Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins.

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity.

Extracellular Proximity Labeling Reveals an Expanded Interactome for the Matrisome Protein TIMP2.

Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

Nucleus accumbens local circuit for cue-dependent aversive learning.

Response to threatening environmental stimuli requires detection and encoding of important environmental features that dictate threat. Aversive events are highly salient, which promotes associative learning about stimuli that signal this threat. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. We describe a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that putative nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor-expressing projection neurons are required for cue-dependent fear learning. Additionally, we find that fear learning and recall is dependent on distinct projection neuron subtypes. Our work demonstrates a critical role for nucleus accumbens substance P in cue-dependent aversive learning.

Andy Golden: Mentorship through the Years.

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Expression of the excitatory opsin ChRERα can be traced longitudinally in rat and nonhuman primate brains with PET imaging.

Optogenetics is a widely used technology with potential for translational research. A critical component of such applications is the ability to track the location of the transduced opsin in vivo. To address this problem, we engineered an excitatory opsin, ChRERα (hChR2(134R)-V5-ERα-LBD), that could be visualized using positron emission tomography (PET) imaging in a noninvasive, longitudinal, and quantitative manner. ChRERα consists of the prototypical excitatory opsin channelrhodopsin-2 (ChR2) and the ligand-binding domain (LBD) of the human estrogen receptor α (ERα). ChRERα showed conserved ChR2 functionality and high affinity for [18F]16α-fluoroestradiol (FES), an FDA-approved PET radiopharmaceutical. Experiments in rats demonstrated that adeno-associated virus (AAV)-mediated expression of ChRERα enables neural circuit manipulation in vivo and that ChRERα expression could be monitored using FES-PET imaging. In vivo experiments in nonhuman primates (NHPs) confirmed that ChRERα expression could be monitored at the site of AAV injection in the primary motor cortex and in long-range neuronal terminals for up to 80 weeks. The anatomical connectivity map of the primary motor cortex identified by FES-PET imaging of ChRERα expression overlapped with a functional connectivity map identified using resting state fMRI in a separate cohort of NHPs. Overall, our results demonstrate that ChRERα expression can be mapped longitudinally in the mammalian brain using FES-PET imaging and can be used for neural circuit modulation in vivo.

Nucleus Accumbens Local Circuit for Cue-Dependent Aversive Learning.

Response to threatening environmental stimuli requires detection and encoding of important environmental features that dictate threat. Aversive events are highly salient which promotes associative learning about stimuli that signal this threat. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. We uncovered a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that putative nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor expressing projection neurons is required for cue-dependent fear learning. Additionally, we found fear learning and recall were dependent on distinct projection-neuron subtypes. Our work demonstrates a critical role for Nucleus Accumbens substance P in cue-dependent aversive learning.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

Novel GLCCI1-BRAF fusion drives kinase signaling in a case of pheochromocytomatosis.

Introduction: Recurrent and metastatic pheochromocytoma (PCC) are rare advanced endocrine neoplasms with limited treatment options. Insight into the pathogenic molecular alterations in patients with advanced PCC can provide therapeutic options for precisely targeting dysregulated pathways. Objective: We report the discovery and characterization of a novel BRAF-containing fusion transcript and its downstream molecular alterations in a patient with recurrent PCC with peritoneal seeding (pheochromocytomatosis). Methods: We reviewed the medical record of a patient with pheochromocytomatosis. A comprehensive pan-cancer molecular profiling using next-generation sequencing (NGS) as well as confirmatory real-time-quantitative PCR were performed on surgical specimens. BRAF rearrangement and downstream molecular changes were assayed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Western blot was used to assess the in vitro activation of the mitogen-activated protein kinase (MAPK) signaling pathway and the EMT markers in transfected HEK-293 cells. Results: The NGS analysis of a specimen from a 72-year-old female patient with pheochromocytomatosis showed an in-frame fusion of exon 3 of Glucocorticoid Induced 1 (GLCCI1) to exon 9 of BRAF. The upstream auto-inhibitory domain of BRAF was excluded from the GLCCI1-BRAF fusion; however, the downstream BRAF kinase domain was intact. A BRAF rearrangement was confirmed via a BRAF-specific break-apart FISH assay. Four separate tumor foci harbored GLCCI1-BRAF fusion. IHC demonstrated increased phosphorylated MEK. HEK-293 cells transfected with the GLCCI1-BRAF fusion demonstrated increased phosphorylated MEK as well as higher expression of EMT markers SNAI1 and ZEB1 in vitro. Conclusion: We demonstrate a novel pathogenic gene fusion of GLCCI1 with the oncogenic kinase domain of BRAF, resulting in an activation of the MAPK signaling pathway and EMT markers. Thus, this patient may benefit from clinically available MEK and/or BRAF inhibitors when systemic therapy is indicated. Summary Statement: This report is the first of GLCCI1 fused to BRAF in a human neoplasm and only the second BRAF-containing fusion transcript in PCC. Detailed molecular characterization of PCC can be a valuable tool in managing patients with recurrent PCC and pheochromocytomatosis that represents a significant clinical challenge.

