Inhibition of Dopamine Neurons Prevents Incentive Value Encoding of a Reward Cue: With Revelations from Deep Phenotyping.

The survival of an organism is dependent on its ability to respond to cues in the environment. Such cues can attain control over behavior as a function of the value ascribed to them. Some individuals have an inherent tendency to attribute reward-paired cues with incentive motivational value, or incentive salience. For these individuals, termed sign-trackers, a discrete cue that precedes reward delivery becomes attractive and desirable in its own right. Prior work suggests that the behavior of sign-trackers is dopamine-dependent, and cue-elicited dopamine in the NAc is believed to encode the incentive value of reward cues. Here we exploited the temporal resolution of optogenetics to determine whether selective inhibition of ventral tegmental area (VTA) dopamine neurons during cue presentation attenuates the propensity to sign-track. Using male tyrosine hydroxylase (TH)-Cre Long Evans rats, it was found that, under baseline conditions, ∼84% of TH-Cre rats tend to sign-track. Laser-induced inhibition of VTA dopamine neurons during cue presentation prevented the development of sign-tracking behavior, without affecting goal-tracking behavior. When laser inhibition was terminated, these same rats developed a sign-tracking response. Video analysis using DeepLabCutTM revealed that, relative to rats that received laser inhibition, rats in the control group spent more time near the location of the reward cue even when it was not present and were more likely to orient toward and approach the cue during its presentation. These findings demonstrate that cue-elicited dopamine release is critical for the attribution of incentive salience to reward cues.SIGNIFICANCE STATEMENT Activity of dopamine neurons in the ventral tegmental area (VTA) during cue presentation is necessary for the development of a sign-tracking, but not a goal-tracking, conditioned response in a Pavlovian task. We capitalized on the temporal precision of optogenetics to pair cue presentation with inhibition of VTA dopamine neurons. A detailed behavioral analysis with DeepLabCutTM revealed that cue-directed behaviors do not emerge without dopamine neuron activity in the VTA. Importantly, however, when optogenetic inhibition is lifted, cue-directed behaviors increase, and a sign-tracking response develops. These findings confirm the necessity of dopamine neuron activity in the VTA during cue presentation to encode the incentive value of reward cues.

Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSCs) that persist after therapy and seed treatment refractory recurrent tumors. GBM tumors display a high degree of intra- and inter-tumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury-response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K targeted gRNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury-response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury-response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of ß1 integrin, FAK, MEK and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity.

Gradient of Developmental and Injury Response transcriptional states defines functional vulnerabilities underpinning glioblastoma heterogeneity.

Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR-Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development.