Functional organization of the midbrain periaqueductal gray for regulating aversive memory formation.

Innately aversive experiences produce rapid defensive responses and powerful emotional memories. The midbrain periaqueductal gray (PAG) drives defensive behaviors through projections to brainstem motor control centers, but the PAG has also been implicated in aversive learning, receives information from aversive-signaling sensory systems and sends ascending projections to the thalamus as well as other forebrain structures which could control learning and memory. Here we sought to identify PAG subregions and cell types which instruct memory formation in response to aversive events. We found that optogenetic inhibition of neurons in the dorsolateral subregion of the PAG (dlPAG), but not the ventrolateral PAG (vlPAG), during an aversive event reduced memory formation. Furthermore, inhibition of a specific population of thalamus projecting dlPAG neurons projecting to the anterior paraventricular thalamus (aPVT) reduced aversive learning, but had no effect on the expression of previously learned defensive behaviors. By contrast, inactivation of dlPAG neurons which project to the posterior PVT (pPVT) or centromedial intralaminar thalamic nucleus (CM) had no effect on learning. These results reveal specific subregions and cell types within PAG responsible for its learning related functions.

Defensive behaviors and brain regional activation changes in rats confronting a snake.

In the present study, we examined behavioral and brain regional activation changes of rats). To a nonmammalian predator, a wild rattler snake (Crotalus durissus terrificus). Accordingly, during snake threat, rat subjects showed a striking and highly significant behavioral response of freezing, stretch attend, and, especially, spatial avoidance of this threat. The brain regional activation patterns for these rats were in broad outline similar to those of rats encountering other predator threats, showing Fos activation of sites in the amygdala, hypothalamus, and periaqueductal gray matter. In the amygdala, only the lateral nucleus showed significant activation, although the medial nucleus, highly responsive to olfaction, also showed higher activation. Importantly, the hypothalamus, in particular, was somewhat different, with significant Fos increases in the anterior and central parts of the ventromedial hypothalamic nucleus (VMH), in contrast to patterns of enhanced Fos expression in the dorsomedial VMH to cat predators, and in the ventrolateral VMH to an attacking conspecific. In addition, the juxtodorsalmedial region of the lateral hypothalamus showed enhanced Fos activation, where inputs from the septo-hippocampal system may suggest the potential involvement of hippocampal boundary cells in the very strong spatial avoidance of the snake and the area it occupied. Notably, these two hypothalamic paths appear to merge into the dorsomedial part of the dorsal premammillary nucleus and dorsomedial and lateral parts of the periaqueductal gray, all of which present significant increases in Fos expression and are likely to be critical for the expression of defensive behaviors in responses to the snake threat.

Projection from the Anterior Cingulate Cortex to the Lateral Part of Mediodorsal Thalamus Modulates Vicarious Freezing Behavior.

Emotional contagion, a primary form of empathy, is present in rodents. Among emotional contagion behaviors, social transmission of fear is the most studied. Here, we modified a paradigm used in previous studies to more robustly assess the social transmission of fear in rats that experienced foot-shock. We used resting-state functional magnetic resonance imaging to show that foot-shock experience enhances the regional connectivity of the anterior cingulate cortex (ACC). We found that lesioning the ACC specifically attenuated the vicarious freezing behavior of foot-shock-experienced observer rats. Furthermore, ablation of projections from the ACC to the mediodorsal thalamus (MDL) bilaterally delayed the vicarious freezing responses, and activation of these projections decreased the vicarious freezing responses. Overall, our results demonstrate that, in rats, the ACC modulates vicarious freezing behavior via a projection to the MDL and provide clues to understanding the mechanisms underlying empathic behavior in humans.

Conditioned stimulus presentations alter anxiety level in fear-conditioned mice.

