Tone Fear Conditioning Research Articles

Effects of nociceptin/orphanin FQ in the acquisition of contextual and tone fear conditioning in rats.

Nociceptin, or orphanin FQ (N/OFQ), the endogenous ligand of NOP receptors, is known to regulate learning and memory processes. To verify the role of N/OFQ in the acquisition of contextual (CFC) and tone fear conditioning (TFC), Wistar male rats received intracerebroventricular injections of N/OFQ (0.1-5.0 nmol) before training, and were tested 24 and 48 hr later to access the freezing response to context and tone, respectively. The intermediate doses (1.0 and 2.5 nmol) impaired the CFC test, sparing TFC. The highest dose (5.0 nmol) reduced freezing during both tests, a result that may be due to nonspecific effects. The posttraining injection of N/OFQ (1 or 5 nmol) did not interfere with CFC and TFC, suggesting a specific effect of the peptide in acquisition processes. Moreover, the impairment observed with N/OFQ (1 nmol) in CFC cannot be attributed to a state-dependent learning because it was not reversed by its pretest administration. The data support the negative role of N/OFQ in the acquisition of aversively motivated tasks, which encompass a spatial component and depend on the hippocampus.

Oxidative Stress Induced by Intense and Exhaustive Exercise Impairs Murine Cognitive Function

It has been shown that exercise is helpful against brain disorders. However, this may not be true for intense exercise (IE). Because it is easy to misadjust exercise intensity with physical condition, it is essential to know the effects of IE on cognitive process because it may have important consequences on people skills and work skills. We investigated the effects of IE on male C57Bl/6 mice, 3-mo-old, undergoing 10 days of intense and exhaustive running program on cognition and its possible relationship with brain oxidative stress. Cognition was evaluated by three different cognitive tests: passive avoidance task, contextual fear conditioning, and tone fear conditioning, performed 24 h after the last exercise session. Brain oxidative stress was evaluated by lipid peroxidation and protein oxidation. There was a remarkable memory reduction of exercised animals in comparison with the control group, associated with increase in the brain oxidative stress, with no alterations in shock sensitivity, locomotion and anxiety parameters. Concurrent vitamin C and E supplementation fully prevented the memory decrement induced by IE and partially recovered both the increased the brain lipid peroxidation and the protein oxidation. In conclusion, IE-induces a high index of brain oxidative stress and impairs memory in murine model that was prevented by vitamin C and E supplementation.

Effects of brief and long maternal separations on the HPA axis activity and the performance of rats on context and tone fear conditioning

Previous studies show that early life events result in neurobehavioural alterations that may be either beneficial or detrimental to the stress response. Given the close relationship between corticosterone secretion and mnemonic processes, the purpose of the present study was to investigate the effects of brief (BMS, 15 min) and long maternal separations (LMS, 180 min) on memory tasks in adult rats, assessed by context and tone fear conditioning. At adulthood, males were evaluated for behavioural and hormonal reaction to the training environment, being tested for context fear conditioning; tone fear conditioning; and learning curve in the context fear conditioning, in which rats were daily re-exposed to the context, followed by a brief footshock and in the last day of the experiment (day 5) animals were exposed to the context. Corticosterone and ACTH plasma levels were determined in naïve rats (basal) or 5, 25 or 45 min after each test. Peak ACTH and corticosterone levels were similar among the groups after context fear conditioning; however, levels of CTL rats remained elevated for a longer time. In the learning curve of context fear conditioning, both BMS and LMS rats exhibited less freezing behaviour than CTL rats, without differences in hormone secretion. There was neither an association between activity of the HPA axis and performance on memory tasks nor different activational properties of the tasks on the HPA axis between BMS and LMS rats, i.e., both manipulations lead to similar performance in hippocampus-dependent and independent memory tasks.

Dissociated roles for the lateral and medial septum in elemental and contextual fear conditioning

Extensive evidence indicates that the septum plays a predominant role in fear learning, yet the direction of this control is still a matter of debate. Increasing data suggest that the medial (MS) and lateral septum (LS) would be differentially required in fear conditioning depending on whether a discrete conditional stimulus (CS) predicts, or not, the occurrence of an aversive unconditional stimulus (US). Here, using a tone CS-US pairing (predictive discrete CS, context in background) or unpairing (context in foreground) conditioning procedure, we show, in mice, that pretraining inactivation of the LS totally disrupted tone fear conditioning, which, otherwise, was spared by inactivation of the MS. Inactivating the LS also reduced foreground contextual fear conditioning, while sparing the higher level of conditioned freezing to the foreground (CS-US unpairing) than to the background context (CS-US pairing). In contrast, inactivation of the MS totally abolished this training-dependent level of contextual freezing. Interestingly, inactivation of the MS enhanced background contextual conditioning under the pairing condition, whereas it reduced foreground contextual conditioning under the unpairing condition. Hence, the present findings reveal a functional dissociation between the LS and the MS in Pavlovian fear conditioning depending on the predictive value of the discrete CS. While the requirement of the LS is crucial for the appropriate processing of the tone CS-US association, the MS is crucial for an appropriate processing of contextual cues as foreground or background information.

