Frequency Specific Optogenetic Stimulation of the Locus Coeruleus Induces Task-Relevant Plasticity in the Motor Cortex.

The locus ceruleus (LC) is the primary source of neocortical noradrenaline, which is known to be involved in diverse brain functions including sensory perception, attention, and learning. Previous studies have shown that LC stimulation paired with sensory experience can induce task-dependent plasticity in the sensory neocortex and in the hippocampus. However, it remains unknown whether LC activation similarly impacts neural representations in the agranular motor cortical regions that are responsible for movement planning and production. In this study, we test whether optogenetic stimulation of the LC paired with motor performance is sufficient to induce task-relevant plasticity in the somatotopic cortical motor map. Male and female TH-Cre + rats were trained on a skilled reaching lever-pressing task emphasizing the use of the proximal forelimb musculature, and a viral approach was used to selectively express ChR2 in noradrenergic LC neurons. Once animals reached criterial behavioral performance, they received five training sessions in which correct task performance was paired with optogenetic stimulation of the LC delivered at 3, 10, or 30 Hz. After the last stimulation session, motor cortical mapping was performed using intracortical microstimulation. Our results show that lever pressing paired with LC stimulation at 10 Hz, but not at 3 or 30 Hz, drove the expansion of the motor map representation of the task-relevant proximal FL musculature. These findings demonstrate that phasic, training-paired activation of the LC is sufficient to induce experience-dependent plasticity in the agranular motor cortex and that this LC-driven plasticity is highly dependent on the temporal dynamics of LC activation.

Electroconvulsive Shock Does Not Impair the Reconsolidation of Cued and Contextual Pavlovian Threat Memory.

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.

Near-Infrared Light Triggered-Release in Deep Brain Regions Using Ultra-photosensitive Nanovesicles.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

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.

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.

Amygdala nuclei critical for emotional learning exhibit unique gene expression patterns.

The amygdala is a heterogeneous, medial temporal lobe structure that has been implicated in the formation, expression and extinction of emotional memories. This structure is composed of numerous nuclei that vary in cytoarchitectonics and neural connections. In particular the lateral nucleus of the amygdala (LA), central nucleus of the amygdala (CeA), and the basal (B) nucleus contribute an essential role to emotional learning. However, to date it is still unclear to what extent these nuclei differ at the molecular level. Therefore we have performed whole genome gene expression analysis on these nuclei to gain a better understanding of the molecular differences and similarities among these nuclei. Specifically the LA, CeA and B nuclei were laser microdissected from the rat brain, and total RNA was isolated from these nuclei and subjected to RNA amplification. Amplified RNA was analyzed by whole genome microarray analysis which revealed that 129 genes are differentially expressed among these nuclei. Notably gene expression patterns differed between the CeA nucleus and the LA and B nuclei. However gene expression differences were not considerably different between the LA and B nuclei. Secondary confirmation of numerous genes was performed by in situ hybridization to validate the microarray findings, which also revealed that for many genes, expression differences among these nuclei were consistent with the embryological origins of these nuclei. Knowing the stable gene expression differences among these nuclei will provide novel avenues of investigation into how these nuclei contribute to emotional arousal and emotional learning, and potentially offer new genetic targets to manipulate emotional learning and memory.

RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives.

Manipulating gene expression within and outside the nervous system is useful for interrogating gene function and developing therapeutic interventions for a variety of diseases. Several approaches exist which enable gene manipulation in preclinical models, and some of these have been approved to treat human diseases. For the last couple of decades, RNA interference (RNAi) has been a leading technique to knockdown (i.e., suppress) specific RNA expression. This has been partly due to the technology's simplicity, which has promoted its adoption throughout biomedical science. However, accumulating evidence indicates that this technology can possess significant shortcomings. This review highlights the overwhelming evidence that RNAi can be prone to off-target effects and is capable of inducing cytotoxicity in some cases. With this in mind, we consider alternative CRISPR/Cas-based approaches, which may be safer and more reliable for gene knockdown. We also discuss the pros and cons of each approach.

The Neurocircuitry of Posttraumatic Stress Disorder and Major Depression: Insights Into Overlapping and Distinct Circuit Dysfunction-A Tribute to Ron Duman.

