United States

Background 
Exosomes are extracellular vesicle (EV) nanocarriers responsible for delivering biologics including lipids, proteins, and nucleic acids. EVs are an attractive therapeutic weapon for delivering drugs and biologics to cells and tissues. Utilizing EVs for clinical applications is based on their low toxicity, ability to target to specific cells and tissues, and can be bioengineered to enhance their activities. Significant challenges remain before exosomes can be widely used in clinical settings. These include developing improved methods for purifying exosomes, enhancing the targeting of exosomes to diseased tissue, and boosting the efficiency of engineering exosomes for disease-specific applications. 
Exosomes need to be engineered in a more efficient way for broad therapeutic application. The lack of efficient intracellular bioengineering technologies to modify produced exosomes is a major obstacle to the widespread use of EVs for therapeutic applications. 
Advances in mammalian synthetic chromosome design and engineering offer a potential improvement in EV manufacturing. Our aim is to adopt the Artificial Chromosome Expression System (ACE System), an autonomous chromosome-based “circuit-board” designed to carry single or multiple copies of genes or DNA elements of interest for EV engineering.

Methods 
We have demonstrated that human miRNAs expressed from the ACE platform residing in a CHO-based cell line failed to process human miRNAs into CHO exosomes. This suggests that the processing of miRNAs from primary gene transcripts may be regulated in a species-specific manner. Thus, the ACE chromosome has been transferred into human HEK293 cells. Engineered miRNAs have been cloned downstream of a modified green fluorescent protein (AcGFP1) expressed from a doxycycline inducible promoter system retrofitted to contain ACE platform recombination system. 

Results 
Our results indicated that the ACE synthetic chromosome platform can be successfully engineered to express miRNAs as part of an initial doxycycline inducible AcGFP1 transcript. Furthermore, isolation of resulting EVs from the ACE engineered HEK293 cells indicated the presence of the expressed miRNAs. 

Conclusion 
From our work, we propose that the ACE synthetic platform has the potential to serve as a genetic circuit-board for modifying EVs of therapeutic value. In addition, the ACE synthetic chromosome is autonomous to the host cellular genome, i.e., non-integrating and non-mutagenic, thus would provide an increasing safety profile for using our developed synthetic chromosome technology in an ex vivo cellular therapy format. Future efforts will focus on enhancing the efficiency of our bioengineering approach for a specific disease indication.

AMA Research Challenge 2023

The Engineering of Human Synthetic Chromosomes for Exosome-based Therapeutics

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Charles Lopresto, DO

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AMA Research Challenge 2022

Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


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AMA would like to thank Laurel Road for sponsoring a $10,000 grand prize for the winner of the AMA Research Challenge. Laurel Road is not involved in the judging or scoring process to select the winner. The winner will be announced on February 4, 2024 during the AMA Research Challenge finals. Payment of the grand prize will be directly from Laurel Road. See the AMA Research Challenge Official Rules for terms and conditions.

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Background
Chromosome-level mutations play an essential role in the mechanisms behind speciation and disease. Analyzing massive genomic data to elucidate orthologous chromosome pairings between primate species is currently limited by computational constraints. Specifically, computational approaches have been limited by their inability to determine balanced chromosomal mutations rapidly, accurately, and cost-effectively. As the Alu family of transposable elements is widely conserved and prevalent (10%) among primate genomes, it is ideal for interspecies comparison and employed in our novel method. Our investigation employs unique, advanced data science techniques to classify orthologous chromosomes across primates, detect chromosomal inversions, and identify chromosomal translocations. We hypothesize that this unique approach will produce accurate results when compared to established literature, perform in less time relative to current methodologies, and be more cost-effective than its counterparts. 

Methods
We obtained and subsequently aligned Alu elements from 11 distinct primate species’ genomes on the University of California Santa Cruz’s Genome Table Browser. After computationally aggregating and statistically analyzing all primate Alu sequences, orthologous chromosome pairs were identified based on the highest frequency of homologous pairings across any two compared primate species. Using the orthologous chromosomes’ loci-specific pairing data, we detected statistically significant balanced inversions and translocations between species. 

