Anna Kiryk

MicroRNA Loss Enhances Learning and Memory in Mice

Dicer-dependent noncoding RNAs, including microRNAs (miRNAs), play an important role in a modulation of translation of mRNA transcripts necessary for differentiation in many cell types. In vivo experiments using cell type-specific Dicer1 gene inactivation in neurons showed its essential role for neuronal development and survival. However, little is known about the consequences of a loss of miRNAs in adult, fully differentiated neurons. To address this question, we used an inducible variant of the Cre recombinase (tamoxifen-inducible CreERT2) under control of Camk2a gene regulatory elements. After induction of Dicer1 gene deletion in adult mouse forebrain, we observed a progressive loss of a whole set of brain-specific miRNAs. Animals were tested in a battery of both aversively and appetitively motivated cognitive tasks, such as Morris water maze, IntelliCage system, or trace fear conditioning. Compatible with rather long half-life of miRNAs in hippocampal neurons, we observed an enhancement of memory strength of mutant mice 12 weeks after the Dicer1 gene mutation, before the onset of neurodegenerative process. In acute brain slices, immediately after high-frequency stimulation of the Schaffer collaterals, the efficacy at CA3-to-CA1 synapses was higher in mutant than in control mice, whereas long-term potentiation was comparable between genotypes. This phenotype was reflected at the subcellular and molecular level by the elongated filopodia-like shaped dendritic spines and an increased translation of synaptic plasticity-related proteins, such as BDNF and MMP-9 in mutant animals. The presented work shows miRNAs as key players in the learning and memory process of mammals.

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.

Transient brain ischemia due to cardiac arrest causes irreversible long-lasting cognitive injury.

Herein, we used a clinically-relevant model of 10 min cardiac arrest (CA) in Wistar rats. Histological analyses of the ischemic brains of old rats showed significant atrophy of CA(1) sector of hippocampus (Nissl and NeuN stainings) corresponding with increase of glial fibrillary acidic protein expression. The long-term behavioral consequences of above manipulation producing global brain ischemia were assessed in young, middle-aged and old rats, i.e., 3-, 6- and 18-months post-treatment, respectively. In young animals no differences were found in the context-dependent memory in Fear Conditioning test. The most striking behavioral abnormalities were found in middle-aged rats (6 months post-ischemia). Ischemic rats showed hyperactivity and decreased level of anxiety in Open Field and problems with spatial learning and memory in a Novel Object Location test, T-maze and Morris Water Maze. In old animals, a decline of motor and cognitive functions was found not only in ischemic but also in sham/control ones. This study describes consequences of global brain ischemia in aging animals.

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.

Impaired rRNA synthesis triggers homeostatic responses in hippocampal neurons

Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimers disease (AD) and may play a role in dementia. Moreover, aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3, and dentate gyrus (DG). Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that 36 transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF) binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the DG a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving neuronal function.

Cognitive abilities of Alzheimer's disease transgenic mice are modulated by social context and circadian rhythm

In the present study, we used a new training paradigm in the intelliCage automatic behavioral assessment system to investigate cognitive functions of the transgenic mice harboring London mutation of the human amyloid precursor protein (APP.V717I). Three groups of animals: 5-, 12- and 18-24-month old were subjected to both Water Maze training and the IntelliCage-based appetitive conditioning. The spatial memory deficit was observed in all three groups of transgenic mice in both behavioral paradigms. However, the APP mice were capable to learn normally when co-housed with the wild-type (WT) littermates, in contrast to clearly impaired learning observed when the transgenic mice were housed alone. Furthermore, in the transgenic mice kept in the Intellicage alone, the cognitive deficit of the young animals was modulated by the circadian rhythm, namely was prominent only during the active phase of the day. The novel approach to study the transgenic mice cognitive abilities presented in this paper offers new insight into cognitive dysfunctions of the Alzheimers disease mouse model.

Towards a computational model of learning and social interactions of mice in IntelliCage.

In [1] we have studied cognitive deficits in transgenic mouse model of Alzheimers Disease. In particular, we used automated mice cages (IntelliCage) to investigate how healthy and transgenic mice learn the location of a reward (sweetened water provided in only one of the four corners of the cage) in different social situations. We showed that co-housing transgenic mice with healthy companions can alleviate learning deficits. We also showed that the ability to find the reward is modulated by the circadian rhythm. A natural continuation of that study is to find specific mechanisms of learning and social interactions responsible for the observed patterns of behavior. To that end we now employ a collection of computational models of learning and decision making [2]. In the models we use the Rescorla-Wagner rule for the learned values of the actions supplemented by a model-specific decision-making rule. In the simplest case we assume the decisions are based purely on the learned rewards, with the probabilities of actions given by the softmax distribution. Such a simple model, in which animals are ignorant of each other, fits the trend of the learning curve well both for healthy (Figure ​(Figure1A)1A) and transgenic (not shown) mice. However, it does not capture the circadian oscillations. Figure 1 Experimental and model learning curves of healthy mice. Left: model without interactions between animals. Right: model with minimal social interactions. Thin lines mark the standard deviation of model curves over 100 repetitions. Next we study a model in which the interactions between animals are included in a minimal form. Specifically, the probability of visiting given corner is reduced if the corner is already occupied by another mouse at the time the choice of the action takes place. In this case we observe that the model learning-curve starts to exhibit oscillations around the trend (Figure ​(Figure1B).1B). Still, the amplitude of these oscillations is significantly smaller than in experimental data, which suggests that other effects come also into play.