A robust activity marking system for exploring active neuronal ensembles

Abstract

Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system’s versatility.DOI: http://dx.doi.org/10.7554/eLife.13918.001

Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq

“…In addition to 11 excitatory neuronal clusters, two inhibitory neuronal clusters (Inh2 and Inh5) and two non-neuronal clusters (Astro and OPC) were significantly associated with expression of activity-regulated genes in response to PTZ treatment. Our analysis suggests that Sst -expressing inhibitory neurons (Inh2) are much more likely to express ARGs than Pvalb -expressing neurons (Inh3) in response to PTZ-induced seizure, which is in agreement with a recent cell-type-specific analysis of inhibitory neuronal response to PTZ using a synthetic IEG reporter system (Sorensen et al., 2016). Interestingly, relatively low-abundant interneuron subtypes, such as Ndnf -expressing cells (Inh5) and oligodendrocyte precursor cells, also exhibited significant transcriptional response to PTZ treatment (Figure 4A), suggesting conserved signaling pathways underlying neuronal activation in these cell-types.…”

Molecular and Circuit-Dynamical Identification of Top-Down Neural Mechanisms for Restraint of Reward Seeking

“…Compared to other dual-viral systems using the doxycycline-tetOff system (Roy et al, 2016), the activity-dependent 4-TM-CreER system allows a more specific time window of labeling (injecting fast-acting tamoxifen as opposed to ceasing doxycycline treatment). In order to efficiently drive a second Cre-dependent virus encoding the opsin (which needed to be expressed at high enough levels to be functionally present in axons), the synthetic E-SARE promoter was chosen to drive CreER for greater induction of Cre expression following neural activity (Kawashima et al, 2013; Sørensen et al, 2016) compared to other IEG promoters such as c-FOS and ARC. This all-virus (and transgenic animal-independent) vCAPTURE strategy may enable future lines of investigation in diverse animal species.…”

Genetic strategies to access activated neurons

“…A Cre-dependent version of RAM (CRAM) effectively labels GABAergic neurons. Since the enhancer sequences are highly conserved, RAM may also be applicable to other species, as was shown in flies and rats (59). …”

“…Furthermore, new reporters that allow users to genetically access multiple experiences in the same brain could directly compare two different experiences and help subtract away non-task relevant background cells. When characterizing new genetic strategies, side-by-side comparisons with existing technologies are particularly informative for potential users (59, 66). Developing methods that combine the advantages of existing tools (Table 1), such as the temporal precision of Ca 2+ -based methods with genetic accessibility, will enhance our ability to interrogate the causal relationships between active populations, circuit function, and animal behavior.…”