Cognitive Tagging of Neurons: CREMediated Genetic Labeling and Characterization of the Cells Involved in Learning and Memory.

In this study, we describe use of Cre-mediated recombination to obtain a permanent genetic labeling of the brain neuronal networks activated during a new experience in animals. This method utilizes bitransgenic Fos-Cre-eGFP mice in which a green fluorescent protein is expressed upon tamoxifen-induced Cre-recombination only in the cells where immediate early gene c-fos expression takes place due to the new experience. We used the classical fear conditioning model to show that ex vivo microscopy of the eGFP protein in Fos-Cre-eGFP mice enables mapping of the neurons of the various brain regions that undergo Cre-recombination during acquisition of a new experience. We exposed the animals to the new environment in brief sessions and demonstrated that double immunohistochemical staining enables a characterization of the types of neocortical and hippocampal neurons that undergo experience-dependent Cre-recombination. Notably, Fos-Cre-eGFP labeled cells appeared to belong to excitatory pyramidal neurons rather than to various types of inhibitory neurons. We also showed that a combination of genetic Cre-eGFP labeling with immunohistochemical staining of the endogenous c-Fos protein allows one to identify and compare the neuronal populations that are activated during two different episodes of new experiences in the same animal. This new approach can be used in a wide spectrum of tasks that require imaging and a comparative analysis of cognitive neuronal networks.

Quantitative cognitive-test characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation.

Cognitive tests on representative groups of freely behaving transgenic mice are shown to enable a quantitative characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation. A systematic analysis of such tests provides a robust quantitative measure for the cognitive effects induced by fiber-optic neurostimulation, validating the performance of fiber-optic neurointerfaces for long-term optogenetic brain stimulations and showing no statistically significant artifacts in the behavior of transgenic mice due to interface implantation.

Lentiviral Transduction of Neurons in Adult Brain: Evaluation of Inflammatory Response and Cognitive Effects in Mice.

We evaluated the effect of hippocampal injection of lentiviral particles p156-CMV-EGFP on behavior, learning, and microglial Iba1(+) cells activation in mice. Testing in the open field and elevated plus-maze revealed higher anxiety levels in lentiviral-injected mice in comparison with animals injected with vehicle. At the same time, lentivirus injection did not change learning and memory of mice in the hippocampal-dependent fear conditioning task. Microglia density in lentivirus-injected mice was significantly higher than in vehicle-injected mice. Thus, hippocampal injection of lentiviral particles with minimum content of transgenes produced evident inflammation process, changed anxiety level of experimental animals, but had no effect on hippocampal-dependent learning and memory.

Waves of c-Fos and Arc Proteins Expression in Neuronal Populations of the Hippocampus in Response to a Single Episode of New Experience.

Accumulation of c-Fos and Arc proteins in neurons in different regions of the hippocampus after single trial of contextual fear conditioning was studied by using immunohistochemical staining. We found that the dynamics of the c-Fos and Arc expression has a biphasic pattern: the first peak was observed in 15-30 min after learning and the second less pronounced peak in 1-3 h. Induction of Arc occurred earlier than c-Fos and the overall dynamics of the two waves slightly varied in the dentate gyrus and hippocampal CA1 and CA3 fields. The findings open the possibility of mapping the cognitive neural networks of the brain with higher temporal resolution and draw attention to fluctuations of hippocampal activity after a single brief episode of new experience.

[Long-Term Contextual Memory in Mice: Persistence and Associability with Reinforcement].

Animals can associate memory of a context with unconditioned stimuli even if there is a long time in- terval between acquisition of contextual memory and its subsequent reinforcement. This phenomenon of context preexposure effect was first described and investigated in rats. Here we studied the possibility of associating previously acquired memory about a context with unconditioned stimulus (immediate shock) in mice. We showed that fear memory in this model was specific for the previously explored context but not for the context of immediate shock. Associative learning was possible when acquisition of contextual memory and presentation unconditioned stimulus (immediate shock) were spaced in the range of 30 min-30 days interval. Resulting memory was stable and persisted for at least 30 days. Our results open new avenues for studies of neuronal mechanisms of associative memory using transgenic re- porter mice.

[Visual Fear Conditioning in Mice: Comparison with Auditory Fear Conditioning].

Up to date, rodent fear conditioning is the most commonly used model of mammalian associative memory in neurobiology. Since the mechanisms of associative memory in this model are mainly studied using audi tory fear conditioning, the question about the generality of this mechanisms in respect to other conditioned stimuli remains open. The aim of this work-was to compare dynamics of visual-and auditory fear memory for- mation and retrieval. We showed that acquisition of freezing in response to visual conditioned stimulus is significantly slower in comparison with the. auditory conditioned stimulus. Moieover, the dynamics of memory retrieval was different in animals, trained to these stimuli, being slower in response to visual conditioned stim- ulus. This may reflect distinct mechanisms of visual and auditory associative memory consolidation or re- trieval in this Model. Our results impose constraints on the generality of conclusiois about the dynamics as- sociative memory formation obtained solely from classical auditory fear conditioning model in mice.

[Inhibition of histone deacetylases in the chick brain modulates expression of c-Fos and ZENK transcription factors and facilitates establishment of long-term memory].

The aim of the work was to examine the role of histone acetylation in memory consolidation in newborn chicks. We studied the effects of histone deacetylase inhibitor trichostatin A (TSA) on a "weak" memory for passive avoidance and on expression of two transcription factors c-Fos and ZENK known to play a role in neuronal plasticity in the chick brain. Intraventricular administration of trichostatin A prior to training produced a dose-dependent enhancement of memory when tested 24 hours after the training. It also increased neuronal expression of c-Fos and ZENK proteins: the density of ZENK immunopositive cells increased in the hippocampus and intermediate medial mesopallium and the density of c-Fos immunopositive cells increased in intermediate arcopallium and dorsocaudal nidopallium. Weak passive avoidance training did not produce further enhancement of c-Fos and ZENK expression in any of these brain areas. These data demonstrate possibility of facilitating long-term memory in day-old chicks by a histone deacetylases inhibitor, thus supporting the hypothesis on the role of histone acetylation in long-term memory formation. They also suggest that these effects might be mediated through modulation of transcriptional response in brain areas involved in consolidation of this form of memory.

Effects of systemic administration of histone deacetylase inhibitor on memory formation and immediate early gene expression in chick brain.

We studied the effects of histone deacetylase inhibitor that stimulates transcriptional activity via histone hyperacetylation on memory formation. Sodium butyrate and sodium valproate enhanced memory in chicks following "weak" training with memory transfer into long-term state. Quantitative analysis of c-Fos and ZENK transcriptional factor gene expression in six structures of chick brain revealed induction of these genes in the structures involved in this type of learning. Sodium valproate administration did not increase this induction, but even reduced it. These findings suggest that sodium butyrate and sodium valproate exert cognitive stimulating action in the "weak" memory formation paradigm, and that this effect is not mediated via enhanced expression of transcriptional factors, which are traditionally considered as "molecular switcher" for memory transfer into long-term state.