The RNA-guided Cas9 nuclease, from the type II prokaryotic clustered regularly interspersed short palindromic repeats (CRISPR) adaptive immune system, has been adapted by scientists to enable site specific genome editing of eukaryotic cells both in vitro and in vivo. Previously, we reported the development of an adeno-associated virus (AAV)-mediated CRISPR Streptococcus pyogenes (Sp) Cas9 system, in which the genome editing function can be regulated by controlling the expression of the guide RNA (sgRNA) in a doxycycline (Dox)-dependent manner. Here, we report the development of an AAV vector tool kit utilizing the Cas9 from Staphylococcus aureus (SaCas9). We demonstrate in vitro genome editing in human derived 293FT cells and mouse derived Neuro2A (N2A) cells and in vivo in neurons of the mouse brain. We also demonstrate the ability to regulate the induction of genome editing temporally with Dox and spatially with Cre-recombinase. The combination of these systems enables AAV-mediated CRISPR/Cas9 genome editing to be regulated both spatially and temporally.

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 STATEMENTMemory 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.

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.

Autism Spectrum Disorders (ASD) are complex neurodevelopmental disorders characterized by repetitive behavior and impaired social communication and interactions. Apart from these core symptoms, a significant number of ASD individuals display higher levels of anxiety and some ASD individuals exhibit impaired emotional learning. We therefore sought to further examine anxiety and emotional learning in an environmentally induced animal model of ASD that utilizes the administration of the known teratogen, valproic acid (VPA) during gestation. Specifically we exposed dams to one of two different doses of VPA (500 and 600 mg/kg) or vehicle on day 12.5 of gestation and examined the resultant progeny. Our data indicate that animals exposed to VPA in utero exhibit enhanced anxiety in the open field test and normal object recognition memory compared to control animals. Animals exposed to 500 mg/kg of VPA displayed normal acquisition of auditory fear conditioning, and exhibited reduced extinction of fear memory and normal litter survival rates as compared to control animals. We observed that animals exposed to 600 mg/kg of VPA exhibited a significant reduction in the acquisition of fear conditioning, a significant reduction in social interaction and a significant reduction in litter survival rates as compared to control animals. VPA (600 mg/kg) exposed animals exhibited similar shock sensitivity and hearing as compared to control animals indicating the fear conditioning deficit observed in these animals was not likely due to sensory deficits, but rather due to deficits in learning or memory retrieval. In conclusion, considering that progeny from dams exposed to rather similar doses of VPA exhibit striking differences in emotional learning, the VPA model may serve as a useful tool to explore the molecular and cellular mechanisms that contribute to not only ASD, but also emotional learning.

Existing memories, when retrieved under certain circumstances, can undergo modification through the protein synthesis-dependent process of reconsolidation. Disruption of this process can lead to the weakening of a memory trace, an approach which is being examined as a potential treatment for disorders characterized by pathological memories, such as Post-Traumatic Stress Disorder. The success of this approach relies upon the ability to robustly attenuate reconsolidation; however, the available literature brings into question the reliability of the various drugs used to achieve such a blockade. The identification of a drug or intervention that can reliably disrupt reconsolidation without requiring intracranial access for administration would be extremely useful. Electroconvulsive shock (ECS) delivered after memory retrieval has been demonstrated in some studies to disrupt memory reconsolidation; however, there exists a paucity of literature characterizing its effects on Pavlovian fear memory. Considering this, we chose to examine ECS as an inexpensive and facile means to impair reconsolidation in rats. Here we show that electroconvulsive seizure induction, when administered after memory retrieval, (immediately, after 30 min, or after 1 h), does not impair the reconsolidation of cued or contextual Pavlovian fear memories. On the contrary, ECS administration immediately after extinction training may modestly impair the consolidation of fear extinction memory.