Results
We successfully classified orthologous chromosomes between two primates with high z-scores (minimum z-score of 2.5 and maximum above 4), confirmed with previously published lists. Graphical representations of primate species, utilizing both subfamily-specific and all Alu elements, allowed for clear, statistically significant detection of chromosomal inversions, which were then validated with previously published genomic differences (10/10). Further loci-specific analysis identified every reported chromosomal translocation or change in orientation greater than 5 Mbp translocations (maximum p < 0.007 and regularly p < 1e–4). 

Conclusion
Our novel approach can be used to detect chromosomal abnormalities in primate genomes, making it a powerful tool for evolutionary and oncological research. Furthermore, it does so in a manner that is far less expensive, lengthy, and resource-intensive, while retaining the accuracy of chromosomal mutation detection. As primates serve as an important model organism for basic science research in various subspecialties of medicine, this approach could have a significant impact on numerous biomedical fields. Future investigations will explore specific data-size constraints in interspecies comparisons and the method’s applicability in detecting mutational patterns in diseases affecting humans and other primate species.

Primate Chromosomal Abnormalities Detection Through Selective Sampling Across Large-Scale Genomic Data

Background
Kidney transplantation is a lifesaving treatment for patients with end-stage renal failure. However, the success of kidney transplantation is hindered by chronic kidney graft rejection. On average, transplanted kidneys last 9 years due to chronic rejection, requiring patients to undergo dialysis or re-transplantation. While advances have reduced the rates of acute rejection, little is known about preventing chronic rejection. In mice and humans, chronic rejection is characterized by a persistent T cell infiltrate. The cytokine IL-15 is elevated in renal graft rejection. However, it is unknown whether IL-15 signaling supports graft-specific T cells that drive chronic rejection. I hypothesize that IL-15 signaling is required for the long-term survival of graft-specific T cells, and that disrupting IL-15 signaling can prevent chronic rejection.

Methods
We transplanted MHC-mismatched kidneys (Balb/c.OVA x B6) to recipient mice (B6) followed by injection of OVA-specific CD8 T cells (OT-I cells). OT-I cells allow for tracking immune response in kidney grafts. Recipient native kidneys were removed during transplantation. Kidney grafts underwent analysis via flow cytometry, immunofluorescence, and histology.

The Cre-loxP system was used for temporal genetic deletion of CD122 or CD215. Anti-CD122 blocking antibody was administered intravenously to transplant mice. Graft rejection was monitored by signs of uremia (lethargy, decreased mobility, ruffled hair), serum creatinine, or death.

Results
We found evidence that IL-15 signaling was taking place in kidney grafts, with cells expressing CD215, a chaperone protein for IL-15, and CD8 T cells expressing CD122, a receptor subunit for sensing IL-15. Using a combination of conditional knockouts of CD215 and CD122, we found that genetic deletion of either CD215 or CD122 significantly reduced graft-specific CD8 T cells. Given the importance of IL-15 signaling, we treated transplant mice with anti-CD122 antibody, which significantly reduced graft-specific CD8 T cells, decreased graft infiltration and fibrosis, and prevented graft rejection.

Conclusion
IL-15 is a key component in the survival and function of graft-specific T cells and, ultimately, graft failure. Disrupting IL-15 signaling with anti-CD122 blocking antibody could serve as a potential approach to the prevention of chronic rejection.

Disrupting the Survival of Pathogenic T cells to Ensure the Survival of Kidney Transplants

Introduction: Metabolic Syndrome (MetS) is a constellation of risk factors including glucose intolerance, central obesity, hypertension, and dyslipidemia. Low-grade inflammation, possibly originating in inflamed fat, has been implicated in the genesis and pathophysiological consequences of MetS. MetS occasionally co-exists with renal artery stenosis (RAS) and aggravates outcomes partly through the release of pro-inflammatory cytokines. Mesenchymal stem/stromal cells (MSCs) are multipotent cells with robust regenerative and immunomodulatory potential. This study tested the hypothesis that MSC delivery would alternate fat inflammation in MetS+RAS.
Methods: Twenty-four pigs were randomly divided into four groups: Lean, MetS, MetS+RAS, and Mets+RAS+MSC (n=6 each). In MetS+RAS+MSC pigs autologous adipose tissue-derived MSCs (107) were injected into the renal artery 6 weeks after RAS induction. Four weeks later, renal function was studied in-vivo using CT. After euthanasia, adipose tissue samples were stained to assess inflammatory markers, regulatory T-cells, resident stem cells, T-bet+ B-cells, adipocyte size, and fat fibrosis. Pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), interleukin-6, and tumor necrosis factor-alpha immunoreactivity and levels were measured in fat and systemic blood, respectively.
Results: Injection of MSCs into MetS+RAS pigs did not affect renal function but slightly decreased blood pressure and circulating MCP-1 levels in systemic blood. Furthermore, in subcutaneous adipose tissue MSCs restored T-regulatory cells and endogenous stem cells, decreased T-bet+ B-cells, and downregulated inflammatory marker immunoreactivity. MSCs also restored adipocyte size and reduced fat fibrosis. 
Conclusion: Adipose tissue-derived MSCs attenuate fat inflammation in pigs with MetS+RAS. Hence, MSCs may represent a promising therapeutic strategy in metabolic disorders.

