Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNß and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

Flexible control of Pavlovian-instrumental transfer based on expected reward value.

The Pavlovian-instrumental transfer (PIT) paradigm is widely used to assay the motivational influence of reward-predictive cues, reflected by their ability to invigorate instrumental behavior. Leading theories assume that a cue's motivational properties are tied to predicted reward value. We outline an alternative view that recognizes that reward-predictive cues may suppress rather than motivate instrumental behavior under certain conditions, an effect termed positive conditioned suppression. We posit that cues signaling imminent reward delivery tend to inhibit instrumental behavior, which is exploratory by nature, in order to facilitate efficient retrieval of the expected reward. According to this view, the motivation to engage in instrumental behavior during a cue should be inversely related to the value of the predicted reward, since there is more to lose by failing to secure a high-value reward than a low-value reward. We tested this hypothesis in rats using a PIT protocol known to induce positive conditioned suppression. In Experiment 1, cues signaling different reward magnitudes elicited distinct response patterns. Whereas the one-pellet cue increased instrumental behavior, cues signaling three or nine pellets suppressed instrumental behavior and elicited high levels of food-port activity. Experiment 2 found that reward-predictive cues suppressed instrumental behavior and increased food-port activity in a flexible manner that was disrupted by post-training reward devaluation. Further analyses suggest that these findings were not driven by overt competition between the instrumental and food-port responses. We discuss how the PIT task may provide a useful tool for studying cognitive control over cue-motivated behavior in rodents. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

CoolMPS for robust sequencing of single-nuclear RNAs captured by droplet-based method.

Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach that relies on nucleotide labeling by re-usable antibodies, but whether it is applicable to snRNA-seq has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS technology. To assess the quality and performance of converted libraries sequenced with CoolMPS, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and libraries that were converted to be compatible with CoolMPS were sequenced on a DNBSEQ-400RS. CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering results, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes during aging. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries.

Reward-predictive cues elicit excessive reward seeking in adolescent rats.

Impulsive behavior during adolescence may stem from developmental imbalances between motivational and cognitive-control systems, producing greater urges to pursue reward and weakened capacities to inhibit such actions. Here, we developed a Pavlovian-instrumental transfer (PIT) protocol to assay rats' ability to suppress cue-motivated reward seeking based on changes in reward expectancy. Traditionally, PIT studies focus on how reward-predictive cues motivate instrumental reward-seeking behavior (lever pressing). However, cues signaling imminent reward delivery also elicit countervailing focal-search responses (food-port entry). We first examined how reward expectancy (cue-reward probability) influences expression of these competing behaviors. Adult male rats increased rates of lever pressing when presented with cues signaling lower probabilities of reward but focused their activity at the food cup on trials with cues that signaled higher probabilities of reward. We then compared adolescent and adult male rats in their responsivity to cues signaling different reward probabilities. In contrast to adults, adolescent rats did not flexibly adjust patterns of responding based on the expected likelihood of reward delivery but increased their rate of lever pressing for both weak and strong cues. These findings indicate that control over cue-motivated behavior is fundamentally dysregulated during adolescence, providing a model for studying neurobiological mechanisms of adolescent impulsivity.