The neurocircuitry that contributes to the pathophysiology of posttraumatic stress disorder and major depressive disorder, psychiatric conditions that exhibit a high degree of comorbidity, likely involves both overlapping and unique structural and functional changes within multiple limbic brain regions. In this review, we discuss neurobiological alterations that are associated with posttraumatic stress disorder and major depressive disorder and highlight both similarities and differences that may exist between these disorders to argue for the existence of a shared neurobiology. We highlight the key contributions based on preclinical studies, emerging from the late Professor Ronald Duman's research, that have shaped our understanding of the neurocircuitry that contributes to both the etiopathology and treatment of major depressive disorder and posttraumatic stress disorder.

A Highly Selective MNK Inhibitor Rescues Deficits Associated with Fragile X Syndrome in Mice.

Fragile X syndrome (FXS) is the most common inherited source of intellectual disability in humans. FXS is caused by mutations that trigger epigenetic silencing of the Fmr1 gene. Loss of Fmr1 results in increased activity of the mitogen-activated protein kinase (MAPK) pathway. An important downstream consequence is activation of the mitogen-activated protein kinase interacting protein kinase (MNK). MNK phosphorylates the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Excessive phosphorylation of eIF4E has been directly implicated in the cognitive and behavioral deficits associated with FXS. Pharmacological reduction of eIF4E phosphorylation is one potential strategy for FXS treatment. We demonstrate that systemic dosing of a highly specific, orally available MNK inhibitor, eFT508, attenuates numerous deficits associated with loss of Fmr1 in mice. eFT508 resolves a range of phenotypic abnormalities associated with FXS including macroorchidism, aberrant spinogenesis, and alterations in synaptic plasticity. Key behavioral deficits related to anxiety, social interaction, obsessive and repetitive activities, and object recognition are ameliorated by eFT508. Collectively, this work establishes eFT508 as a potential means to reverse deficits associated with FXS.

Hippocampal mitogen-activated protein kinase phosphatase-1 regulates behavioral and systemic effects of chronic corticosterone administration.

Clinical reports indicate a bidirectional relationship between mental illness and chronic systemic diseases. However, brain mechanisms linking chronic stress and development of mood disorders to accompanying peripheral organ dysfunction are still not well characterized in animal models. In the current study, we investigated whether activation of hippocampal mitogen-activated protein kinase phosphatase-1 (MKP-1), a key factor in depression pathophysiology, also acts as a mediator of systemic effects of stress. First, we demonstrated that treatment with the glucocorticoid receptor (GR) agonist dexamethasone or acute restraint stress (ARS) significantly increased Mkp-1 mRNA levels within the rat hippocampus. Conversely, administration of the GR antagonist mifepristone 30 min before ARS produced a partial blockade of Mkp-1 upregulation, suggesting that stress activates MKP-1, at least in part, through upstream GR signaling. Chronic corticosterone (CORT) administration evoked comparable increases in hippocampal MKP-1 protein levels and produced a robust increase in behavioral emotionality. In addition to behavioral deficits, chronic CORT treatment also produced systemic pathophysiological effects. Elevated levels of renal inflammation protein markers (NGAL and IL18) were observed suggesting tissue damage and early kidney impairment. In a rescue experiment, the effects of CORT on development of depressive-like behaviors and increased NGAL and IL18 protein levels in the kidney were blocked by CRISPR-mediated knockdown of hippocampal Mkp-1 prior to CORT exposure. In sum, these findings further demonstrate that MKP-1 is necessary for development of enhanced behavioral emotionality, while also suggesting a role in stress mechanisms linking brain dysfunction and systemic illness such as kidney disease.

Identification of plasticity-associated genes regulated by Pavlovian fear conditioning in the lateral amygdala.