It is generally believed that fear is rapidly triggered by a distinct cue while anxiety onset is less precise and not associated with a distinct cue. Although it has been claimed that both processes can be measured with certain independence of each other, it is unclear how exactly they differ. In this study, we measured anxiety in mice that received discriminative fear conditioning using behavioral, heart rate and calcium (Ca2+) responses in the ventral hippocampal CA1 (vCA1) neurons. We found that the occurrence of fear significantly interfered with anxiety measurements under various conditions. Diazepam reduced basal anxiety level but had no effect during the presentation of conditioned stimulus (CS). Injection of an inhibitory peptide of PKMzeta (ZIP) into the basolateral amygdala almost entirely abolished CS-triggered fear expression and reduced anxiety to basal level. Heart rate measures suggested a small reduction in anxiety during CS-. Calcium responses in the lateral hypothalamus-projecting vCA1 neurons showed a steady decay during CS suggesting a reduced anxiety. Thus, under our experimental conditions, CS presentations likely reduce anxiety level in the fear-conditioned mice.

Electrolytic post-training lesions of the bed nucleus of the stria terminalis block startle potentiation in a cued fear conditioning procedure.

Existing neuroanatomical models argue that the bed nucleus of the stria terminalis (BST) principally mediates sustained, long-lasting fear or anxiety responses, but not shorter, phasic fear responses, although recent studies paint a more complex picture. In the current study, we evaluated the effect of post-training electrolytic BST lesions in a cued fear conditioning protocol with relatively short (10 s) tones. We hypothesized that the BST would not play a crucial role in the expression of fear upon re-exposure to the conditioned tones. Tone fear memory was primarily assessed through fear-potentiated startle. In addition, freezing measurements were obtained throughout the test sessions. In a series of three experiments, we explored the effects of BST lesions, taking into consideration contextual influences on cued fear expression (using (dis)similar training and test contexts) and temporal involvement of the BST in the consolidation of fear learning (lesion induction 3 or 27 h after fear conditioning). In all three experiments, we found that post-training electrolytic lesions of the BST significantly reduced fear-potentiated startle, implying a deficit in differentiation between tone and context. These results are surprising and challenge the general consensus on the lack of BST involvement in cued fear. We discuss several alternative explanations that may account for these unexpected findings.

Response-specific sex difference in the retention of fear extinction.

Fear conditioning studies in rodents allow us to assess vulnerability factors which might underlie fear-based psychopathology such as post-traumatic stress disorder (PTSD). Despite PTSD being more prevalent in females than males, very few fear conditioning studies in rodents have tested females. Our study assessed fear conditioning and extinction in male and female rats using both fear-potentiated startle and freezing behavior as measures. Rats were trained to fear cues that predicted the occurrence of shock and then subsequently exposed to an extinction training procedure where the cue was presented repeatedly in the absence of shock. Retention of the extinction memory was assessed the next day. Our results showed that females exhibited less retention of fear extinction, but only when measured by fear-potentiated startle. Our results highlight the importance of using multiple indices of fear behavior, particularly when comparing sexes on measures of extinction learning.

Auditory conditioned stimulus presentation during NREM sleep impairs fear memory in mice.

Externally manipulating memories by presenting conditioned stimuli (CS) during sleep is a new approach to investigating memory processing during sleep. However, whether presenting a CS during REM or NREM sleep enhances or extinguishes fear memory has not been clearly delineated. In this study, mice underwent trace fear conditioning consisting of an auditory CS paired with a foot shock, and the auditory CS was re-presented during subsequent REM or NREM sleep. Mice that received auditory cueing during NREM but not REM sleep showed impaired fear memory upon later presentation of the auditory CS. These findings have implications for the use of cueing during sleep and advance our understanding of the role of REM and NREM sleep in memory consolidation.

Acoustic startle response in rats predicts inter-individual variation in fear extinction.

