Roles of testosterone and amygdaloid LTP induction in determining sex differences in fear memory magnitude

Women are thought to form fear memory more robust than men do and testosterone is suspected to play a role in determining such a sex difference. Mouse cued fear freezing was used to study the sex-related susceptibility and the role of testosterone in fear memory in humans. A 75-dB tone was found to provoke weak freezing, while 0.15-mA and 0.20-mA footshock caused strong freezing responses. No sex differences were noticed in the tone- or footshock-induced (naïve fear) freezing. Following the conditionings, female mice exhibited greater tone (cued fear)-induced freezing than did male mice. Nonetheless, female mice demonstrated indistinctive cued fear freezing across the estrous phases and ovariectomy did not affect such freezing in female mice. Orchidectomy enhanced the cued fear freezing in male mice. Systemic testosterone administrations and an intra-lateral nucleus of amygdala (LA) testosterone infusion diminished the cued fear freezing in orchidectomized male mice, while pretreatment with flutamide (Flu) eradicated these effects. Long-term potentiation (LTP) magnitude in LA has been known to correlate with the strength of the cued fear conditioning. We found that LA LTP magnitude was indeed greater in female than male mice. Orchidectomy enhanced LTP magnitude in males' LA, while ovariectomy decreased LTP magnitude in females' LA. Testosterone decreased LTP magnitude in orchidectomized males' LA and estradiol enhanced LTP magnitude in ovariectomized females' LA. Finally, male mice had lower LA GluR1 expression than female mice and orchidectomy enhanced the GluR1 expression in male mice. These findings, taken together, suggest that testosterone plays a critical role in rendering the sex differences in the cued fear freezing and LA LTP. Testosterone is negatively associated with LA LTP and the cued fear memory in male mice. However, ovarian hormones and LA LTP are loosely associated with the cued fear memory in female mice.     

Altered expression of synaptotagmin 13 mRNA in adult mouse brain after contextual fear conditioning

Contextual fear memory processing requires coordinated changes in neuronal activity and molecular networks within brain. A large number of fear memory-related genes, however, still remain to be identified. Synaptotagmin 13 (Syt13), an atypical member of synaptotagmin family, is highly expressed in brain, but its functional roles within brain have not yet been clarified. Here, we report that the expression of Syt13 mRNA in adult mouse brain was altered following contextual fear conditioning. C57BL/6 mice were exposed to a novel context and stimulated by strong electrical footshock according to a contextual fear conditioning protocol. After 24h, the mice were re-exposed to the context without electrical footshock for the retrieval of contextual fear memory. To investigate the relationship between Syt13 and contextual fear memory, we carried out in situ hybridization and analyzed gene expression patterns for Syt13 at four groups representing temporal changes in brain activity during contextual fear memory formation. Contextual fear conditioning test induced significant changes in mRNA levels for Syt13 within various brain regions, including lateral amygdala, somatosensory cortex, piriform cortex, habenula, thalamus, and hypothalamus, during both acquisition and retrieval sessions. Our data suggest that Syt13 may be involved in the process of contextual fear memory.     

Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning

We recorded neurons from the hippocampus of freely behaving rats during an auditory fear conditioning task. Rats received either paired or unpaired presentations of an auditory conditioned stimulus (CS) and an electric shock unconditioned stimulus (US). Hippocampal neurons (place and theta cells) acquired responses to the auditory CS in the paired but not in the unpaired group. After CS-US pairing, rhythmic firing of theta cells became synchronized to the onset of the CS. Conditioned responses of place cells were gated by their location-specific firing, so that after CS-US pairing, place cells responded to the CS only when the rat was within the cell's place field. These findings may help to elucidate how the hippocampus contributes to context-specific memory formation during associative learning.     

Molecular mechanisms of fear learning and memory

Pavlovian fear conditioning is a particularly useful behavioral paradigm for exploring the molecular mechanisms of learning and memory because a well-defined response to a specific environmental stimulus is produced through associative learning processes. Synaptic plasticity in the lateral nucleus of the amygdala (LA) underlies this form of associative learning. Here, we summarize the molecular mechanisms that contribute to this synaptic plasticity in the context of auditory fear conditioning, the form of fear conditioning best understood at the molecular level. We discuss the neurotransmitter systems and signaling cascades that contribute to three phases of auditory fear conditioning: acquisition, consolidation, and reconsolidation. These studies suggest that multiple intracellular signaling pathways, including those triggered by activation of Hebbian processes and neuromodulatory receptors, interact to produce neural plasticity in the LA and behavioral fear conditioning. Collectively, this body of research illustrates the power of fear conditioning as a model system for characterizing the mechanisms of learning and memory in mammals and potentially for understanding fear-related disorders, such as PTSD and phobias.     