Upregulation of polysialylated neural cell adhesion molecule in the dorsal hippocampus after contextual fear conditioning is involved in long-term memory formation.

The role of the hippocampus in pavlovian fear conditioning is controversial. Although lesion and pharmacological inactivation studies have suggested a key role for the dorsal hippocampus in contextual fear conditioning, the involvement of the ventral part is still uncertain. Likewise, the debate is open with regard to the putative implication of each hippocampal subdivision in fear conditioning to a discrete conditioned stimulus. We explored the potential existence of dissociations occurring in the dorsal versus ventral hippocampus at the cellular level while dealing with either contextual or cued fear conditioning and focused in a molecular "signature" linked to structural plasticity, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). We found an upregulation of PSA-NCAM expression in the dorsal (but not ventral) dentate gyrus at 24 h after contextual (but not tone) fear conditioning. Specific removal of PSA through microinfusion of the enzyme endoneuraminidase-N in the dorsal (but not ventral) hippocampus reduced freezing responses to the conditioned context. Therefore, we present evidence for a specific role of PSA-NCAM in the dorsal hippocampus in the plasticity processes occurring during consolidation of the context representation after "standard" contextual fear conditioning. Interestingly, we also found that exposing animals just to the context induced an activation of PSA-NCAM in both dorsal and ventral dentate gyrus. Altogether, these findings highlighting the distinctive occurrence of these neuroplastic processes in the dorsal hippocampus during the standard contextual fear-conditioning task enlighten the ongoing debate about the involvement of these hippocampal subdivisions in pavlovian fear conditioning.

Extracellular Hippocampal Acetylcholine Level Controls Amygdala Function and Promotes Adaptive Conditioned Emotional Response

Ample data indicate that tone and contextual fear conditioning differentially require the amygdala and the hippocampus. However, mechanisms subserving the adaptive selection among environmental stimuli (discrete tone vs context) of those that best predict an aversive event are still elusive. Because the hippocampal cholinergic neurotransmission is thought to play a critical role in the coordination between different memory systems leading to the selection of appropriate behavioral strategies, we hypothesized that this cholinergic signal may control the competing acquisition of amygdala-mediated tone and contextual conditioning. Using pavlovian fear conditioning in mice, we first show a higher level of hippocampal acetylcholine release and a specific pattern of extracellular signal-regulated kinase 1/2 (ERK1/2) activation within the lateral (LA) and basolateral (BLA) amygdala under conditions in which the context is a better predictor than a discrete tone stimulus. Second, we demonstrate that levels of hippocampal cholinergic neurotransmission are causally related to the patterns of ERK1/2 activation in amygdala nuclei and actually determine the selection among the context or the simple tone the stimulus that best predicts the aversive event. Specifically, decreasing the hippocampal cholinergic signal not only impaired contextual conditioning but also mimicked conditioning to the discrete tone, both in terms of the behavioral outcome and the LA/BLA ERK1/2 activation pattern. Conversely, increasing this cholinergic signal not only disrupted tone conditioning but also promoted contextual fear conditioning. Hence, these findings highlight that hippocampal cholinergic neurotransmission controls amygdala function, thereby leading to the selection of relevant emotional information.

