquisition or retrieval (CS-I test) a profound corticosterone secretion was also detected. Systemic administration of the MR antagonist spironolactone altered the behavioral coping style irrespective the antagonist was administered 60 min prior to the acquisition or before the retrieval session. Surprisingly, the MR agonist fludrocortisone given 60 min prior to acquisition or retrieval of OFC had similar effects as the antagonist. In addition, post-training administration of fludrocortisone, following a weak training procedure, facilitated the consolidation of OFC. Fludrocortisone rather than spironolactone reduced serum corticosterone levels, suggesting that, at least in part, the effects of the MR agonist may derive from additional GR-mediated HPA-axis suppression. In conclusion, the present study suggests the involvement of the MR in the fine-tuning of behavioral adaptation necessary for optimal information storage and expression, as revealed by the marked alterations in the risk assessment behavior.

The usefulness of olfactory fear conditioning for the study of early emotional and cognitive impairment in reserpine model.

Due to the ability for depleting neuronal storages of monoamines, the reserpine model is a suitable approach for the investigation of the neurobiology of neurodegenerative diseases. However, the behavioral effects of low doses of reserpine are not always detected by classic animal tests of cognition, emotion, and sensory ability. In this study, the effects of reserpine (0.5-1.0mg/kg) were evaluated in olfactory fear conditioning, inhibitory avoidance, open-field, elevated plus-maze, and olfactory discrimination. Possible protective effects were also investigated. We found that single administration of reserpine impaired the acquisition of olfactory fear conditioning (in both doses) as well as olfactory discrimination (in the higher dose), while no effects were seen in all other tests. Additionally, we demonstrated that prior exposure to environmental enrichment prevented effects of reserpine in animals tested in olfactory fear conditioning. Altogether, these findings suggest that a combined cognitive, emotional and sensory-dependent task would be more sensitive to the effects of the reserpine model. In addition, the present data support the environmental enrichment as an useful approach for the study of resilience mechanisms in neurodegenerative processes.

Infusion of cannabidiol into infralimbic cortex facilitates fear extinction via CB1 receptors.

Previous studies have implicated cannabinoids in extinction of conditioned fear. We have recently showed that intraventricular infusion of the phytocannabinoid cannabidiol (CBD) facilitates fear extinction, but the brain regions underlying this effect remained unknown. Here we demonstrate that repeated microinjections of CBD into the infralimbic cortex (IL) facilitated fear extinction, as indicated by reduced levels of freezing during extinction test. Systemic administration of the CB1 receptor antagonist rimonabant blocked the effects of intra-IL CBD, suggesting that CBD acts through CB1 receptors to facilitate fear extinction. Our findings suggest a potential therapeutic use of CBD for extinction-based therapies of aversive memories in humans.

Systemic or intra-prelimbic cortex infusion of prazosin impairs fear memory reconsolidation.

The alpha-1 adrenergic antagonist prazosin has been used to alleviate the symptoms of PTSD, but the mechanism remains unclear. One possibility is that prazosin may disrupt fear memory reconsolidation, leading to attenuation of fear responses. To test this hypothesis, we administered a single systemic injection of prazosin during the reconsolidation of olfactory fear conditioning in rats. We found that a post-retrieval injection of prazosin disrupted subsequent retrieval of fear. Similarly, intra-prelimbic cortex infusion of prazosin during the reconsolidation period also disrupted subsequent retrieval of fear. These findings suggest that fear memory undergoes reconsolidation through activation of alpha-1 adrenergic receptors in the prelimbic cortex.