Fear memory formation involves p190 RhoGAP and ROCK proteins through a GRB2-mediated complex

We used fear conditioning, which is known to alter synaptic efficacy in lateral amygdala (LA), to study molecular mechanisms underlying long-term memory. Following fear conditioning, the tyrosine phosphorylated protein p190 RhoGAP becomes associated with GRB2 in LA significantly more in conditioned than in control rats. RasGAP and Shc were also found to associate with GRB2 in LA significantly more in the conditioned animals. Inhibition of the p190 RhoGAP-downstream kinase ROCK in LA during fear conditioning impaired long- but not short-term memory. Thus, the p190 RhoGAP/ROCK pathway, which regulates the morphology of dendrites and axons during neural development, plays a central role, through a GRB2-mediated molecular complex, in fear memory formation in the lateral amygdala.     

Memorization of contextual and CS conditioned fear response (freezing) in a one-trial acquisition paradigm.

In fear-conditioned Wistar rats freezing was induced by the delivery of a series of footshocks paired to tones (CS) in a specific conditioning chamber (context). CS and contextual fear were acquired in the same single conditioning session without preexposition to the conditioning chamber (day 1). Different groups of animals were conditioned employing three increasing US (footshock) intensities (0.25, 0.5, 0.75 mA). During the retention sessions context and CS conditioned freezing (fear response) were measured using a paradigm that fulfilled the following conditions: i) CS freezing retention was measured in a context different from the conditioning one; ii) CS and context freezing were measured at increased delays after the training session (days 3 and 4, 14 and 15, 28 and 29). The results show that there are significant differences between CS and context freezing retention, which are clearly related to delay after the initial session and to US intensity. In particular: 1) conditioned freezing to a discrete tone is better retained than conditioned freezing to context (irrespective of US intensity); 2) context freezing is directly related to US intensity much more than to tone freezing; 3) context freezing is easier to extinguish than tone freezing. The results are discussed in relation to previous ones and to their relevance to freezing genesis neural correlates.     

Molecular mechanisms underlying emotional learning and memory in the lateral amygdala

Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The lateral nucleus of the amygdala (LA) is a crucial site of neural changes that occur during fear conditioning. Pharmacological manipulations of the LA, strategically timed with respect to training and testing, have shed light on the molecular events that mediate the acquisition of fear associations and the formation and maintenance of long-term memories of those associations. Similar mechanisms have been found to underlie long-term potentiation (LTP) in LA, an artificial means of inducing synaptic plasticity and a physiological model of learning and memory. Thus, LTP-like changes in synaptic plasticity may underlie fear conditioning. Given that the neural circuit underlying fear conditioning has been implicated in emotional disorders in humans, the molecular mechanisms of fear conditioning are potential targets for psychotherapeutic drug development.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

Environmental variables that ameliorate extinction learning deficits in the 129S1/SvlmJ mouse strain

Fear conditioning is an associative learning process by which organisms learn to avoid environmental stimuli that are predictive of aversive outcomes. Fear extinction learning is a process by which avoidance of fear‐conditioned stimuli is attenuated when the environmental stimuli is no longer predictive of the aversive outcome. Aberrant fear conditioning and extinction learning are key elements in the development of several anxiety disorders. The 129S1 inbred strain of mice is used as an animal model for maladaptive fear learning because this strain has been shown to generalize fear to other nonaversive stimuli and is less capable of extinguishing fear responses relative to other mouse strains, such as the C57BL/6. Here we report new environmental manipulations that enhance fear and extinction learning, including the ability to discriminate between an aversively paired tone and a neutral tone, in both the 129S1 and C57BL/6 strains of mice. Specifically, we show that discontinuous (“pipped”) tone stimuli significantly enhance within‐session extinction learning and the discrimination between neutral and aversively paired stimuli in both strains. Furthermore, we find that extinction training in novel contexts significantly enhances the consolidation and recall of extinction learning for both strains. Cumulatively, these results underscore how environmental changes can be leveraged to ameliorate maladaptive learning in animal models and may advance cognitive and behavioral therapeutic strategies.     

Olfactory conditioning of proboscis activity in Drosophila melanogaster

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.     