Behavioral effects of corpus callosum transection and environmental enrichment in adult rats

A common assumption about the corpus callosum transection (CCX) is that it only affects behaviors heavily relying on interhemispheric communication. However, cerebral laterality is ubiquitous across motor and perceptual, cognitive and emotional domains, and the corpus callosum is important for its establishment. Several recent studies showed that the partial denervation of the sensorimotor isocortex through CCX derepressed neural growth processes that were sensitive to motor demand (experience-dependent neural plasticity). We investigated whether the facilitatory effects of CCX on cortical neural plasticity, shaped by differential housing, extended beyond the motor domain. Adult rats were housed in enriched (EE), standard (SE) or impoverished environments (IE) for 10 weeks, that is, 2 weeks before they underwent CCX or sham surgery, and, then, 8 weeks throughout the experiments. After they recovered from surgery, the behavioral performance of rats was tested using open-field, spontaneous alternation in the T-maze, paw preference, Morris water maze, and tone fear conditioning. The results indicated that the effects of CCX and housing on open-field behavior were independent, with CCX increasing the time spent in the center of the field at the beginning of the observation (i.e., emotionality), and EE and IE increasing rearing (emotionality) and reducing teeth-chattering (habituation), respectively. CCX reduced the frequency of spontaneous alternation, denoting spatial working memory deficits, while housing did not influence this performance. Neither CCX, nor housing significantly affected paw preference lateralization, although CCX was associated with a leftward bias in paw preference. In the Morris water maze, housing had effects on spatial acquisition, while CCX reduced activity, without interfering with spatial memory. CCX did not influence tone fear conditioning, but context fear conditioning seemed to benefit from EE. We conclude that CCX in adult rats has subtle, but specific behavioral effects pertaining to emotionality, spatial working memory, and, possibly, aversively motivated exploration, and these effects are either independent or only peripherally interact with the effects of housing.

A mutant mouse with a highly specific contextual fear-conditioning deficit found in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen

Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has generated memory mutants in the mouse. We tested a group of N-ethyl-N-nitrosourea (ENU) mutagenized mice in the conditioned fear paradigm. We screened for both dominant and recessive mutations that caused impairments in contextual or tone fear conditioning. Heritability testing confirmed three fear conditioning mutants, i.e., Forgetful, Slowlearner, and Scatterbrain. All three have a learning or short-term memory deficit in contextual fear conditioning. Forgetful was further characterized and showed a highly specific phenotype. The contextual fear-conditioning deficit was apparent when Forgetful was trained with tone-shock pairings, but not when trained with shock alone. The deficit was not due to changes in shock sensitivity or anxiety. Forgetful was not impaired in two other memory tests (hidden platform version of Morris water maze and object recognition). Our data show that a mutagenesis screen can generate mutant mice with highly specific memory phenotypes that can supplement existing mice with targeted mutations. Mapping of Slowlearner found linkage to a region of chromosome 12 (LOD score of 6.5 close to D12Mit171), which suggests that ENU mutants should enable the positional cloning of genes involved in memory formation.

WIN 55212-2 impairs contextual fear conditioning through the activation of CB1 cannabinoid receptors

The memory deficits induced by cannabinoid agonists have been found in several behavioral paradigms. Nevertheless, there is evidence that not all types of memory are impaired after cannabinoid administration. The aim of this study was to investigate whether the cannabinoid agonist WIN 55212-2 (WIN) is able to influence the acquisition of fear conditioning using tone and contextual versions. For tone-fear conditioning, male Wistar rats were placed in the conditioning chamber and after 3 min, a sound (CS) was presented for 10 s that terminated with a 1-s electric footshock (1.5 mA). For contextual-fear conditioning, a similar procedure was used but no sound was presented. Twenty-four hours after, the animals were re-exposed to the respective CS (tone or conditioning chamber) and the freezing behavior was registered. A subsequent experiment investigated a possible state-dependent effect of WIN by administering WIN or control solution 30 min before conditioning and before testing. WIN (2.5 and 5.0 mg/kg) administered i.p. 30 min before impaired contextual fear conditioning but did not modify the freezing behavior elicited by tone presentation. These animals did not show any state-dependent effects of WIN. Further, the impaired contextual conditioning was prevented by preadministration of SR141716A (1.0 mg/kg, i.p.) or SR147778 (1.0 mg/kg, i.p.), selective cannabinoid CB1 receptor antagonists. The present findings highlight that cannabinoid agonists effects are selective for the hippocampus-dependent aversive memories in rats. This effect appears to be related to the activation of CB1 cannabinoid receptors and confirms that cannabinoids might provide a novel approach for the treatment of unpleasant memories.

