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Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

Drug-associated sensory cues increase motivation for drug and the orexin system is importantly involved in this stimulus-enhanced motivation. Ventral tegmental area (VTA) is a major target by which orexin signaling modulates reward behaviors, but it is unknown whether this circuit is necessary for cue-driven motivation for cocaine. Here, we investigated the role of VTA orexin signaling in cue-driven motivation for cocaine using a behavioral economics (BE) paradigm. We found that infusion of the orexin-1 receptor (Ox1R) antagonist SB-334867 (SB) into VTA prior to BE testing reduced motivation when animals were trained to self-administer cocaine with discrete cues and tested on BE with those cues. SB had no effect when animals were trained to self-administer cocaine without cues or tested on BE without cues, indicating that learning to associate cues with drug delivery during self-administration training was necessary for cues to recruit orexin signaling in VTA. These effects were specific to VTA, as injections of SB immediately dorsal had no effect. Moreover, intra-VTA SB did not have an impact on locomotor activity, or low- or high-effort consumption of sucrose. Finally, we microinjected a novel retrograde adeno-associated virus (AAVretro) containing an orexin-specific short hairpin RNA (OxshRNA) into VTA to knock down orexin in the hypothalamus-VTA circuit. These injections significantly reduced orexin expression in lateral hypothalamus (LH) and decreased cue-driven motivation. These studies demonstrate a role for orexin signaling in VTA, specifically when cues predict drug reward.

In the FOUNdational Data INitiative for Parkinson’s Disease (FOUNDIN-PD) we sought to produce a multi-layered molecular dataset in a large cohort of 95 Induced pluripotent stem cells (iPSC) lines at multiple timepoints during differentiation to dopaminergic (DA) neurons, a major affected cell type in Parkinson’s Disease (PD). The lines are derived from the Parkinson’s Progression Markers Initiative (PPMI) study that includes both people with PD and unaffected individuals across a wide range of polygenic risk scores (PRS) with both risk variants identified by genome-wide association studies (GWAS), and monogenic causal alleles. We generated genetic, epigenetic, regulatory, transcriptomic, proteomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand key molecular relationships between disease associated genetic variation and proximate molecular events in a PD relevant cell-type. Analyses of all data modalities collected in FOUNDIN-PD suggest that the differentiation to DA neurons, while not fully mature, was successful and robust. Interrogation of PD genetic risk in this relevant cellular context may elucidate the functional effects of some of these risk variants alone or in combination with other variants. These data reveal that DA neurons derived from human iPSC provide a valuable cellular context and foundational atlas for modeling PD-related genetic risk. In addition to making the data and analyses for this molecular atlas readily available, we have integrated these data into the browsable FOUNDIN-PD data portal (https://www.foundinpd.org) to be used as a resource for understanding the molecular pathogenesis of PD.

Cues associated with negative reinforcement can stimulate relapse and contribute to opioid use disorder., Opioid use disorder (OUD) is a debilitating disorder that affects millions of people. Neutral cues can acquire motivational properties when paired with the positive emotional effects of drug intoxication to stimulate relapse. However, much less research has been devoted to cues that become conditioned to the aversive effects of opioid withdrawal. We argue that environmental stimuli promote motivation for opioids when cues are paired with withdrawal (conditioned withdrawal) and generate opioid consumption to terminate conditioned withdrawal (conditioned negative reinforcement). We review evidence that cues associated with pain drive opioid consumption, as patients with chronic pain may misuse opioids to escape physical and emotional pain. We highlight sex differences in withdrawal-induced stress reactivity and withdrawal cue processing and discuss neurocircuitry that may underlie withdrawal cue processing in dependent individuals. These studies highlight the importance of studying cues associated with withdrawal in dependent individuals and point to areas for exploration in OUD research.