Mesenchymal Stem Cells Reverse Inflammatory Changes in the Adipose Tissue of Swine with Metabolic Syndrome and Renovascular Hypertension

Background
Prior information is routinely integrated in neocortex during decision-making. The orbitofrontal and posterior parietal cortex (OFC, PPC) directly represent and use stimulus history to efficiently guide ongoing behavior. To integrate prior stimulus information selectively and direct behavior flexibly, a mechanism for routing information is necessary. Prior evidence suggests critical structures in the mesial temporal lobe (MTL) route information during navigation. The neural dynamics underlying the behavioral effects of prior experiences remain poorly understood. Through studying the evolution of electrophysiologic signatures in human intracranial recordings during learning, we investigate how experiences are routed to modify processing in neocortex and direct behavior.

Methods
We trained eleven treatment-refractory epilepsy participants with a reward-based decision-making task with reversal learning in two contexts. We used a Bayesian framework to infer expectation for reward, a computation that depends on prior choices and reward outcomes. We simultaneously recorded neural ensemble activity from high frequency (HF, 30-120 Hz) neural power in intracranial electrodes implanted across the brain. To measure the representation of history-dependent computations, we performed a trial-wise regression of HF neural power on expectation. We monitored the effect of experience on neocortical processing by measuring how variations in representations changed with variations in expectation using representational similarity and multivariate pattern analyses. We tracked how much representations are predictive of choice behavior throughout learning using a linear decoder to measure the influence of prior experiences on behavior.

Results
Representations of expectation in MTL were conditioned on the history of rewards and were greater when current reward outcomes were consistent with prior compared to inconsistent. This feature was not observed in neocortical regions. These history-dependent computations in MTL reliably explained increases in the separation of HF neural power in the OFC. Representations of expectation across several neocortical regions were predictive of choice behavior significantly above chance. Choice prediction accuracy increased with learning using representations in OFC and not other neocortical regions.

Conclusion
Our findings suggest that prior reward experiences are represented in MTL structures, which then direct the formation of neocortical representations. OFC representations during learning are associated with MTL dynamics and are behaviorally relevant, suggesting that the OFC is a critical locus for mediating effects of prior experience on behavior. These findings add to accumulating evidence that flexible behavior depends on interactions between MTL and neocortex. Our study offers electrophysiological signatures for history-dependent computations to supervise the generation of representations that can redirect behavior.


Prior experiences fine-tune representations in human neocortex to direct behavior

Background
Diffuse midline glioma (DMG) is an aggressive, almost universally lethal form of high-grade glioma that occurs in children. One reason that DMG are so difficult to treat is that epigenetically unstable tumor cells evolve to resist radiation, chemotherapy, and other forms of treatment. Treating DMG cells with HDAC inhibitors (HDACi) reduces cell viability in mouse models, yet HDACi do not improve clinical outcomes when provided as monotherapies. The overall goal of this research is to identify combination therapies to synergistically reduce the growth and survival of DMG cells. A CRISPR-based genetic screen in DMG cell lines revealed that knockout of polo-like kinase 1 (PLK1), a key regulator of cell cycle progression, may sensitize DMG cells to HDACi. The purpose of this study is to develop PLK1i+HDACi combination therapies for DMG, which have few treatment options.