Behavioral changes resulting from the administration of cycloheximide in the pilocarpine model of epilepsy

Cycloheximide influences synaptic reorganization resulting from pilocarpine-induced status epilepticus (SE). To investigate the possible behavioral consequences of this effect, we subjected animals to pilocarpine-induced SE either in the absence (Pilo group) or presence of cycloheximide (Chx group). Animals were further divided regarding the occurrence of spontaneous recurrent seizures (SRS). Two months after SE induction animals were exposed to different behavioral tests. Age-matched naïve animals were used as controls. All epileptic groups showed a significantly diminished freezing time in contextual and tone fear conditioning, performed poorly in the Morris water maze and present less seconds in immobility position as compared to controls. Only Pilo animals explored more extensively the open arms of the elevated plus maze and showed increased in horizontal exploratory activity in the open field as compared to controls. With the exception of Pilo animals without recorded SRS, all other groups had extensive tissue shrinkage in central nucleus of the amygdala as compared to controls. Cycloheximide-treated animals differed from Pilo animals in the extent of hilar loss and supragranular mossy fiber sprouting as well as tissue shrinkage in the dorsal hippocampus. Despite the histological differences seen in the dorsal hippocampus between experimental groups, no differences were encountered in the cognitive tests used to evaluate dorsal hippocampal function. The encountered histological differences between Chx and Pilo animals, however, might underlie the different emotional responses between the two groups.

Effects of pre- or post-training entorhinal cortex AP5 injection on fear conditioning

Fear conditioning is one of the most studied paradigms to assess the neural basis of emotional memory. The circuitry involves NMDA receptor activation in the amygdala and, in the case of contextual conditioning, in the hippocampus. Entorhinal cortex is one of the major input/output structures to the hippocampus and also projects to the amygdala, both through glutamatergic transmission. Other learning tasks involving hippocampus and amygdala, such as inhibitory avoidance, require entorhinal cortex during acquisition and consolidation. However, the involvement of NMDA receptors mediated transmission in entorhinal cortex in fear conditioning acquisition and consolidation is not clear. To investigate that issue, rats were trained in fear conditioning to both contextual and tone conditioned stimulus. Immediately before, immediately, 30 or 90 min after training they received NMDA antagonist AP5 or saline injections bilaterally in the entorhinal cortex (AP—6.8 mm, L ± 5.0 mm DV—6.8 mm). Contextual fear conditioning was measured 24 h after training, and tone fear conditioning 48 h after training. AP5 injections selectively impaired contextual fear conditioning only when injected pre-training. Post-training injections had no effect. These findings suggest that entorhinal cortex NMDA receptors are necessary for acquisition, but not for consolidation, of contextual fear conditioning. On the other hand, both acquisition and consolidation of tone fear conditioning seem to be independent of NMDA receptors in the entorhinal cortex.

Lack of phenotype for LTP and fear conditioning learning in calpain 1 knock-out mice

We previously proposed the hypothesis that calpain activation played an important role in long-term potentiation (LTP) of synaptic transmission in hippocampus. Two forms of calpain are predominant in brain tissues, calpain 1 (μ-calpain), activated by micromolar calcium concentration and calpain 2 (m-calpain), activated by millimolar calcium concentration in vitro. In the present study, we tested the role of calpain 1 in LTP and in learning and memory using calpain 1 knock-out mice. Changes in learning and memory were assessed using both context and tone fear conditioning. No differences in freezing responses were observed between the knock-out and the wild-type animals during the acquisition phase of the training, eliminating the possibility that the knock-out animals could be differentially affected by the foot shock. Likewise, no differences in freezing responses elicited by either the context or the tone were observed during the retention phase. No differences in short-term potentiation (STP) or LTP were observed in hippocampal slices from the knock-out and matched wild-type mice. Several interpretations might explain these negative results. First, it is conceivable that calpain 2 plays a more dominant role in neurons, and that calpain 1 makes a minor contribution as opposed to its suspected predominant role in the hematopoietic system. Alternatively, it is conceivable that some as yet unknown compensatory mechanisms take effect, and that calpain 2 or another calpain isoform substitutes for the missing calpain 1.

Effects of dorsal striatum lesions in tone fear conditioning and contextual fear conditioning

It has been suggested that the striatum mediates hippocampus-independent memory tasks. Classical fear conditioning to a discrete stimulus such as a tone is not affected by hippocampal lesion, whereas contextual fear conditioning is an hippocampus dependent task. The purpose of the present study was to verify the effect of dorsal striatal lesions on tone and contextual fear conditioning. The lesioned rats were not impaired in contextual fear conditioning but in tone fear conditioning both electrolytically and neurotoxically lesioned animals showed less freezing compared with controls. The lesion effect was observed after a postoperative recovery period of 14 days but not after 2 months. The results support the hypothesis that the dorsal striatum is involved in hippocampus-independent memory tasks, but, in spite of this involvement, it does not seem to be a critical structure.