Addiction is a chronic disorder that consists of a three-stage cycle of binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. These stages involve, respectively, neuroadaptations in brain circuits involved in incentive salience and habit formation, stress surfeit and reward deficit, and executive function. Much research on addiction focuses on the neurobiology underlying single drug use. However, alcohol use disorder (AUD) can be co-morbid with substance use disorder (SUD), called dual dependence. The limited epidemiological data on dual dependence indicates that there is a large population of individuals suffering from addiction who are dependent on more than one drug and/or alcohol, yet dual dependence remains understudied in addiction research. Here, we review neurobiological data on neurotransmitter and neuropeptide systems that are known to contribute to addiction pathology and how the involvement of these systems is consistent or divergent across drug classes. In particular, we highlight the dopamine, opioid, corticotropin-releasing factor, norepinephrine, hypocretin/orexin, glucocorticoid, neuroimmune signaling, endocannabinoid, glutamate, and GABA systems. We also discuss the limited research on these systems in dual dependence. Collectively, these studies demonstrate that the use of multiple drugs can produce neuroadaptations that are distinct from single drug use. Further investigation into the neurobiology of dual dependence is necessary to develop effective treatments for addiction to multiple drugs.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs.Graphical abstract

Addiction is a chronic disorder that consists of a three-stage cycle of binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. These stages involve, respectively, neuroadaptations in brain circuits involved in incentive salience and habit formation, stress surfeit and reward deficit, and executive function. Much research on addiction focuses on the neurobiology underlying single drug use. However, alcohol use disorder (AUD) can be co-morbid with substance use disorder (SUD), called dual dependence. The limited epidemiological data on dual dependence indicates that there is a large population of individuals suffering from addiction who are dependent on more than one drug and/or alcohol, yet dual dependence remains understudied in addiction research. Here, we review neurobiological data on neurotransmitter and neuropeptide systems that are known to contribute to addiction pathology and how the involvement of these systems is consistent or divergent across drug classes. In particular, we highlight the dopamine, opioid, corticotropin-releasing factor, norepinephrine, hypocretin/orexin, glucocorticoid, neuroimmune signaling, endocannabinoid, glutamate, and GABA systems. We also discuss the limited research on these systems in dual dependence. Collectively, these studies demonstrate that the use of multiple drugs can produce neuroadaptations that are distinct from single drug use. Further investigation into the neurobiology of dual dependence is necessary to develop effective treatments for addiction to multiple drugs.

Signaling at the orexin-1 receptor (OxR1) is important for motivated drug taking. Using a within-session behavioral economics (BE) procedure, we previously found that pharmacologic blockade of the OxR1 decreased motivation (increased demand elasticity) for the potent and short-acting opioid remifentanil and reduced low-effort remifentanil consumption. However, the mechanism through which orexin regulates remifentanil demand is currently unknown. Previous work implicated OxR1 signaling within ventral pallidum (VP) as a potential target. VP is densely innervated by orexin fibers and is known to regulate opioid reward. Accordingly, this study sought to determine the role of VP OxR1 signaling in remifentanil demand and cue-induced reinstatement of remifentanil seeking in male rats. Intra-VP microinjections of the OxR1 antagonist SB-334867 (SB) decreased motivation (increased demand elasticity; α) for remifentanil without affecting remifentanil consumption at low effort. Baseline α values predicted the degree of cue-induced remifentanil seeking, and microinjection of SB into VP attenuated this behavior without affecting extinction responding. Baseline α values also predicted SB efficacy, such that SB was most effective in attenuating reinstatement behavior in highly motivated rats. Together, these findings support a selective role for VP OxR1 signaling in motivation for the opioid remifentanil. Our findings also highlight the utility of BE in predicting relapse propensity and efficacy of treatment with OxR1 antagonists.SIGNIFICANCE STATEMENTAbuse of opioids has risen rapidly and continues to be a major health crisis. Thus, there is an urgent need to better understand the neurobiological and behavioral mechanisms underlying opioid addiction. Here, we investigate the role of orexin-1 receptor signaling (OxR1) within ventral pallidum (VP) in remifentanil demand and cue-induced reinstatement of remifentanil seeking. Using a within-session behavioral economics procedure, we show that intra-VP microinjections of the OxR1 antagonist SB-334867 decreased motivation (increased demand elasticity) without affecting remifentanil consumption at low effort. We also found that SB microinjected intra-VP attenuated cue-induced reinstatement of remifentanil seeking. Together, our results support a role for VP OxR1 signaling in opioid reward.