Although a large portion of the population is exposed to a traumatic event at some point, only a small percentage of the population develops post-traumatic stress disorder (PTSD), suggesting the presence of predisposing factors. Abnormal acoustic startle response (ASR) has been shown to be associated with PTSD, implicating it as a potential predictor of the development of PTSD-like behavior. Since poor extinction and retention of extinction learning are characteristic of PTSD patients, it is of interest to determine if abnormal ASR is predictive of development of such deficits. To determine whether baseline ASR has utility in predicting the development of PTSD-like behavior, the relationship between baseline ASR and freezing behavior following Pavlovian fear conditioning was examined in a group of adult, male Sprague-Dawley rats. Baseline acoustic startle response (ASR) was assessed preceding exposure to a Pavlovian fear conditioning paradigm where freezing behavior was measured during fear conditioning, extinction training, and extinction testing. Although there was no relationship between baseline ASR and fear memory following conditioning, rats with low baseline ASR had significantly lower magnitude of retention of the extinction memory than rats with high baseline ASR. The results suggest that baseline ASR has value as a predictive index of the development of a PTSD-like phenotype.

Secondary task performance during challenging walking tasks and freezing episodes in Parkinson's disease.

Parkinson's disease (PD) patients likely use attentional strategies to compensate for their gait deficits, which increases the cognitive challenge of walking. The interplay between cognitive functions and gait can be investigated by evaluating the subject's attendance to a secondary task during walking. We hypothesized that the ability to attend to a secondary task decreases during challenging walking conditions in PD, particularly during freezing of gait (FOG)-episodes. Twenty-nine PD patients and 14 age-matched controls performed a simple reaction task that involved squeezing a ball as fast as possible in response to an auditory stimulus. Participants performed this reaction task during four conditions: (1) walking at preferred speed; (2) walking with short steps at preferred speed; (3) walking with short steps, as rapidly as possible; (4) making rapid full turns. We used surface electromyography to determine reaction times, and a pressure sensor located within the ball to determine movement onset. Reaction times of PD patients were slower (on average by 42 ms) compared to controls, regardless of the walking task. In both groups, reaction times were significantly longer during the turning condition compared to all other conditions. FOG-episodes were most often seen during the turning condition. In PD patients, reaction times were significantly longer during FOG-episodes compared to trials without FOG. Our results suggest that turning requires more attentional resources than other walking tasks. The observation of delayed reaction times during FOG-episodes compared to trials without FOG suggests that freezers use additional resources to overcome their FOG-episodes.

Modification of hippocampal markers of synaptic plasticity by memantine in animal models of acute and repeated restraint stress: implications for memory and behavior.

Stress is any condition that impairs the balance of the organism physiologically or psychologically. The response to stress involves several neurohormonal consequences. Glutamate is the primary excitatory neurotransmitter in the central nervous system, and its release is increased by stress that predisposes to excitotoxicity in the brain. Memantine is an uncompetitive N-methyl D-aspartate glutamatergic receptors antagonist and has shown beneficial effect on cognitive function especially in Alzheimer's disease. The aim of the work was to investigate memantine effect on memory and behavior in animal models of acute and repeated restraint stress with the evaluation of serum markers of stress and the expression of hippocampal markers of synaptic plasticity. Forty-two male rats were divided into seven groups (six rats/group): control, acute restraint stress, acute restraint stress with Memantine, repeated restraint stress, repeated restraint stress with Memantine and Memantine groups (two subgroups as positive control). Spatial working memory and behavior were assessed by performance in Y-maze. We evaluated serum cortisol, tumor necrotic factor, interleukin-6 and hippocampal expression of brain-derived neurotrophic factor, synaptophysin and calcium-/calmodulin-dependent protein kinase II. Our results revealed that Memantine improved spatial working memory in repeated stress, decreased serum level of stress markers and modified the hippocampal synaptic plasticity markers in both patterns of stress exposure; in ARS, Memantine upregulated the expression of synaptophysin and brain-derived neurotrophic factor and downregulated the expression of calcium-/calmodulin-dependent protein kinase II, and in repeated restraint stress, it upregulated the expression of synaptophysin and downregulated calcium-/calmodulin-dependent protein kinase II expression.

Independent hypothalamic circuits for social and predator fear.

The neural circuits mediating fear to naturalistic threats are poorly understood. We found that functionally independent populations of neurons in the ventromedial hypothalamus (VMH), a region that has been implicated in feeding, sex and aggression, are essential for predator and social fear in mice. Our results establish a critical role for VMH in fear and have implications for selective intervention in pathological fear in humans.