Pre-Training Reversible Inactivation of the Basal Amygdala (BA) Disrupts Contextual,but Not Auditory,Fear Conditioning,in Rats

The basolateral amygdala complex (BLA), including the lateral (LA), basal (BA) and accessory basal (AB) nuclei, is involved in acquisition of contextual and auditory fear conditioning. The BA is one of the main targets for hippocampal information, a brain structure critical for contextual learning, which integrates several discrete stimuli into a single configural representation. Congruent with the hodology, selective neurotoxic damage to the BA results in impairments in contextual, but not auditory, fear conditioning, similarly to the behavioral impairments found after hippocampal damage. This study evaluated the effects of muscimol-induced reversible inactivation of the BA during a simultaneous contextual and auditory fear conditioning training on later fear responses to both the context and the tone, tested separately, without muscimol administration. As compared to control rats micro-infused with vehicle, subjects micro-infused with muscimol before training exhibited, during testing without muscimol, significant reduction of freezing responses to the conditioned context, but not to the conditioned tone. Therefore, reversible inactivation of the BA during training impaired contextual, but not auditory fear conditioning, thus confirming and extending similar behavioral observations following selective neurotoxic damage to the BA and, in addition, revealing that this effect is not related to the lack of a functional BA during testing.     

Natural amyloid-beta oligomers acutely impair the formation of a contextual fear memory in mice

Memory loss is one of the hallmark symptoms of Alzheimer's disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss.     

Mapping and Deciphering Neural Codes of NMDA Receptor-Dependent Fear Memory Engrams in the Hippocampus

Mapping and decoding brain activity patterns underlying learning and memory represents both great interest and immense challenge. At present, very little is known regarding many of the very basic questions regarding the neural codes of memory: are fear memories retrieved during the freezing state or non-freezing state of the animals? How do individual memory traces give arise to a holistic, real-time associative memory engram? How are memory codes regulated by synaptic plasticity? Here, by applying high-density electrode arrays and dimensionality-reduction decoding algorithms, we investigate hippocampal CA1 activity patterns of trace fear conditioning memory code in inducible NMDA receptor knockout mice and their control littermates. Our analyses showed that the conditioned tone (CS) and unconditioned foot-shock (US) can evoke hippocampal ensemble responses in control and mutant mice. Yet, temporal formats and contents of CA1 fear memory engrams differ significantly between the genotypes. The mutant mice with disabled NMDA receptor plasticity failed to generate CS-to-US or US-to-CS associative memory traces. Moreover, the mutant CA1 region lacked memory traces for “what at when” information that predicts the timing relationship between the conditioned tone and the foot shock. The degraded associative fear memory engram is further manifested in its lack of intertwined and alternating temporal association between CS and US memory traces that are characteristic to the holistic memory recall in the wild-type animals. Therefore, our study has decoded real-time memory contents, timing relationship between CS and US, and temporal organizing patterns of fear memory engrams and demonstrated how hippocampal memory codes are regulated by NMDA receptor synaptic plasticity.     

Contextual fear conditioning differs for infant, adolescent, and adult rats

Contextual fear conditioning was tested in infant, adolescent, and adult rats in terms of Pavlovian-conditioned suppression. When a discrete auditory-conditioned stimulus (CS) was paired with footshock (unconditioned stimulus, US) within the largely olfactory context, infants and adolescents conditioned to the context with substantial effectiveness, but adult rats did not. When unpaired presentations of the CS and US occurred within the context, contextual fear conditioning was strong for adults, weak for infants, but about as strong for adolescents as when pairings of CS and US occurred in the context. Nonreinforced presentations of either the CS or context markedly reduced contextual fear conditioning in infants, but, in adolescents, CS extinction had no effect on contextual fear conditioning, although context extinction significantly reduced it. Neither CS extinction nor context extinction affected responding to the CS–context compound in infants, suggesting striking discrimination between the compound and its components. Female adolescents showed the same lack of effect of component extinction on response to the compound as infants, but CS extinction reduced responding to the compound in adolescent males, a sex difference seen also in adults. Theoretical implications are discussed for the development of perceptual-cognitive processing and hippocampus role.     

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.     

Muscarinic Receptors in Amygdala Control Trace Fear Conditioning

Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.     

Auditory-evoked spike firing in the lateral amygdala and Pavlovian fear conditioning: mnemonic code or fear bias?

Amygdala neuroplasticity has emerged as a candidate substrate for Pavlovian fear memory. By this view, conditional stimulus (CS)-evoked activity represents a mnemonic code that initiates the expression of fear behaviors. However, a fear state may nonassociatively enhance sensory processing, biasing CS-evoked activity in amygdala neurons. Here we describe experiments that dissociate auditory CS-evoked spike firing in the lateral amygdala (LA) and both conditional fear behavior and LA excitability in rats. We found that the expression of conditional freezing and increased LA excitability was neither necessary nor sufficient for the expression of conditional increases in CS-evoked spike firing. Rather, conditioning-related changes in CS-evoked spike firing were solely determined by the associative history of the CS. Thus, our data support a model in which associative activity in the LA encodes fear memory and contributes to the expression of learned fear behaviors.