Author Correction: Hippocampus-driven feed-forward inhibition of the prefrontal cortex mediates relapse of extinguished fear.

In the version of this article initially published, the traces in Fig. 1j and in Fig. 1k, right, were duplicated from the corresponding traces in Fig. 1c, bottom, and Fig. 1d, bottom right. The error has been corrected in the HTML and PDF versions of the article.

Hippocampus-driven feed-forward inhibition of the prefrontal cortex mediates relapse of extinguished fear.

The medial prefrontal cortex (mPFC) has been implicated in the extinction of emotional memories, including conditioned fear. We found that ventral hippocampal (vHPC) projections to the infralimbic (IL) cortex recruited parvalbumin-expressing interneurons to counter the expression of extinguished fear and promote fear relapse. Whole-cell recordings ex vivo revealed that optogenetic activation of vHPC input to amygdala-projecting pyramidal neurons in the IL was dominated by feed-forward inhibition. Selectively silencing parvalbumin-expressing, but not somatostatin-expressing, interneurons in the IL eliminated vHPC-mediated inhibition. In behaving rats, pharmacogenetic activation of vHPC→IL projections impaired extinction recall, whereas silencing IL projectors diminished fear renewal. Intra-IL infusion of GABA receptor agonists or antagonists, respectively, reproduced these effects. Together, our findings describe a previously unknown circuit mechanism for the contextual control of fear, and indicate that vHPC-mediated inhibition of IL is an essential neural substrate for fear relapse.

Kctd13 deletion reduces synaptic transmission via increased RhoA.

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.

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.

Increasing the GluN2A/GluN2B Ratio in Neurons of the Mouse Basal and Lateral Amygdala Inhibits the Modification of an Existing Fear Memory Trace.

UNLABELLED: Reconsolidation updating is a form of memory modification in which an existing memory can become destabilized upon retrieval and subsequently be modified via protein-synthesis-dependent reconsolidation. However, not all memories appear to destabilize upon retrieval and thus are not modifiable via reconsolidation updating approaches and the neurobiological basis for this remains poorly understood. Here, we report that auditory fear memories created with 10 tone-shock pairings are resistant to retrieval-dependent memory destabilization and are associated with an increase in the synaptic GluN2A/GluN2B ratio in neurons of the basal and lateral amygdala (BLA) compared with weaker fear memories created via one or three tone-shock pairings. To increase the GluN2A/GluN2B ratio after learning, we generated a line of mice that expresses an inducible and doxycycline-dependent GFP-GluN2A transgene specifically in α-CaMKII-positive neurons. Our findings indicate that increasing the GluN2A/GluN2B ratio in BLA α-CaMKII-positive neurons after a weak fear memory has consolidated inhibits retrieval-dependent memory destabilization and modification of the fear memory trace. This was associated with a reduction in retrieval-dependent AMPA receptor trafficking, as evidenced by a reduction in retrieval-dependent phosphorylation of GluR1 at serine-845. In addition, we determined that increasing the GluN2A/GluN2B ratio before fear learning significantly impaired long term memory consolidation, whereas short-term memory remained unaltered. An increase in the GluN2A/GluN2B ratio after fear learning had no influence on fear extinction or expression. Our results underscore the importance of NMDAR subunit composition for memory destabilization and suggest a mechanism for why some memories are resistant to modification. SIGNIFICANCE STATEMENT: Memory modification using reconsolidation updating is being examined as one of the potential treatment approaches for attenuating maladaptive memories associated with emotional disorders. However, studies have shown that, whereas weak memories can be modified using reconsolidation updating, strong memories can be resistant to this approach. Therefore, treatments targeting the reconsolidation process are unlikely to be clinically effective unless methods are devised to enhance retrieval-dependent memory destabilization. Currently, little is known about the cellular and molecular events that influence the induction of reconsolidation updating. Here, we determined that an increase in the GluN2A/GluN2B ratio interferes with retrieval-dependent memory destabilization and inhibits the initiation of reconsolidation updating.

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.

The production of viral vectors designed to express large and difficult to express transgenes within neurons.

BACKGROUND: Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons. RESULTS: We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains. CONCLUSIONS: We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.

Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala.

BACKGROUND: In recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype's ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (αCaMKII). RESULTS: We determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5's decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined. CONCLUSIONS: For laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.