Ewelina Knapska

Reciprocal Patterns of c-Fos Expression in the Medial Prefrontal Cortex and Amygdala after Extinction and Renewal of Conditioned Fear.

After extinction of conditioned fear, memory for the conditioning and extinction experiences becomes context dependent. Fear is suppressed in the extinction context, but renews in other contexts. This study characterizes the neural circuitry underlying the context-dependent retrieval of extinguished fear memories using c-Fos immunohistochemistry. After fear conditioning and extinction to an auditory conditioned stimulus (CS), rats were presented with the extinguished CS in either the extinction context or a second context, and then sacrificed. Presentation of the CS in the extinction context yielded low levels of conditioned freezing and induced c-Fos expression in the infralimbic division of the medial prefrontal cortex, the intercalated nuclei of the amygdala, and the dentate gyrus (DG). In contrast, presentation of the CS outside of the extinction context yielded high levels of conditioned freezing and induced c-Fos expression in the prelimbic division of the medial prefrontal cortex, the lateral and basolateral nuclei of the amygdala, and the medial division of the central nucleus of the amygdala. Hippocampal areas CA1 and CA3 exhibited c-Fos expression when the CS was presented in either context. These data suggest that the context specificity of extinction is mediated by prefrontal modulation of amygdala activity, and that the hippocampus has a fundamental role in contextual memory retrieval.

Hippocampal and prefrontal projections to the basal amygdala mediate contextual regulation of fear after extinction.

Knowing when and where to express fear is essential to survival. Recent work in fear extinction paradigms reveals that the contextual regulation of fear involves a neural network involving the hippocampus, medial prefrontal cortex, and amygdala. The amygdaloid basal nuclei (BA) receive convergent input from the ventral hippocampus (VH) and prelimbic (PL) prefrontal cortex and may integrate VH and PL input to regulate fear expression. To examine the functional organization of this neural circuit, we used cellular imaging of c-fos expression in anatomically defined neuronal populations and circuit disconnections to identify the pathways involved in the contextual control of extinguished fear. Before behavioral testing, we infused a retrograde tracer into the amygdala to label BA-projecting neurons in VH and PL. Rats then underwent fear conditioning and extinction and were tested for their fear to the extinguished conditioned stimulus (CS) in either the extinction context or in another context; freezing behavior served as the index of conditional fear. CS presentation outside the extinction context renewed conditional freezing and was associated with significantly more c-fos expression in BA-projecting neurons in the VH and PL than that induced by CS presentation in the extinction context. We next examined whether direct or indirect projections of VH to BA mediate fear renewal. Interestingly, disconnections of the VH from either the BA or PL eliminated renewal. These findings suggest that convergent inputs from both the VH and PL in the BA mediate the contextual control of fear after extinction.

New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

Functional anatomy of neural circuits regulating fear and extinction

The memory of fear extinction is context dependent: fear that is suppressed in one context readily renews in another. Understanding of the underlying neuronal circuits is, therefore, of considerable clinical relevance for anxiety disorders. Prefrontal cortical and hippocampal inputs to the amygdala have recently been shown to regulate the retrieval of fear memories, but the cellular organization of these projections remains unclear. By using anterograde tracing in a transgenic rat in which neurons express a dendritically-targeted PSD-95:Venus fusion protein under the control of a c-fos promoter, we found that, during the retrieval of extinction memory, the dominant input to active neurons in the lateral amygdala was from the infralimbic cortex, whereas the retrieval of fear memory was associated with greater hippocampal and prelimbic inputs. This pattern of retrieval-related afferent input was absent in the central nucleus of the amygdala. Our data show functional anatomy of neural circuits regulating fear and extinction, providing a framework for therapeutic manipulations of these circuits.

Social modulation of learning in rats

It is well known that emotions participate in the regulation of social behaviors and that the emotion displayed by a conspecific influences the behavior of other animals. In its simplest form, empathy can be characterized as the capacity to be affected by and/or share the emotional state of another. However, to date, relatively little is known about the mechanisms by which animals that are not in direct danger share emotions. In the present study, we used a model of between-subject transfer of fear to characterize the social interaction during which fear is transmitted, as well as the behavioral effects of socially transmitted fear. We found that (1) during social interaction with a recently fear-conditioned partner, observers and demonstrators exhibit social exploratory behaviors rather than aggressive behaviors; (2) learning and memory in a shock-motivated shuttle avoidance task are facilitated in rats that underwent a social interaction with a partner that had been fear conditioned; and (3) a brief social interaction with a recently fear-conditioned partner immediately before fear conditioning increases conditioned freezing measured on the next day. The observed effects were not due to a stress-induced increase in pain sensitivity or analgesia. Collectively, these data suggest that a brief social interaction with a cage mate that has undergone an aversive learning experience promotes aversive learning in an otherwise naïve animal. We argue that socially transferred fear is an adaptation that promotes defensive behavior to potentially dangerous situations in the environment.

Reward Learning Requires Activity of Matrix Metalloproteinase-9 in the Central Amygdala

Learning how to avoid danger and pursue reward depends on negative emotions motivating aversive learning and positive emotions motivating appetitive learning. The amygdala is a key component of the brain emotional system; however, an understanding of how various emotions are differentially processed in the amygdala has yet to be achieved. We report that matrix metalloproteinase-9 (MMP-9, extracellularly operating enzyme) in the central nucleus of the amygdala (CeA) is crucial for appetitive, but not for aversive, learning in mice. The knock-out of MMP-9 impairs appetitively motivated conditioning, but not an aversive one. MMP-9 is present at the excitatory synapses in the CeA with its activity greatly enhanced after the appetitive training. Finally, blocking extracellular MMP-9 activity with its inhibitor TIMP-1 provides evidence that local MMP-9 activity in the CeA is crucial for the appetitive, but not for aversive, learning.

Behavioral Characterization of GLT1 (+/-) Mice as a Model of Mild Glutamatergic Hyperfunction

GLT1 is one of the major transporters responsible for maintenance of glutamate homeostasis in the brain. In the present study, glutamate transporter 1-deficient GLT1 homozygous (-/-) and heterozygous (+/-) mice were investigated with the intention that they may provide a model of hyperglutamatergic state resulting in various behavioral alterations. The GLT1 (-/-) mice had lower body and brain weight, mild neuronal loss in CA1 hippocampal region as well as focal gliosis and severe focal neuronal paucity in layer II of the neocortex. The short life-span of GLT1 (-/-) precluded us from systematic behavioral studies in these mice. In contrast, GLT1 (+/-) mice exhibiting a 59% decrease in GLT1 immunoreactivity in their brain tissue, showed no apparent morphological brain abnormalities, and their life-span was not markedly different from controls. Behavior ally, GLT1 (+/-) presented moderate behavioral alterations compared to their wildtype littermates, such as: mild sensorimotor impairment, hyperlocomotion (at 3 month of age only), lower anxiety (at 6 months), better learning of cue-based fear conditioning but worse context-based fear conditioning. Our results suggest that GLT1 (+/-) mice may serve as a potentially useful model to study neurodegenerative disease conditions with mild hyperglutamatergic activity.

Social modulation in extinction of aversive memories.

Return of fear after extinction is a considerable challenge for the efficacy of exposure-based therapies. Fear recovery is most often modeled in the laboratory by changing the experimental context and studied in isolated animals. Since social context is an important factor affecting behavior, the question arises how it influences the recovery of extinguished fear. Here we present two novel behavioral models that allow studying social modulation of extinction memory retrieval. We show that the presence of a fearful cage mate results in a robust renewal of freezing as well as avoidance responses that were previously successfully extinguished.

Sex differences in social modulation of learning in rats.

In its simplest form, empathy can be characterized as the capacity to share the emotional experiences among individuals, a phenomenon known as emotional contagion. Recent research shows that emotional contagion and its adaptive role can be studied in rodents. However, it is not known whether sex differences observed in human empathy extend to its more primitive forms. In the present study, we used a rat model of emotional contagion to compare the behavioral consequences of social transfer of information about threat, and the subsequent neural activation patterns in male and female rats. We found that: (1) males and females display a similar behavioral pattern during the interaction with either a fear-conditioned or a control rat; (2) interaction with a fear-conditioned conspecific positively modulates two-way avoidance learning in male and diestral female rats but not in estral females; and (3) such interaction results in increased c-Fos expression in the central and lateral nuclei of the amygdala and the prelimbic and infralimbic cortex in males, whereas in females no such changes were observed. Collectively, our results point to the occurrence of sex and estrus cycle phase differences in susceptibility to emotional contagion and underlying neuronal activation in rodents.

A novel automated behavioral test battery assessing cognitive rigidity in two genetic mouse models of autism

Repetitive behaviors are a key feature of many pervasive developmental disorders, such as autism. As a heterogeneous group of symptoms, repetitive behaviors are conceptualized into two main subgroups: sensory/motor (lower-order) and cognitive rigidity (higher-order). Although lower-order repetitive behaviors are measured in mouse models in several paradigms, so far there have been no high-throughput tests directly measuring cognitive rigidity. We describe a novel approach for monitoring repetitive behaviors during reversal learning in mice in the automated IntelliCage system. During the reward-motivated place preference reversal learning, designed to assess cognitive abilities of mice, visits to the previously rewarded places were recorded to measure cognitive flexibility. Thereafter, emotional flexibility was assessed by measuring conditioned fear extinction. Additionally, to look for neuronal correlates of cognitive impairments, we measured CA3-CA1 hippocampal long term potentiation (LTP). To standardize the designed tests we used C57BL/6 and BALB/c mice, representing two genetic backgrounds, for induction of autism by prenatal exposure to the sodium valproate. We found impairments of place learning related to perseveration and no LTP impairments in C57BL/6 valproate-treated mice. In contrast, BALB/c valproate-treated mice displayed severe deficits of place learning not associated with perseverative behaviors and accompanied by hippocampal LTP impairments. Alterations of cognitive flexibility observed in C57BL/6 valproate-treated mice were related to neither restricted exploration pattern nor to emotional flexibility. Altogether, we showed that the designed tests of cognitive performance and perseverative behaviors are efficient and highly replicable. Moreover, the results suggest that genetic background is crucial for the behavioral effects of prenatal valproate treatment.