Kandov, E. | Search bioRxiv

bioRxiv2024

Medial prefrontal dopamine dynamics reflect allocation of selective attention

The mesocortical dopamine system is comprised of midbrain dopamine neurons that predominantly innervate the medial prefrontal cortex (mPFC) and exert a powerful neuromodulatory influence over this region1,2. mPFC dopamine activity is thought to be critical for fundamental neurobiological processes including valence coding and decision-making3,4. Despite enduring interest in this pathway, the stimuli and conditions that engage mPFC dopamine release have remained enigmatic due to inherent limitations in conventional methods for dopamine monitoring which have prevented real-time in vivo observation5. Here, using a fluorescent dopamine sensor enabling time-resolved recordings of cortical dopamine activity in freely behaving mice, we reveal the coding properties of this system and demonstrate that mPFC dopamine dynamics conform to a selective attention signal. Contrary to the long-standing theory that mPFC dopamine release preferentially encodes aversive and stressful events6-8, we observed robust dopamine responses to both appetitive and aversive stimuli which dissipated with increasing familiarity irrespective of stimulus intensity. We found that mPFC dopamine does not evolve as a function of learning but displays striking temporal precedence with second-to-second changes in behavioral engagement, suggesting a role in allocation of attentional resources. Systematic manipulation of attentional demand revealed that quieting of mPFC dopamine signals the allocation of attentional resources towards an expected event which, upon detection triggers a sharp dopamine transient marking the transition from decision-making to action. The proposed role of mPFC dopamine as a selective attention signal is the first model based on direct observation of time-resolved dopamine dynamics and reconciles decades of competing theories.

neuroscience↗

bioRxiv2020

IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an open system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues. Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

immunology↗