Most recent studies aimed at defining the cellular and molecular mechanisms of Pavlovian fear conditioning have focused on protein kinase signaling pathways and the transcription factor cAMP-response element binding protein (CREB) that promote fear memory consolidation in the lateral nucleus of the amygdala (LA). Despite this progress, there still remains a paucity of information regarding the genes downstream of CREB that are required for long-term fear memory formation in the LA. We have adopted a strategy of using microarray technology to initially identify genes induced within the dentate gyrus following in vivo long-term potentiation (LTP) followed by analysis of whether these same genes are also regulated by fear conditioning within the LA. In the present study, we first identified 34 plasticity-associated genes that are induced within 30 min following LTP induction utilizing a combination of DNA microarray, qRT-PCR, and in situ hybridization. To determine whether these genes are also induced in the LA following Pavlovian fear conditioning, we next exposed rats to an auditory fear conditioning protocol or to control conditions that do not support fear learning followed by qRT-PCR on mRNA from microdissected LA samples. Finally, we asked whether identified genes induced by fear learning in the LA are downstream of the extracellular-regulated kinase/mitogen-activated protein kinase signaling cascade. Collectively, our findings reveal a comprehensive list of genes that represent the first wave of transcription following both LTP induction and fear conditioning that largely belong to a class of genes referred to as 'neuronal activity dependent genes' that are likely calcium, extracellular-regulated kinase/mitogen-activated protein kinase, and CREB-dependent.

Genetically Engineering the Nervous System with CRISPR-Cas.

The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for genetic interrogation of the normal and diseased nervous system. Additionally, we discuss limitations of the CRISPR editing system and suggest how future modifications to existing platforms may advance our understanding of the brain.

The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is required for memory consolidation of pavlovian fear conditioning in the lateral amygdala.

The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene that has been widely implicated in hippocampal-dependent learning and memory and is believed to play an integral role in synapse-specific plasticity. Here, we examined the role of Arc/Arg3.1 in amygdala-dependent Pavlovian fear conditioning. We first examined the regulation of Arc/Arg3.1 mRNA and protein after fear conditioning and LTP-inducing stimulation of thalamic inputs to the lateral amygdala (LA). Quantitative real-time PCR, in situ hybridization, Western blotting and immunohistochemistry revealed a significant upregulation of Arc/Arg3.1 mRNA and protein in the LA relative to controls. In behavioral experiments, intra-LA infusion of an Arc/Arg3.1 antisense oligodeoxynucleotide (ODN) was observed to be anatomically restricted to the LA, taken up by LA cells, and to promote significant knockdown of Arc/Arg3.1 protein. Rats given intra-LA infusions of multiple doses of the Arc/Arg3.1 ODN showed an impairment of LTM (tested approximately 24 later), but no deficit in STM (tested 3 h later) relative to controls infused with scrambled ODN. Finally, to determine whether upregulation of Arc/Arg3.1 occurs downstream of ERK/MAPK activation, we examined Arc/Arg3.1 expression in rats given intra-LA infusion of the MEK inhibitor U0126. Relative to vehicle controls, infusion of U0126 impaired training-induced increases in Arc/Arg3.1 expression. These findings suggest that Arc/Arg3.1 expression in the amygdala is required for fear memory consolidation, and further suggest that Arc/Arg3.1 regulation in the LA is downstream of the ERK/MAPK signaling pathway.

A mechanism of nucleocytoplasmic trafficking for the homeodomain protein PRH.

Proline-rich homeodomain (PRH)/hematopoietically expressed homeodomain (Hex) is a homeodomain protein that plays an important role in early embryonic patterning and hematopoiesis. PRH can act as either a tumor suppressor or an oncogene and its expression is dysregulated in certain types of lymphoid and myeloid leukemias. Aberrant exclusion of PRH from the nuclei has been associated with thyroid and breast cancers and a subset of myeloid leukemias. Accordingly, nuclear localization of PRH was found to be necessary for the inhibition of eIF4E-dependent transformation. Since PRH's nuclear-cytoplasmic localization has been associated with neoplastic transformation we sought to better understand how PRH is transported to the nuclear compartment. Here, we report an essential element that controls the mechanism of PRH nucleocytoplasmic trafficking, namely that it is imported into the nuclei by Karyopherin/Importin 7. Kap7 was identified as a binding partner for PRH in a GST-pull down from a HeLa cell protein lysate, followed by mass-spectrometry. The Kap7-PRH complex is dissociated in the presence of RanGTP, as expected for a nuclear import complex. Kap7 can bind directly to PRH in a GST-pull down assay with purified proteins, as well as mediates the transport of PRH to the nuclear compartment in a digitonin permeabilized cells assay. Finally, in vivo depletion of Kap7 dramatically reduces accumulation of PRH in the nucleus. Our data open the way for investigations of the mechanism of perturbed PRH localization in tumors and possible therapeutic interventions.

The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase.

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade.

The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase.

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.

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.

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.

Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.