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.

Reconnectable fiberscopes for chronic in vivo deep-brain imaging.

Reconnectable bundles consisting of thousands of optical fibers are shown to enable high-quality image transmission, offering a platform for the creation of implantable fiberscopes for minimally invasive in vivo brain imaging. Experiments on various lines of transgenic mice verify the performance of this fiberscope as a powerful tool for chronic in vivo neuroimaging using genetically encoded calcium indicators, neuronal activity markers as well as axon growth regulators and brain-specific protein drivers in deep regions of live brain.

Two-photon imaging of fiber-coupled neurons.

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two-photon imaging is shown to enable a quantitative in situ analysis of such fiber-single-neuron coupling in the live brain of transgenic mice. Fiber-optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two-photon imaging. These results pave the way for fiber-optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber-optic single-neuron stimulation into the optical imaging framework.

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.

Three-dimensional fiber-optic readout of single-neuron-resolved fluorescence in living brain of transgenic mice.

A bundle of individually addressable optical fibers is shown to enable three-dimensional optical readout from single neurons in live brain, as well as in intact brain extracted from transgenic mice. With individual fibers in the bundle being only a few microns in diameter, single neurons are readily resolved in brain images transmitted by the fiber bundle. The third dimension is added by scanning the fiber in the longitudinal direction, with the fluorescence return read out from only one of the fibers in the bundle.

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.

Formation of spatial and nonspatial memory in different condensed versions of short-term learning in Morris water maze.

We studied the formation of spatial and nonspatial memory in mice during learning in three different condensed versions of Morris water maze task. Learning in combined version caused the formation of both spatial and nonspatial memory, whereas learning in condensed versions (spatial and nonspatial) led to memory formation specific for the version.

Implantable fiber-optic interface for parallel multisite long-term optical dynamic brain interrogation in freely moving mice.

Seeing the big picture of functional responses within large neural networks in a freely functioning brain is crucial for understanding the cellular mechanisms behind the higher nervous activity, including the most complex brain functions, such as cognition and memory. As a breakthrough toward meeting this challenge, implantable fiber-optic interfaces integrating advanced optogenetic technologies and cutting-edge fiber-optic solutions have been demonstrated, enabling a long-term optogenetic manipulation of neural circuits in freely moving mice. Here, we show that a specifically designed implantable fiber-optic interface provides a powerful tool for parallel long-term optical interrogation of distinctly separate, functionally different sites in the brain of freely moving mice. This interface allows the same groups of neurons lying deeply in the brain of a freely behaving mouse to be reproducibly accessed and optically interrogated over many weeks, providing a long-term dynamic detection of genome activity in response to a broad variety of pharmacological and physiological stimuli.

Increased 5-bromo-2'-deoxyuridine incorporation in various brain structures following passive avoidance training in mice.

We studied the effects of training on DNA synthesis intensity in mouse brain. Brain cells where DNA synthesis-associated processes took place under the influence of training were detected by immunohistochemical labeling of DNA molecules with synthetic thymine analogue 5-bromo-2'-deoxyuridine. The number of 5-bromo-2'-deoxyuridine-positive cell increased in various structures of the brain under the influence of training.

Increased 5-bromo-2'-deoxyuridine incorporation in various brain structures following passive avoidance training in mice.

We studied the effects of training on DNA synthesis intensity in mouse brain. Brain cells where DNA synthesis-associated processes took place under the influence of training were detected by immunohistochemical labeling of DNA molecules with synthetic thymine analogue 5-bromo-2'-deoxyuridine. The number of 5-bromo-2'-deoxyuridine-positive cell increased in various structures of the brain under the influence of training.

Immunohistochemical detection of two neuronal populations involved in two different episodes of animal cognitive activity.

We developed a method to detect and superimpose neuronal populations involved in two different episodes of activity in the brain of single animal. This method is based on usage of two transcriptional factors with different accumulation curves, c-Fos and pCREB, as the markers. The overlap of neuronal populations involved in performance of spatial and nonspatial tasks in Morris water maze has been shown to occur at "chance" level.