Methods
We conducted dose curve analyses to analyze changes in DMG cell growth and survival in response to increasing doses of HDACi combined with PLK1 inhibitors using the CellTiterGlo assay to measure cell viability. We also conducted immunofluorescence (IF) imaging of DMG cell lines treated with different HDACi and PLK1i to monitor cell cycle (phos-H3-Ser10) and DNA damage (yH2AX) markers.

Results
We found that co-treatment with PLK1i sensitizes DMG cells to HDAC inhibitors, as shown in the dose curve analyses where the addition of PLK1i (GSK461364, BI2536) lowered the IC50 values for two different HDACi (entinostat and panobinostat) compared to HDACi alone. Analysis of the IF images showed higher %(+) signals for both yH2AX and phos-H3-Ser10 in PLK1i and HDACi treated cells compared to either vehicle-treated controls, or DMG cells treated with HDACi and PLK1i alone. 

Conclusion
We conclude that PLK1i + HDACi drug combinations synergistically reduce DMG viability. We also observe that PLK1i + HDACi combinations affect DMG cell phenotypes via increased DNA damage and induced mitotic arrest. The importance of this work is that it potentially opens more therapeutic approaches for patients with DMG who would otherwise have a poor prognosis and limited treatment options. Future steps for this study are to determine the optimal PLK inhibitors to best sensitize DMG to HDAC inhibitors and to detect changes in signal transduction pathways and gene transcription downstream of PLK1i+HDACi combination therapy by phospho-proteomics and RNA sequencing.

Combinatorial targeting of kinase signaling and chromatin regulation to treat pediatric high grade glioma

Background:
Among the genes related to familial Parkinson’s disease (PD), the D620N VPS35 mutation is associated with an increase in -synuclein (αS) aggregates and neuronal death. Reducing the αS levels in neurons is a potential strategy for reducing αS accumulation and consequent dopaminergic neuronal damage. Recent studies suggest that VPS35 regulates αS degradation by impacting the endosomal-lysosomal system (ELS), macroautophagy (MA), chaperone-mediated autophagy (CMA), and the ubiquitin-proteasome system (UPS). Our aim is to investigate the role of VPS35 in increasing αS clearance and to test a novel VPS35 stabilizer, guanidine hydrazone (2a). 

Methods:
We overexpressed exogenous mutant A53T αS by lentiviral infection or transient transfection in SH-SY5Y cells, co-treated this group with 2a (or saline control) at 20 uM for 3, 4 or 5 days, and analyzed the levels of αS, VPS35, and αS degradation pathway markers by western blot. We evaluated lysosomal function through the Cathepsin-D (CTSD) activity assay. We then looked at the effect of 2a on αS levels after selective blockage of αS degradation pathways. Macroautophagy was blocked after 1 day with bafilomycin A1 (BfA1) at 50 nM and UPS was inhibited after 8 hours with MG-132 at 10 uM. 

Results:
2a increased VPS35 protein levels (~75%) and retromer function, with an increased level of the VPS35 cargo VMAT2 (~50%). This correlates with a ~ 50% reduction of Phospho-Ser129 αS with transient transfection and total αS with lentiviral infection, in the soluble fraction. Furthermore, we found an increase in ELS markers (LAMP1, Rab5), CMA markers (LAMP2a, Hsc70), macroautophagy markers (LC3b), UPS markers (PSMB5), highlighting the effect of 2a on boosting these pathways. Interestingly, CTSD activity was decreased after 2a treatment, which could be explained by a CTSD “burnout” due to 2a-mediated ELS boosting. After blocking macroautophagy with BfA1 and UPS with MG-132, the western blot data showed ~75 % reduction of Phospho-Ser129 αS in the infected group treated with 2a plus BfA1 or MG-132 compared to the infected cells treated only with BfA1 or MG-132. Thus, 2a can decrease αS levels even when macroautophagy or UPS are inhibited by BfA1 or MG-132 respectively. This result could suggest that other degradation pathways predominate in mediating the 2a-induced αS clearance, but it is also possible that compensatory upregulation occurs in the non-blocked pathways. 

Conclusion:
These findings highlight Vps35 as a novel pharmacological target for PD treatment and the role ELS and UPS play in αS degradation and, potentially, neuroprotection in PD patients.