Lateral hypothalamus (LH) orexin neuron signaling has been implicated in the motivation to seek and take drugs of abuse. The number of LH orexin neurons has been shown to vary with behavioral state and can be upregulated with exposure to drugs of abuse. We sought to determine if the number of LH orexin neurons related to individual differences in motivation (demand) for cocaine in our behavioral economics (BE) paradigm, and whether knockdown of these cells predicted changes in economic demand. We quantified LH orexin cell numbers in animals immediately following our BE paradigm, as well as BE-experienced animals after a two-week period of abstinence to relate the number of LH orexin cells to economic demand for cocaine. We also unilaterally knocked down LH orexin expression prior to BE with an orexin morpholino antisense to determine how reduced orexin numbers impacted cocaine demand. Animals with greater motivation for cocaine (lower demand elasticity) had more LH orexin neurons. Following a two-week abstinence from BE, the number of LH orexin neurons predicted economic demand for cocaine prior to abstinence. Reducing LH orexin cell numbers with antisense decreased motivation for cocaine (increased demand elasticity) without affecting baseline consumption. In addition, the number of spared LH orexin neurons after antisense treatment correlated with individual demand for cocaine. These studies point to a role for the endogenous number of LH orexin neurons in individual differences in motivation for cocaine.

The orexin system is a potential treatment target for drug addiction. Orexin-1 receptor (OxR1) antagonism reduces demand for cocaine and remifentanil, indicating that orexin-based therapies may reduce demand for many classes of abused drugs. However, pharmacokinetics vary greatly among opioids and it is unclear if OxR1 antagonism would reduce demand for all opioids, particularly ones with high abuse liability. Here, we established a behavioral economics (BE) procedure to assess the effects of OxR1 antagonism on demand for the highly abused opioid fentanyl. We also investigated the utility of our procedure to predict OxR1 antagonism efficacy and relapse propensity. Demand parameters α (demand elasticity or price sensitivity of consumption, an inverse measure of drug motivation) and Q(o) (drug consumption at null cost) were assessed. The OxR1 antagonist SB-334867 (SB) decreased motivation (increased α) for fentanyl without affecting Q(o). Baseline α values predicted SB efficacy, such that SB was most effective at reducing motivation (increasing α) in highly motivated rats. Baseline α values predicted the amount of cued reinstatement of fentanyl seeking; this reinstatement behavior was attenuated by SB administration. These results highlight the promise of the orexin system as a treatment target for opioid addiction and emphasize the usefulness of BE procedures in the study of opioid abuse.

The lateral septum (LS) is a brain region implicated in motivation, addiction, anxiety, and affect. We recently found that LS is necessary for cocaine-seeking behaviors including conditioned place preference and reinstatement of extinguished drug seeking, which involve LS input to limbic regions including ventral tegmental area (VTA) and orexin neurons in hypothalamus. Here, we microinjected baclofen-muscimol (B-M) in LS prior to testing in a behavioral economics (BE) paradigm. We found that intra-LS B-M decreased motivation (increased demand elasticity; α) for cocaine, but did not change consumption at low effort (Q0 ). We also compared the effects of LS inhibition with the effects of treatment with the benzodiazepine diazepam, which has been shown to facilitate reward pathways and disinhibit VTA dopamine neurons. Pretreatment with diazepam blocked the effects of LS inhibition and restored cocaine demand to that following vehicle treatment. These changes in cocaine demand after LS inhibition or diazepam were not due to effects on anxiety, as both manipulations produced similar effects on anxiety measures but opposing effects on drug taking. Collectively, these studies point to LS as a critical region driving motivation for cocaine, likely through its interactions with the mesolimbic dopamine system.

