Two photon stimulation in freely moving animals to understand the causal role of sleep reactivation in learning

Abstract:

Understanding the causal role of sleep-associated neuronal reactivations in memory consolidation requires simultaneous observation and precise manipulation of functionally defined ensembles in freely behaving animals. To address this gap, this project aims at integrating patterned two-photon optogenetic stimulation with miniature two-photon microscopy (Mini2P) in mice, enabling cellular-resolution imaging and targeted activation of prefrontal cortex (PFC) neurons during natural sleep. The first goal will be to identify PFC ensembles reactivated during post-learning sleep following auditory-reward conditioning. By artificially enhancing the reactivation probability of these memory-specific ensembles via optogenetic stimulation, we will test whether this manipulation improves behavioral recall, thereby probing the direct causal contribution of sleep reactivations to memory consolidation. This work will establish a transformative methodology for interrogating neural circuit dynamics in vivo during natural behaviors.

Description of the proposed project :

The goal of this project is to combine two-photon stimulation with two-photon imaging in our head-mounted microscope to study the role of ensembles of neurons that are reactivated in prefrontal cortex during sleep.

To understand the mechanisms underlying cognition and behavior, it is essential to obtain broad and precise insights into neuronal activity during the performance of complex tasks in natural settings. Over the past twenty years, two-photon (2P) microscopy has significantly advanced the limits of the number of neurons that can be simultaneously recorded in awake, behaving animals. This technique offers unparalleled precision in terms of the location and genetic identity of neurons. However, 2P microscopy traditionally requires head-fixation of the animal, which imposes substantial limitations on the range of behaviors that can be studied and their relevance to natural conditions.

To address this challenge, we use a miniature two-photon microscope (Mini2P), to image neuronal activity of hundreds of neurons in the frontal cortex in freely moving mice (Zong et al., 2022). This system enables the observation of cellular activity under various conditions and behaviors, including sleep. Nevertheless, establishing a causal link between brain activity and behavior necessitates the ability to control neuronal dynamics and observe the resulting effects on animal behavior.

To achieve this, a method of choice is optogenetics, a technique based on the expression of a light-modulated actuator in genetically defined cell type allowing for the modification of neuronal activity with light. Recent developments of optogenetics use cutting-edge optical methods to shape the light into arbitrary spatial patterns allowing to activate ensembles of neurons not only selected based on their genetic identity but also about their functional specificity (Accanto et al., 2023; Lorca-Cámara et al., 2024).

As a first step, under the supervision of specialists from the Bioimaging facility, the student will contribute to setting up a cutting-edge system combining two-photon imaging and photostimulation in our miniature microscope. Then the student will use this novel tool to identify the ensemble of neurons that are activated spontaneously during sleep in the mouse prefrontal cortex. These reactivations are thought to be important for memory consolidation. To test this hypothesis the student will image neuronal reactivations in the prefrontal cortex before and after the memorization of a behavioral association between a sound and a reward. Thanks to this longitudinal observation, the reactivated neural ensembles specific to this memory will be identified. To study the causal role of these ensembles, the reactivation probability of this specific neuronal ensemble will be artificially increased with targeted optogenetic stimulations. We will then test if this manipulation improves memorization of the behavioral association.

Main scopes of the project:

- Establish a cutting-edge tool for combined imaging and photo-stimulation of neuronal activity on the prefrontal cortex.

- Study of the causal role of sleep reactivations for memory consolidation.

The Hearing Institute (Institut de l’Audition, IdA, Paris 12eme) is a new center for basic and translational neuroscience research in the field of hearing, which opened in 2020 at the initiative of the Fondation Pour l'Audition and the Institut Pasteur. The overarching goal of the Institute is to elucidate the principles underpinning the workings of the auditory system, auditory perception and cognition from the molecular to the cognitive level. Optical techniques are key for this endeavor.

https://www.institut-audition.fr/

Experience required: We expect the candidate to have strong knowledge in neuroscience, evidenced by outstanding university grades. A background in physics and in particular with knowledge in optics and microscopy or a clear motivation to learn these techniques will be an asset A publication/preprint of the candidate’s previous lab work is highly desirable. Strong supporting letters from two (associate) professors are mandatory. The candidate's interest, enthusiasm, commitment, dedication, and ability to learn and share will be key for this project.