Effects of Withdrawal from Cocaine Self-Administration on Rat Orbitofrontal Cortex Parvalbumin Neurons Expressing Cre recombinase: Sex-Dependent Changes in Neuronal Function and Unaltered Serotonin Signaling.

The orbitofrontal cortex (OFC) is a brain region involved in higher-order decision-making. Rodent studies show that cocaine self-administration (CSA) reduces OFC contribution to goal-directed behavior and behavioral strategies to avoid drug intake. This change in OFC function persists for many weeks after cocaine withdrawal, suggesting involvement in the process of addiction. The mechanisms underlying impaired OFC function by cocaine are not well-understood. However, studies implicate altered OFC serotonin (5-HT) function in disrupted cognitive processes during addiction and other psychiatric disorders. Thus, it is hypothesized that cocaine impairment of OFC function involves changes in 5-HT signaling, and previous work shows that 5-HT1A and 5-HT2A receptor-mediated effects on OFC pyramidal neurons (PyNs) are impaired weeks after cocaine withdrawal. However, 5-HT effects on other contributors to OFC circuit function have not been fully investigated, including the parvalbumin-containing, fast-spiking interneurons (OFCPV), whose function is essential to normal OFC-mediated behavior. Here, 5-HT function in naive rats and those withdrawn from CSA were evaluated using a novel rat transgenic line in which the rat parvalbumin promoter drives Cre-recombinase expression to permit identification of OFCPV cells by fluorescent reporter protein expression. We find that whereas CSA altered basal synaptic and membrane properties of the OFCPV neurons in a sex-dependent manner, the effects of 5-HT on these cells were unchanged by CSA. These data suggest that the behavioral effects of dysregulated OFC 5-HT function caused by cocaine experience are primarily mediated by changes in 5-HT signaling at PyNs, and not at OFCPV neurons.

Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.

Abstinence-dependent dissociable central amygdala microcircuits control drug craving.

We recently reported that social choice-induced voluntary abstinence prevents incubation of methamphetamine craving in rats. This inhibitory effect was associated with activation of protein kinase-Cδ (PKCδ)-expressing neurons in central amygdala lateral division (CeL). In contrast, incubation of craving after forced abstinence was associated with activation of CeL-expressing somatostatin (SOM) neurons. Here we determined the causal role of CeL PKCδ and SOM in incubation using short-hairpin RNAs against PKCδ or SOM that we developed and validated. We injected two groups with shPKCδ or shCtrlPKCδ into CeL and trained them to lever press for social interaction (6 d) and then for methamphetamine infusions (12 d). We injected two other groups with shSOM or shCtrlSOM into CeL and trained them to lever press for methamphetamine infusions (12 d). We then assessed relapse to methamphetamine seeking after 1 and 15 abstinence days. Between tests, the rats underwent either social choice-induced abstinence (shPKCδ groups) or homecage forced abstinence (shSOM groups). After test day 15, we assessed PKCδ and SOM, Fos, and double-labeled expression in CeL and central amygdala medial division (CeM). shPKCδ CeL injections decreased Fos in CeL PKCδ-expressing neurons, increased Fos in CeM output neurons, and reversed the inhibitory effect of social choice-induced abstinence on incubated drug seeking on day 15. In contrast, shSOM CeL injections decreased Fos in CeL SOM-expressing neurons, decreased Fos in CeM output neurons, and decreased incubated drug seeking after 15 forced abstinence days. Our results identify dissociable central amygdala mechanisms of abstinence-dependent expression or inhibition of incubation of craving.

Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression.

The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1α (EF1α) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.

Escalated Alcohol Self-Administration and Sensitivity to Yohimbine-Induced Reinstatement in Alcohol Preferring Rats: Potential Role of Neurokinin-1 Receptors in the Amygdala.

Genetic factors significantly contribute to the risk for developing alcoholism. To study these factors and other associated phenotypes, rodent lines have been developed using selective breeding for high alcohol preference. One of these models, the alcohol preferring (P) rat, has been used in hundreds of preclinical studies over the last few decades. However, very few studies have examined relapse-like behavior in this rat strain. In this study, we used operant self-administration and yohimbine-induced reinstatement models to examine relapse-like behavior in P rats. Our previous work has demonstrated that P rats show increased expression of the neurokinin-1 receptor (NK1R) in the central nucleus of the amygdala (CeA), and this functionally contributes to escalated alcohol consumption in this strain. We hypothesized that P rats would show increased sensitivity to yohimbine-induced reinstatement that is also mediated by NK1R in the CeA. Using Fos staining, site-specific infusion of NK1R antagonist, and viral vector overexpression, we examined the influence of NK1R on the sensitivity to yohimbine-induced reinstatement of alcohol seeking. We found that P rats displayed increased sensitivity to yohimbine-induced reinstatement as well as increased neuronal activation in the CeA after yohimbine injection compared to the control Wistar strain. Intra-CeA infusion of NK1R antagonist attenuates yohimbine-induced reinstatement in P rats. Conversely, upregulation of NK1R within the CeA of Wistar rats increases alcohol consumption and sensitivity to yohimbine-induced reinstatement. These findings suggest that NK1R upregulation in the CeA contributes to multiple alcohol-related phenotypes in the P rat, including alcohol consumption and sensitivity to relapse.

Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.

MANF deletion abrogates early larval Caenorhabditis elegans stress response to tunicamycin and Pseudomonas aeruginosa.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is the only human neurotrophic factor with an evolutionarily-conserved C. elegans homolog, Y54G2A.23 or manf-1. MANF is a small, soluble, endoplasmic-reticulum (ER)-resident protein that is secreted upon ER stress and promotes survival of target cells such as neurons. However, the role of MANF in ER stress and its mechanism of cellular protection are not clear and the function of MANF in C. elegans is only beginning to emerge. In this study, we show that depletion of C. elegans manf-1 causes a slight decrease in lifespan and brood size; furthermore, combined depletion of manf-1 and the IRE-1/XBP-1 ER stress/UPR pathway resulted in sterile animals that did not produce viable progeny. We demonstrate upregulation of markers of ER stress in L1 larval nematodes, as measured by hsp-3 and hsp-4 transcription, upon depletion of manf-1 by RNAi or mutation; however, there was no difference in tunicamycin-induced expression of hsp-3 and hsp-4 between wild-type and MANF-deficient worms. Surprisingly, larval growth arrest observed in wild-type nematodes reared on tunicamycin is completely prevented in the manf-1 (tm3603) mutant. Transcriptional microarray analysis revealed that manf-1 mutant L1 larvae exhibit a novel modulation of innate immunity genes in response to tunicamycin. The hypothesis that manf-1 negatively regulates the innate immunity pathway is supported by our finding that the development of manf-1 mutant larvae compared to wild-type larvae is not inhibited by growth on P. aeruginosa. Together, our data represent the first characterization of C. elegans MANF as a key modulator of organismal ER stress and immunity.

Ultrastructural localization of DREADDs in monkeys.

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

Ventral Pallidum Is the Primary Target for Accumbens D1 Projections Driving Cocaine Seeking.

Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.

Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.

Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.