Improved reversal learning and altered fear conditioning in transgenic mice with regionally restricted p25 expression.

Cleavage of the cyclin-dependent kinase 5 activator p35 generates the protein fragment p25, which accumulates in the forebrain of patients with Alzheimer's disease. Although p25 expression has been suggested to affect learning and memory, this hypothesis has not been tested to date. To investigate the role of p25 in hippocampus-dependent learning and memory we have generated transgenic mice expressing p25 preferentially in postnatal forebrain. p25 expression was highest in hippocampus where it averaged approximately 33% of endogenous p35 expression. This low level of p25 expression did not seem to result in hyperphosphorylation of tau, but increased the phosphorylation of neurofilament M and enhanced the expression of tau protein. These molecular changes did not correlate with neurodegeneration or motor abnormalities. In the Morris water maze the p25 mutants were normal in learning an initial platform location, but surprisingly reversal learning was improved when the platform position was changed. The p25 mutants were normal in contextual fear conditioning. However, when trained with a tone presentation the mutants showed reduced contextual conditioning and enhanced tone fear conditioning. We conclude that low p25 expression has pleiotropic effects on learning and memory. As p25 expression can improve learning and memory, p25 formation could be a compensatory mechanism for learning and memory deficits in Alzheimer's disease.

Chasing "fear memories" to the cerebellum.

The cerebellum has long been known as a center of fine motor control and sensory-motor integration. It also is appreciated as essential for the acquisition and expression of conditioned eye-blink responses, a type of discrete sensory-motor learning (1, 2). Recently, the cerebellum also has been implicated in acquiring (3–5) and expressing (6) “emotional” associative learning, which is commonly believed to depend entirely on forebrain regions. Specifically, the learning and memories for an association between neutral and fear-eliciting stimuli, also known as “fear conditioning”, often are said to reside in the basolateral amygdala (ref. 7, for an alternative view see ref. 8). An elegant study by Sacchetti and colleagues in this issue of PNAS (9) adds a new dimension to the recently described role of the cerebellum in emotional learning and memory by revealing that the cerebellum is essential for consolidating memories for both tone and contextual fear conditioning for several days longer than the basolateral amygdala. Thus, their findings are not only relevant to the current debate regarding the sites of permanent storage of fear memories, but more importantly, they expand our knowledge of the topographical and chronological composition of the brain network(s) responsible for consolidating this type of memory.

Circadian modulation of learning and memory in fear-conditioned mice

Endogenous processes referred to as circadian oscillators generate many of the daily rhythms in physiology and behavior of a variety of animals including humans. We investigated the possible circadian regulation of acquisition, recall and extinction in two strains of mice (C-57/6J and C-3H). Mice were trained in either the day or night with a tone and context fear conditioning protocol. The mice were then tested over the course of several days for their ability to recall the training. When comparing the performance of animals in the day and night, the mice acquired the conditioning faster in the day than in the night. Furthermore, the recall for context and tone consistently peaked during the day for at least 3 days after training, irrespective of the time of training. Finally, the loss of this training (or extinction) exhibited a rhythm in that mice trained in night exhibited a greater degree of extinction than mice trained in the day. For all of these rhythms in acquisition, recall, and extinction the phase of the rhythm was controlled by the prior light–dark (LD) cycle. When we reversed the phase of the LD cycle, the phase of the rhythm also reversed. Importantly, all three of the rhythms also continued in constant darkness demonstrating the endogenous, and presumably circadian nature, of the rhythms.

Effects of the selective M1 muscarinic receptor antagonist dicyclomine on emotional memory.

The nonselective muscarinic antagonist scopolamine is known to impair the acquisition of some learning tasks such as inhibitory avoidance. There has been recent research into the effects of this drug in contextual fear conditioning and tone fear conditioning paradigms. The purpose of the present study was to assess the role of the selective M1 muscarinic antagonist dicyclomine in these paradigms and in the inhibitory avoidance test. Rats were administered different doses of dicyclomine or saline 30 min before acquisition training. The animals were tested 24 hr later, and it was observed that 16 mg/kg of dicyclomine impaired both contextual fear conditioning and inhibitory avoidance. However, dicyclomine (up to 64 mg/kg) did not affect tone fear conditioning. These results suggest that the selective M1 muscarinic antagonist dicyclomine differentially affects aversively motivated tasks known to be dependent on hippocampal integrity (such as contextual fear conditioning and inhibitory avoidance) but does not affect similar hippocampus-independent tasks.