Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum suppresses interaction with familiar mice.

Social preference, the decision to interact with one member of the same species over another, is critical to optimize social interactions. Thus, adult rodents favor interacting with novel conspecifics over familiar ones, but whether this social preference stems from neural circuits facilitating interactions with novel individuals or suppressing interactions with familiar ones remains unknown. Here, we identify neurons in the infra-limbic area (ILA) of the mouse prefrontal cortex that express the neuropeptide corticotropin-releasing hormone (CRH) and project to the dorsal region of the rostral lateral septum (rLS). We show how release of CRH during familiar encounters disinhibits rLS neurons, thereby suppressing social interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression in ILA during the first 2 post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.

CPEB3 low-complexity motif regulates local protein synthesis via protein-protein interactions in neuronal ribonucleoprotein granules.

Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs) are a type of biomolecular condensate necessary for local protein synthesis and are involved in synaptic plasticity and long-term memory. Most of the proteins in RNPs possess low-complexity motifs (LCM), allowing for increased promiscuity of protein-protein interactions. Here, we describe the importance of protein-protein interactions mediated by the LCM of RNA-binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3). CPEB3 is necessary for long-term synaptic plasticity and memory persistence, but the mechanisms involved are still not completely elucidated. We now present key mechanisms involved in its regulation of synaptic plasticity. We find that CPEB3-LCM plays a role in appropriate local protein synthesis of messenger ribonucleic acid (mRNA) targets, through crucial protein-protein interactions that drive localization to neuronal Decapping protein 1 (DCP1)-bodies. Translation-promoting CPEB3 and translation-inhibiting CPEB1 are packaged into neuronal RNP granules immediately after chemical long-term potentiation is induced, but only translation-promoting CPEB3 is repackaged to these organelles at later time points. This localization to neuronal RNP granules is critical for functional influence on translation as well as overall local protein synthesis (measured as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) insertion into the membrane and localization to the synapse). We therefore conclude that protein-protein interaction between the LCM of CPEB3 plays a critical role in local protein synthesis by utilizing neuronal RNP granules.

A direct lateral entorhinal cortex to hippocampal CA2 circuit conveys social information required for social memory.

The hippocampus is essential for different forms of declarative memory, including social memory, the ability to recognize and remember a conspecific. Although recent studies identify the importance of the dorsal CA2 region of the hippocampus in social memory storage, little is known about its sources of social information. Because CA2, like other hippocampal regions, receives its major source of spatial and non-spatial information from the medial and lateral subdivisions of entorhinal cortex (MEC and LEC), respectively, we investigated the importance of these inputs for social memory. Whereas MEC inputs to CA2 are dispensable, the direct inputs to CA2 from LEC are both selectively activated during social exploration and required for social memory. This selective behavioral role of LEC is reflected in the stronger excitatory drive it provides to CA2 compared with MEC. Thus, a direct LEC → CA2 circuit is tuned to convey social information that is critical for social memory.

Enkephalin release from VIP interneurons in the hippocampal CA2/3a region mediates heterosynaptic plasticity and social memory.

The hippocampus contains a diverse array of inhibitory interneurons that gate information flow through local cortico-hippocampal circuits to regulate memory storage. Although most studies of interneurons have focused on their role in fast synaptic inhibition mediated by GABA release, different classes of interneurons express unique sets of neuropeptides, many of which have been shown to exert powerful effects on neuronal function and memory when applied pharmacologically. However, relatively little is known about whether and how release of endogenous neuropeptides from inhibitory cells contributes to their behavioral role in regulating memory formation. Here we report that vasoactive intestinal peptide (VIP)-expressing interneurons participate in social memory storage by enhancing information transfer from hippocampal CA3 pyramidal neurons to CA2 pyramidal neurons. Notably, this action depends on release of the neuropeptide enkephalin from VIP neurons, causing long-term depression of feedforward inhibition onto CA2 pyramidal cells. Moreover, VIP neuron activity in the CA2 region is increased selectively during exploration of a novel conspecific. Our findings, thus, enhance our appreciation of how GABAergic neurons can regulate synaptic plasticity and mnemonic behavior by demonstrating that such actions can be mediated by release of a specific neuropeptide, rather than through classic fast inhibitory transmission.

Is Arc mRNA Unique: A Search for mRNAs That Localize to the Distal Dendrites of Dentate Gyrus Granule Cells Following Neural Activity.

There have been several attempts to identify which RNAs are localized to dendrites; however, no study has determined which RNAs localize to the dendrites following the induction of synaptic activity. We sought to identify all RNA transcripts that localize to the distal dendrites of dentate gyrus granule cells following unilateral high frequency stimulation of the perforant pathway (pp-HFS) using Sprague Dawley rats. We then utilized laser microdissection (LMD) to very accurately dissect out the distal 2/3rds of the molecular layer (ML), which contains these dendrites, without contamination from the granule cell layer, 2 and 4 h post pp-HFS. Next, we purified and amplified RNA from the ML and performed an unbiased screen for 27,000 RNA transcripts using Affymetrix microarrays. We determined that Activity Regulated Cytoskeletal Protein (Arc/Arg3.1) mRNA, exhibited the greatest fold increase in the ML at both timepoints (2 and 4 h). In total, we identified 31 transcripts that increased their levels within the ML following pp-HFS across the two timepoints. Of particular interest is that one of these identified transcripts was an unprocessed micro-RNA (pri-miR132). Fluorescent in situ hybridization and qRT-PCR were used to confirm some of these candidate transcripts. Our data indicate Arc is a unique activity dependent gene, due to the magnitude that its activity dependent transcript localizes to the dendrites. Our study determined other activity dependent transcripts likely localize to the dendrites following neural activity, but do so with lower efficiency compared to Arc.

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.

The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.

Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning.

The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13-1E13 GC/mL; 1 µl/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1 µl/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1 µl/side), designed to express shArc, shLuc, shCntrl or shRNAs designed to target Egr1 (shEgr1), or GluN2A (shGluN2A), or no shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene knockdown of Egr1 and GluN2A compared to the other groups examined respectively, but Arc was not knocked down in the shArc group under these conditions. Differences in fear conditioning among the shLuc, shCntrl, shArc and shEgr1 groups were not detected under these circumstances; however, the shGluN2A group exhibited significantly impaired fear conditioning compared to most of the groups, indicating that gene specific deficits in fear conditioning could be observed utilizing viral mediated delivery of shRNA. Collectively, these data indicate that viral mediated shRNA expression was toxic to neurons in vivo, under all viral titers examined and this toxicity in some cases may be masking gene specific changes in learning. Therefore, the use of this technology in behavioral neuroscience warrants a heightened level of careful consideration and potential methods to alleviate shRNA induced toxicity are discussed.