The endosomal-lysosomal and ubiquitin-proteasome systems control alpha-synuclein degradation in PD

Background
The number of Alzheimer's disease (AD) patients continues to escalate annually. Alzheimer’s patients are starting to gain access to FDA-approved disease-modifying therapies. However, those come with major risks and hence additional therapeutic strategies are needed. In addition to memory deficits, sleep impairments have been identified in Alzheimer’s patients. Disruptions of sleep-dependent brain rhythms and slow oscillations prevalent during NREM sleep were observed in patients with AD as well as in the mouse models, including APP/PS1 mice. Reduced slow wave power was due to the dysfunction of GABAergic interneurons and low GABA. Thus, we hypothesized that transplantation of healthy Medial Ganglionic Eminence (MGE) inhibitory interneuron progenitors could restore inhibition and rescue cortico-thalamic circuit function driving slow oscillations in APP/PS1 hosts.

Methods
To that end, healthy donor MGE progenitors were harvested from VGAT-Venus or VGAT-ChR2-EYFP mice and were transplanted into the cortices of host APP/PS1 mice. The viability, maturation, and differentiation of the transplanted donor MGE progenitors were assessed via immunohistochemistry. Furthermore, to determine the functionality of donor inhibitory neurons and their effect on slow oscillations, voltage-sensitive dye imaging was conducted 2 months post-transplantation once the donor cells had time to mature and incorporate into host circuitry. Additionally, light activation of ChR2 in APP/PS1 mice transplanted with VGAT-ChR2-EYFP cells were undertaken to measure the effect of optogenetic stimulation on slow oscillations.

Results
We found that the transplanted MGE progenitors not only survived but also differentiated into mature interneurons expressing somatostatin and parvalbumin markers within the host mice. Importantly, transplantation of healthy MGE progenitors restored the slow wave power in APP/PS1 mice two months post-transplantation. Optogenetic activation of donor interneurons further increased the power of slow oscillations.

Conclusion
These results suggest that stem cell therapy with MGE progenitors warrants further investigation and could hold promise as a therapeutic strategy to combat the progression of Alzheimer's disease.

Mouse-derived Interneuron Progenitors Rescue Host Circuit Function in an Alzheimer's Model

Background: Hydrodynamic transfection was recently demonstrated to deliver recombinant arylsulfatase A, the enzyme deficient in metachromatic leukodystrophy, to diverse tissues in murine models of this lysosomal storage disorder (LSD). Late-infantile neuronal ceroid lipofuscinosis (LINCL) is another LSD, caused by the deficiency of the lysosomal protease tripeptidyl-peptidase 1 (TPP1), whose existing therapy could be improved by endogenously synthesizing and trafficking extra-chromosomally-encoded recombinant TPP1 (rTPP1) across the blood-brain barrier (BBB). We sought to determine if hydrodynamic transfection could target therapeutic quantities of rTPP1 across the BBB in mouse models of LINCL.

Methods: Previously-designed rTPP1 constructs (wild-type human TPP1 [WT TPP1], TPP1R465G W470G, and TPP1R465G W470G-(GS)5-D10-(GS)5-K16ApoE [TPP1R465G W470G DKA]) were cloned onto plasmid vectors: (1) N1, a multipurpose vector and (2) CAG, a vector optimized for hydrodynamic transfection. Four plasmid constructs were synthesized: N1 WT TPP1, N1 TPP1R465G W470G, N1 TPP1R465G W470G DKA, and CAG WT TPP1. Plasmid constructs were purified from bacterial cultures and LINCL model mice lacking TPP1 activity were injected with 50 μg of plasmids in 2.5 mL of phosphate-buffered saline (PBS). Blood plasma samples were drawn at 6, 24, and 48 hours post-transfection, after which point the mice were sacrificed and brains and livers were harvested. Plasma samples and brain and liver homogenates were assayed for TPP1 activity, which was compared with non-transfected LINCL mice. Activity was normalized to that of non-injected WT mice. Welch’s ANOVA test was used to determine enzymatic activity differences between non-transfected LINCL mice and those transfected with synthesized constructs, and the resulting p-values were adjusted for multiple comparisons using Dunnett’s T3 test.