Accumbal opioid receptors modulate cue competition in one-trial overshadowing.

The contribution of opioid receptors in the nucleus accumbens to contextual and auditory fear conditioning was examined. Impairment in contextual fear conditioning was found when training occurred under accumbal infusions of the opioid receptor agonist morphine in a dose-dependent and receptor specific fashion, only when shock onset coincided with auditory stimulus offset. Contextual fear conditioning was spared, however when the delivery of shock was not signalled by an auditory stimulus, the auditory stimulus was of low intensity (70dB), or an interval (10s or 30s) was interpolated between auditory stimulus offset and shock onset. These results provide evidence that opioid receptors in the nucleus accumbens regulate competition between contextual and discrete auditory stimuli for association formation.

Neurons activated during fear memory consolidation and reconsolidation are mapped to a common and new topography in the lateral amygdala.

A key question in neuroscience is how memory is selectively allocated to neural networks in the brain. This question remains a significant research challenge, in both rodent models and humans alike, because of the inherent difficulty in tracking and deciphering large, highly dimensional neuronal ensembles that support memory (i.e., the engram). In a previous study we showed that consolidation of a new fear memory is allocated to a common topography of amygdala neurons. When a consolidated memory is retrieved, it may enter a labile state, requiring reconsolidation for it to persist. What is not known is whether the original spatial allocation of a consolidated memory changes during reconsolidation. Knowledge about the spatial allocation of a memory, during consolidation and reconsolidation, provides fundamental insight into its core physical structure (i.e., the engram). Using design-based stereology, we operationally define reconsolidation by showing a nearly identical quantity of neurons in the dorsolateral amygdala (LAd) that expressed a plasticity-related protein, phosphorylated mitogen-activated protein kinase, following both memory acquisition and retrieval. Next, we confirm that Pavlovian fear conditioning recruits a stable, topographically organized population of activated neurons in the LAd. When the stored fear memory was briefly reactivated in the presence of the relevant conditioned stimulus, a similar topography of activated neurons was uncovered. In addition, we found evidence for activated neurons allocated to new regions of the LAd. These findings provide the first insight into the spatial allocation of a fear engram in the LAd, during its consolidation and reconsolidation phase.

Prepulse inhibition predicts working memory performance whilst startle habituation predicts spatial reference memory retention in C57BL/6 mice.

Prepulse inhibition (PPI) of the acoustic startle reflex refers to the attenuation of the startle response to an intense pulse stimulus when it is shortly preceded by a weak non-startling prepulse stimulus. It is a well-established high-throughput translational measure of pre-attentive sensory gating, and its impairment is detected in several neuropsychiatric diseases including schizophrenia. It has been hypothesized that PPI might be associated with, or predictive of, cognitive deficiency in such diseases, and therefore provide an efficient assay for screening drugs with potential pro-cognitive efficacy. Free from any predetermined disease model, the present study evaluated in a homogeneous cohort of inbred C57BL/6 mice the presence of a statistical link between PPI expression and cognitive performance. Performance indices in a spatial reference memory test and a working memory test conducted in the Morris water maze, and contextual fear conditioning were correlated against pre-existing baseline PPI expression. A specific correlative link between working memory and PPI induced by weak (but not strong) prepulse was revealed. In addition, a correlation between habituation of the startle reflex and reference memory was identified for the first time: a stronger overt habituation effect was associated with superior spatial search accuracy. The PPI paradigm thus provides two independent predictors of dissociable cognitive traits in normal C57BL/6 mice; and they might serve as potential markers for high-throughput evaluation of potential cognitive enhancers, especially in the context of schizophrenia where deficits in startle habituation and PPI co-exist.

Brain region-specific activity patterns after recent or remote memory retrieval of auditory conditioned fear.

Memory is thought to be sparsely encoded throughout multiple brain regions forming unique memory trace. Although evidence has established that the amygdala is a key brain site for memory storage and retrieval of auditory conditioned fear memory, it remains elusive whether the auditory brain regions may be involved in fear memory storage or retrieval. To investigate this possibility, we systematically imaged the brain activity patterns in the lateral amygdala, MGm/PIN, and AuV/TeA using activity-dependent induction of immediate early gene zif268 after recent and remote memory retrieval of auditory conditioned fear. Consistent with the critical role of the amygdala in fear memory, the zif268 activity in the lateral amygdala was significantly increased after both recent and remote memory retrieval. Interesting, however, the density of zif268 (+) neurons in both MGm/PIN and AuV/TeA, particularly in layers IV and VI, was increased only after remote but not recent fear memory retrieval compared to control groups. Further analysis of zif268 signals in AuV/TeA revealed that conditioned tone induced stronger zif268 induction compared to familiar tone in each individual zif268 (+) neuron after recent memory retrieval. Taken together, our results support that the lateral amygdala is a key brain site for permanent fear memory storage and suggest that MGm/PIN and AuV/TeA might play a role for remote memory storage or retrieval of auditory conditioned fear, or, alternatively, that these auditory brain regions might have a different way of processing for familiar or conditioned tone information at recent and remote time phases.

Post-exposure sleep deprivation facilitates correctly timed interactions between glucocorticoid and adrenergic systems, which attenuate traumatic stress responses.

Reliable evidence supports the role of sleep in learning and memory processes. In rodents, sleep deprivation (SD) negatively affects consolidation of hippocampus-dependent memories. As memory is integral to post-traumatic stress symptoms, the effects of post-exposure SD on various aspect of the response to stress in a controlled, prospective animal model of post-traumatic stress disorder (PTSD) were evaluated. 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. Urine samples were collected daily for corticosterone levels, and heart rate (HR) was also measured. Finally, the impact of manipulating the hypothalamus-pituitary-adrenal axis and adrenergic activity before SD was assessed. Mifepristone (MIFE) and epinephrine (EPI) were administered systemically 10-min post-stress exposure and behavioral responses and response to trauma reminder were measured on days 7-8. Hippocampal expression of glucocorticoid receptors (GRs) and morphological assessment of arborization and dendritic spines were subsequently evaluated. Post-exposure SD effectively ameliorated long-term, stress-induced, PTSD-like behavioral disruptions, reduced trauma reminder freezing responses, and decreased hippocampal expression of GR compared with exposed-untreated controls. Although urine corticosterone levels were significantly elevated 1 h after SD and the HR was attenuated, antagonizing GRs with MIFE or stimulation of adrenergic activity with EPI effectively abolished the effect of SD. MIFE- and EPI-treated animals clearly demonstrated significantly lower total dendritic length, fewer branches and lower spine density along dentate gyrus dendrites with increased levels of GR expression 8 days after exposure, as compared with exposed-SD animals. Intentional prevention of sleep in the early aftermath of stress exposure may well be beneficial in attenuating traumatic stress-related sequelae. Post-exposure SD may disrupt the consolidation of aversive or fearful memories by facilitating correctly timed interactions between glucocorticoid and adrenergic systems.

Temporal factors control hippocampal contributions to fear renewal after extinction.

Fear can be extinguished by repeated exposure to a cue that signals threat. However, extinction does not erase fear, as an extinguished cue presented in a context distinct from that of extinction results in renewed fear of that cue. The hippocampus, which is involved in the formation of contextual representations, is a natural candidate structure for investigations into the neural circuitry underlying fear renewal. Thus far, studies examining the necessity of the hippocampus for fear renewal have produced mixed results. We isolated the conditions under which the hippocampus may be required for renewal. Rats received lesions of the dorsal hippocampus either prior to tone fear conditioning or following extinction. Fear renewal was measured using discrete tone presentations or a long, continuous tone. The topography of fear responding at test was assessed by comparing "early" and "sustained" renewal, where early fear was determined by freezing to the first discrete tone or the equivalent initial segment of a continuous tone and sustained fear was determined by freezing averaged across all discrete tones or the entire continuous tone. We found that following pretraining damage of the hippocampus, early renewal remained intact regardless of lesion condition. However, sustained renewal only persisted in discrete, but not continuous, tone-tested animals. A more extensive analysis of the topography of fear responding revealed that the disruption of renewal was generated when the tone duration at test began to violate that used during extinction, suggesting that the hippocampus is sensitive to mismatches in CS-duration. Postextinction lesions resulted in an overall reduction of fear renewal. This pattern of results is consistent with those observed for contextual fear conditioning, wherein animals display a resistance to anterograde amnesia despite the presence of a strong retrograde amnesia for the same contextual information. Furthermore, the data support a role for the hippocampus in sustaining renewal when the CS duration at test does not match that used during extinction.