Hippocampal oscillations arise from coordinated activity among distinct populations of neurons and are associated with cognitive functions. Much progress has been made toward identifying the contribution of specific neuronal populations in hippocampal oscillations, but less is known about the role of hippocampal area CA2, which is thought to support social memory. Furthermore, the little evidence on the role of CA2 in oscillations has yielded conflicting conclusions. Therefore, we sought to identify the contribution of CA2 to oscillations using a controlled experimental system. We used excitatory and inhibitory DREADDs to manipulate CA2 neuronal activity and studied resulting hippocampal-prefrontal cortical network oscillations. We found that modification of CA2 activity bidirectionally regulated hippocampal and prefrontal cortical low-gamma oscillations and inversely modulated hippocampal ripple oscillations in mice. These findings support a role for CA2 in low-gamma generation and ripple modulation within the hippocampus and underscore the importance of CA2 in extrahippocampal oscillations.

Signaling at the orexin-1 receptor (Ox1R) is important for motivation for various drugs of abuse. Recently, our laboratory showed that systemic blockade of Ox1Rs decreased motivation for the potent and short-acting opioid remifentanil, as well as remifentanil consumption at null cost (Porter-Stransky et al., 2017). However, the central site(s) through which orexin acts to regulate opioid-seeking behaviors are not known. We investigated ventral pallidum (VP) as a potential site of orexin action, as VP is a known mediator of opioid reward and is densely innervated by orexin-immunoreactive fibers. We used a within-session behavioral economics (BE) paradigm in which remifentanil price (responses/ μg iv remifentanil) was sequentially increased throughout the session. Rats were implanted with bilateral cannulae into caudal VP (cVP), through which microinjections of SB334867 (SB), an Ox1R antagonist, were given prior to BE and reinstatement testing. Contrary to systemic SB treatment, intra-cVP SB preferentially reduced motivation (increased demand elasticity and reinstatement) for reminfentanil, without affecting remifentanil consumption at null cost. These effects were specific to cVP, as control microinjections of SB immediately dorsal to cVP did not affect remifentanil-seeking. Baseline demand for remifentanil predicted cued reinstatement of remifentanil seeking and the efficacy with which intra-cVP SB administration reduced motivation for remifentanil. These findings indicate a specific role for cVP Ox1R signaling in mediating the motivational properties of the opioid remifentanil, distinct from its consumption at null cost. Our findings also highlight the BE paradigm as an effective biomarker for predicting opioid addiction behaviors and treatment efficacy.

Hippocampal oscillations arise from coordinated activity among distinct populations of neurons and are associated with cognitive functions and behaviors. Although much progress has been made toward identifying the relative contribution of specific neuronal populations in hippocampal oscillations, far less is known about the role of hippocampal area CA2, which is thought to support social aspects of episodic memory. Furthermore, the little existing evidence on the role of CA2 in oscillations has led to conflicting conclusions. Therefore, we sought to identify the specific contribution of CA2 pyramidal neurons to brain oscillations using a controlled experimental system. We used excitatory and inhibitory DREADDs in transgenic mice to acutely and reversibly manipulate CA2 pyramidal cell activity. Here, we report on the role of CA2 in hippocampal-prefrontal cortical network oscillations and social behavior. We found that excitation or inhibition of CA2 pyramidal cells bidirectionally regulated hippocampal and prefrontal cortical low gamma oscillations and inversely modulated hippocampal ripple oscillations. Further, CA2 inhibition impaired social approach behavior. These findings support a role for CA2 in low gamma generation and ripple modulation within the hippocampus and underscore the importance of CA2 neuronal activity in extrahippocampal oscillations and social behavior.

Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1–Tmod3 are expressed in brain cells. We investigated the role of Tmod’s C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2’s LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1’s LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2’s LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant.