Effects of prolonged exposure to context following contextual fear conditioning on synaptic properties in rat hippocampal slices

Acute stressful events enhance plasma corticosterone release and profoundly affect synaptic functions, which are involved in the development of stress-related cognitive and mental disorders. However, how exposure to stressful context immediately after str

Involvement of the insular cortex in the consolidation and expression of contextual fear conditioning

The insular cortex (IC) has been reported to be involved in the modulation of memory and autonomic and defensive responses. However, there is conflicting evidence about the role of the IC in fear conditioning. To explore the IC involvement in both behavioral and autonomic responses induced by contextual fear conditioning, we evaluated the effects of the reversible inhibition of the IC neurotransmission through bilateral microinjections of the non-selective synapse blocker CoCl2 (1 mm) 10 min before or immediately after the conditioning session or 10 min before re-exposure to the aversive context. In the conditioning session, rats were exposed to a footshock chamber (context) and footshocks were used as the unconditioned stimulus. Forty-eight hours later, the animals were re-exposed to the aversive context for 10 min, but no shock was given. Behavioral (freezing) as well as cardiovascular (arterial pressure and heart rate increases) responses induced by re-exposure to the aversive context were analysed. It was observed that the local IC neurotransmission inhibition attenuated freezing and the mean arterial pressure and heart rate increase of the groups that received the CoCl2 either immediately after conditioning or 10 min before re-exposure to the aversive context, but not when the CoCl2 was injected before the conditioning session. These findings suggest the involvement of the IC in the consolidation and expression of contextual aversive memory. However, the IC does not seem to be essential for the acquisition of memory associated with aversive context. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Cannabidiol injected into the bed nucleus of the stria terminalis reduces the expression of contextual fear conditioning via 5-HT1A receptors

Systemic administration of cannabidiol (CBD) attenuates cardiovascular and behavioral changes induced by re-exposure to a context that had been previously paired with footshocks. Previous results from our group using cFos immunohistochemistry suggested that the bed nucleus of the stria terminalis (BNST) is involved in this effect. The mechanisms of CBD effects are still poorly understood, but could involve 5-HT1A receptor activation. Thus, the present work investigated if CBD administration into the BNST would attenuate the expression of contextual fear conditioning and if this effect would involve the activation of 5-HT1A receptors. Male Wistar rats with cannulae bilaterally implanted into the BNST were submitted to a 10 min conditioning session (six footshocks, 1.5 mA/3 s). Twenty-four hours later freezing and cardiovascular responses (mean arterial pressure and heart rate) to the conditioning box were measured for 10 min. CBD (15, 30 or 60 nmol) or vehicle was administered 10 min before the re-exposure to the aversive context. The second experiment was similar to the first one except that animals received microinjections of the 5-HT1A receptor antagonist WAY100635 (0.37 nmol) 5 min before CBD (30 nmol) treatment. The results showed that CBD (30 and 60 nmol) treatment significantly reduced the freezing and attenuated the cardiovascular responses induced by re-exposure to the aversive context. Moreover, WAY100635 by itself did not change the cardiovascular and behavioral response to context, but blocked the CBD effects. These results suggest that CBD can act in the BNST to attenuate aversive conditioning responses and this effect seems to involve 5-HT1A receptor-mediated neurotransmission.

Spectral characteristics of the hippocampal LFP during contextual fear conditioning

OBJECTIVE: The hippocampus has an important role in the acquisition and recall of aversive memories. The objective of this study was to investigate the relationship among hippocampal rhythms. METHODS: Microeletrodes arrays were implanted in the hippocampus of Wistar rats. The animals were trained and tested in a contextual fear conditioning task. The training consisted in applying shocks in the legs. The memory test was performed 1 day (recent memory) or 18 days (remote memory) after training. We proposed a measure based on the FFT power spectrum, denominated "delta-theta ratio", to characterize the different behaviors (active exploration and freezing) and the memories types. RESULTS: The delta-theta ratio was able to distinguish recent and remote memories. In this study, the ratio for the 18-day group was smaller than for the 1-day group. Moreover, this measure was useful to distinguish the different behavior states active exploration and freezing. CONCLUSIONS: The results suggest delta-theta oscillations could reflect the demands on information processing during recent and remote memory recalls.

Basolateral amygdala is not critical for cognitive memory of contextual fear conditioning

Evidence that lesions of the basolateral amygdala complex (BLC) impair memory for fear conditioning in rats, measured by lack of “freezing” behavior in the presence of cues previously paired with footshocks, has suggested that the BLC may be a critical locus for the memory of fear conditioning. However, evidence that BLC lesions may impair unlearned as well as conditioned freezing makes it difficult to interpret the findings of studies assessing conditioned fear with freezing. The present study investigated whether such lesions prevent the expression of several measures of memory for contextual fear conditioning in addition to freezing. On day 1, rats with sham lesions or BLC lesions explored a Y maze. The BLC-lesioned rats (BLC rats) displayed a greater exploratory activity. On day 2, each of the rats was placed in the “shock” arm of the maze, and all of the sham and half of the BLC rats received footshocks. A 24-hr retention test assessed the freezing, time spent per arm, entries per arm, and initial entry into the shock arm. As previously reported, shocked BLC rats displayed little freezing. However, the other measures indicated that the shocked BLC rats remembered the fear conditioning. They entered less readily and less often and spent less time in the shock arm than did the control nonshocked BLC rats. Compared with the sham rats, the shocked BLC rats entered more quickly and more often and spent more time in the shock arm. These findings indicate that an intact BLC is not essential for the formation and expression of long-term cognitive/explicit memory of contextual fear conditioning.

Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein δ

CCAAT/enhancer binding protein δ (C/EBPδ) is a transcriptional regulator implicated in the hepatic acute phase response and in adipogenic and myeloid cell differentiation. We found that C/EBPδ is widely expressed in the peripheral and central nervous systems, including neurons of the hippocampal formation, indicating a role in neural functions. To examine the role of C/EBPδ in vivo, we generated mice with a targeted deletion of the C/EBPδ gene. This mutation does not interfere with normal embryonic and postnatal development. Performance in a battery of behavioral tests indicates that basic neurological functions are normal. Furthermore, performance in a Morris water maze task suggests that C/EBPδ mutant mice have normal spatial learning. However, in the contextual and auditory-cue-conditioned fear task, C/EBPδ null mice displayed significantly more conditioned freezing to the test context than did wild-type controls, but equivalent conditioning to the auditory cue. These data demonstrate a selectively enhanced contextual fear response in mice carrying a targeted genomic mutation and implicate C/EBPδ in the regulation of a specific type of learning and memory.

Contextual, but not auditory, fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats

The basolateral amygdala complex (BLA) is involved in acquisition of contextual and auditory fear conditioning. However, the BLA is not a single structure but comprises a group of nuclei, including the lateral (LA), basal (BA) and accessory basal (AB) nuclei. While it is consensual that the LA is critical for auditory fear conditioning, there is controversy on the participation of the BA in fear conditioning. Hodological and neurophysiological findings suggest that each of these nuclei processes distinct information in parallel; the BA would deal with polymodal or contextual representations, and the LA would process unimodal or elemental representations. Thus, it seems plausible to hypothesize that the BA is required for contextual, but not auditory, fear conditioning. This hypothesis was evaluated in Wistar rats submitted to multiple-site ibotenate-induced damage restricted to the BA and then exposed to a concurrent contextual and auditory fear conditioning training followed by separated contextual and auditory conditioning testing. Differing from electrolytic lesion and lidocaine inactivation, this surgical approach does not disturb fibers of passage originating in other brain areas, restricting damage to the aimed nucleus. Relative to the sham-operated controls, rats with selective damage to the BA exhibited disruption of performance in the contextual, but not the auditory, component of the task. Thus, while the BA seems required for contextual fear conditioning, it is not critical for both an auditory-US association, nor for the expression of the freezing response. (C) 2009 Elsevier Inc. All rights reserved.

The structure of Pavlovian fear conditioning in the amygdala

Do different brains forming a specific memory allocate the same groups of neurons to encode it? One way to test this question is to map neurons encoding the same memory and quantitatively compare their locations across individual brains. In a previous study, we used this strategy to uncover a common topography of neurons in the dorsolateral amygdala (LAd) that expressed a learning-induced and plasticity-related kinase (p42/44 mitogen-activated protein kinase; pMAPK), following auditory Pavlovian fear conditioning. In this series of experiments, we extend our initial findings to ask to what extent this functional topography depends upon intrinsic neuronal structure. We first showed that the majority (87 %) of pMAPK expression in the lateral amygdala was restricted to principal-type neurons. Next, we verified a neuroanatomical reference point for amygdala alignment using in vivo magnetic resonance imaging and in vitro morphometrics. We then determined that the topography of neurons encoding auditory fear conditioning was not exclusively governed by principal neuron cytoarchitecture. These data suggest that functional patterning of neurons undergoing plasticity in the amygdala following Pavlovian fear conditioning is specific to memory formation itself. Further, the spatial allocation of activated neurons in the LAd was specific to cued (auditory), but not contextual, fear conditioning. Spatial analyses conducted at another coronal plane revealed another spatial map unique to fear conditioning, providing additional evidence that the functional topography of fear memory storing cells in the LAd is non-random and stable. Overall, these data provide evidence for a spatial organizing principle governing the functional allocation of fear memory in the amygdala.

Acute restraint stress alters subsequent auditory fear conditioning in the rat

The neural basis of Pavlovian fear conditioning is well understood and depends upon neural processes within the amygdala. Stress is known to play a role in the modulation of fear-related behavior, including Pavlovian fear conditioning. Chronic restraint stress has been shown to enhance fear conditioning to discrete and contextual stimuli; however, the time course and extent of restraint that is essential for this modulation of fear learning remains unclear. Thus, we tested the extent to which a single exposure to 1 hr of restraint would alter subsequent auditory fear conditioning in rats.

Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

Both alpha(1)- and beta(1)-adrenoceptors in the bed nucleus of the stria terminalis are involved in the expression of conditioned contextual fear

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Both alpha(1)- and beta(1)-adrenoceptors in the bed nucleus of the stria terminalis are involved in the expression of conditioned contextual fear

BACKGROUND AND PURPOSE The bed nucleus of the stria terminalis (BNST) is a limbic structure that is involved in the expression of conditioned contextual fear. Among the numerous neural inputs to the BNST, noradrenergic synaptic terminals are prominent and some evidence suggests an activation of this noradrenergic neurotransmission in the BNST during aversive situations. Here, we have investigated the involvement of the BNST noradrenergic system in the modulation of behavioural and autonomic responses induced by conditioned contextual fear in rats. EXPERIMENTAL APPROACH Male Wistar rats with cannulae bilaterally implanted into the BNST were submitted to a 10 min conditioning session (6 footshocks, 1.5 ma/ 3 s). Twenty-four hours later freezing and autonomic responses (mean arterial pressure, heart rate and cutaneous temperature) to the conditioning box were measured for 10 min. The adrenoceptor antagonists were administered 10 min before the re-exposure to the aversive context. KEY RESULTS L-propranolol, a non-selective beta-adrenoceptor antagonist, and phentolamine, a non-selective a-adrenoceptor antagonist, reduced both freezing and autonomic responses induced by aversive context. Similar results were observed with CGP20712, a selective beta 1-adrenoceptor antagonist, and WB4101, a selective a1-antagonist, but not with ICI118,551, a selective beta 2-adrenoceptor antagonist or RX821002, a selective a2-antagonist. CONCLUSIONS AND IMPLICATIONS These findings support the idea that noradrenergic neurotransmission in the BNST via a1- and beta 1-adrenoceptors is involved in the expression of conditioned contextual fear.

Are two threats worse than one? The effects of face race and emotional expression on fear conditioning

Facial cues of racial outgroup or anger mediate fear learning that is resistant to extinction. Whether this resistance is potentiated if fear is conditioned to angry, other race faces has not been established. Two groups of Caucasian participants were conditioned with two happy and two angry face conditional stimuli (CSs). During acquisition, one happy and one angry face were paired with an aversive unconditional stimulus whereas the second happy and angry faces were presented alone. CS face race (Caucasian, African American) was varied between groups. During habituation, electrodermal responses were larger to angry faces regardless of race and declined less to other race faces. Extinction was immediate for Caucasian happy faces, delayed for angry faces regardless of race, and slowest for happy racial outgroup faces. Combining the facial cues of other race and anger does not enhance resistance to extinction of fear.

Mice selectively bred for High and Low fear behavior show differences in the number of pMAPK (p44/42 ERK) expressing neurons in lateral amygdala following Pavlovian fear conditioning

Individual variability in the acquisition, consolidation and extinction of conditioned fear potentially contributes to the development of fear pathology including posttraumatic stress disorder (PTSD). Pavlovian fear conditioning is a key tool for the study of fundamental aspects of fear learning. Here, we used a selected mouse line of High and Low Pavlovian conditioned fear created from an advanced intercrossed line (AIL) in order to begin to identify the cellular basis of phenotypic divergence in Pavlovian fear conditioning. We investigated whether phosphorylated MAPK (p44/42 ERK/MAPK), a protein kinase required in the amygdala for the acquisition and consolidation of Pavlovian fear memory, is differentially expressed following Pavlovian fear learning in the High and Low fear lines. We found that following Pavlovian auditory fear conditioning, High and Low line mice differ in the number of pMAPK-expressing neurons in the dorsal sub nucleus of the lateral amygdala (LAd). In contrast, this difference was not detected in the ventral medial (LAvm) or ventral lateral (LAvl) amygdala sub nuclei or in control animals. We propose that this apparent increase in plasticity at a known locus of fear memory acquisition and consolidation relates to intrinsic differences between the two fear phenotypes. These data provide important insights into the micronetwork mechanisms encoding phenotypic differences in fear. Understanding the circuit level cellular and molecular mechanisms that underlie individual variability in fear learning is critical for the development of effective treatment of fear-related illnesses such as PTSD.