Towards chronic contextual conditioning in rats: the effects of different numbers of unpaired tone-shock presentations on freezing time and startle.

Contextual conditioning in rats is typically quantified using freezing time or startle amplitude. In this study, we combined both anxiety measures in one procedure and systematically examined the effect of training with 0, 5, 10 or 15 unpaired tone-shock (0.8 mA - 250 ms) presentations on the expression of contextual conditioning in a chronic protocol with two training and testing days. Such a chronic procedure may be valuable as a chronic anxiety model. Training with 5, 10 or 15 explicitly unpaired shocks resulted in significant contextual freezing. There was no significant increase in freezing time from post-test 1 to post-test 2 and there were no differences between the three shocked groups, implying that the different numbers of shocks did not affect the degree of contextual freezing, probably because the ceiling freezing value had already been reached. Surprisingly, we observed no potentiated startle in the conditioned context. To summarize, our protocol produced consistent contextual freezing over two testing days.

Maternal dietary loads of α-tocopherol differentially influence fear conditioning and spatial learning in adult offspring.

α-Tocopherol, the main component of vitamin E, is well known to be a radical scavenger, so an increased intake of vitamin E is recommended in complicated pregnancy, to prevent possible fetus damage by free radical. In a previous work, we found that maternal α-tocopherol supplementation affects PKC-mediated cellular signaling and hippocampal synaptic plasticity in developing brain; the latter effect persists in adulthood. Here, adult rats maternally exposed to supranutritional doses of α-tocopherol were evaluated for Contextual Fear Conditioning and spatial learning in Morris Water Maze, two different hippocampus-dependent learning tasks. Moreover, anxiety, spontaneous activity, and explorative drive were also evaluated as factors potentially affecting learning performance. Treated rats showed a different behavior with respect to controls: performance in Contextual Fear Conditioning was improved, while spatial learning tested in Morris Water Maze, was impaired. The improvement of fear response was not ascribable to differences in anxiety level and/or spontaneous activity; thus it appears to be a specific effect of α-tocopherol overloading during brain development. On the contrary, the impaired performance in Morris Water Maze exhibited by treated rats can be in part explained by their enhanced explorative drive. Although extrapolation from rats to humans is difficult, a caveat in assuming supranutritional doses of vitamin E in pregnancy arises from this study.

A block to pre-prepared movement in gait freezing, relieved by pedunculopontine nucleus stimulation.

Gait freezing and postural instability are disabling features of Parkinsonian disorders, treatable with pedunculopontine nucleus stimulation. Both features are considered deficits of proximal and axial musculature, innervated predominantly by reticulospinal pathways and tend to manifest when gait and posture require adjustment. Adjustments to gait and posture are amenable to pre-preparation and rapid triggered release. Experimentally, such accelerated release can be elicited by loud auditory stimuli--a phenomenon known as 'StartReact'. We observed StartReact in healthy and Parkinsonian controls. However, StartReact was absent in Parkinsonian patients with severe gait freezing and postural instability. Pedunculopontine nucleus stimulation restored StartReact proximally and proximal reaction times to loud stimuli correlated with gait and postural disturbance. These findings suggest a relative block to triggered, pre-prepared movement in gait freezing and postural instability, relieved by pedunculopontine nucleus stimulation.