Results: All constructs exhibited high levels of expression in murine hepatocytes (p<0.0001) and were successfully secreted into the bloodstream (p<0.0001). No TPP1 activity was detected in non-injected LINCL mice and PBS-injected LINCL mice. N1 TPP1R465G W470G (mean [95% confidence interval]: 5.0% [2.1%, 8.0%], p=0.0352) and CAG WT TPP1 (3.8% [1.9%, 5.6%], p=0.0198) exhibited increased brain delivery at the 0.05 level of significance, while brain delivery of N1 WT TPP1 (4.8% [0.9%, 8.6%], p=0.1712) and N1 TPP1R465G W470G DKA (1.5% [1.0%, 2.0%], p=0.0518) were not statistically-significant.

Conclusion: N1 TPP1R465G W470G and CAG WT TPP1 exhibited modest ability to cross the BBB. Although this result is encouraging, further study is required to evaluate the therapeutic efficacy of these constructs. In addition, it will be necessary to determine the potential to translate these findings to humans.

Evaluation of Hydrodynamic Transfection for Brain Enzyme Replacement Therapy for Late-Infantile Neuronal Ceroid Lipofuscinosis

Brain network organization varies between individuals, and individual differences in intrinsic brain networks have been linked to differences in cognitive ability. Individual differences in brain connectivity may be especially evident in brain networks that support executive functions such as cognitive control and attention such as the frontoparietal and salience networks. The frontoparietal network is theorized to play a role in cognitive control, which refers to the selection of thoughts and behaviors in context of task demands. Specifically, the frontoparietal network is necessary to flexibly coordinate behavior in a goal-driven manner. Meanwhile, the salience network has been hypothesized to modulate the relationship between the default mode network, which contributes to self-oriented cognition, and the frontoparietal network.
Prior work suggests that the maturation of executive functions during development parallels the integration of the salience networks with the frontoparietal network. Additionally, increased frontoparietal and salience intranetwork connectivity has been associated with development from periadolescence to adulthood. However, associations between frontoparietal or salience intranetwork connectivity and executive functions abilities have not been tested in periadolescent children. Here we tested whether intranetwork connectivity of frontoparietal and salience networks was correlated with executive function measures in periadolescent children. Participants in the NIA-funded Polygenic Risk of Alzheimer’s disease in Nebraska Kids (PRANK) study (N = 94) completed the NIH toolbox cognition battery as well as a functional MRI (fMRI) scan including resting-state data collection. Intranetwork connectivity was measured using the Human Connectome Project’s connectome workbench software. Results of the data analysis indicated a positive correlation between scores on fluid cognition measures and frontoparietal intranetwork connectivity, r(73) = .29, p = .01. A positive correlation was also observed with flanker task scores and frontoparietal intranetwork connectivity, r(73) = .25, p = .03. Finally, we observed a positive correlation with salience intranetwork connectivity and age-adjusted dimensional card sorting task scores, r(91) = .22, p = .03. Our preliminary findings show an association of executive function measures with intranetwork connectivity of two key intrinsic functional networks, and they suggest that frontoparietal and salience intranetwork connectivity may reflect increased efficiency of cognitive control during development.

Associations between salience and frontoparietal intranetwork connectivity and executive functions tasks in periadolescent children.

Cocaine use disorder (CUD) is a prevalent neuropsychiatric disorder with few existing treatments. Thus, there is an unmet need for the identification of new pharmacological targets for CUD. Previous studies using environmental enrichment versus isolation paradigms have found that the latter induces increased cocaine self-administration with correlative increases in the excitability of medium spiny neurons (MSN) of the nucleus accumbens shell (NAcSh). Expanding upon these findings, we sought in the present investigation to elucidate molecular determinants of these phenomena.

To identify potential new drug targets for CUD, we mined a previous large-scale differential transcriptomic dataset derived from the NAcSh of rats in environmentally enriched or isolated conditions that self-administered either saline or cocaine. Bioinformatic analyses revealed that fibroblast growth factor 13 (FGF13) mRNA, which codes for a prominent auxiliary protein of the voltage-gated Na+ (Nav) channel, is differentially expressed in the NAcSh by cocaine versus saline self-administration. Based upon this finding, we used in vivo genetic silencing to study the functional and behavioral consequences of knocking down FGF13 in the NAcSh. Functional studies revealed that knockdown of FGF13 in the NAcSh augmented excitability of MSNs by increasing the activity of Nav channels. Behaviorally, these electrophysiological changes were shown to correlate with knockdown of FGF13 in the NAcSh decreasing cocaine demand elasticity. 


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