Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice

Always stressed The integrated stress response (ISR) plays a role in proteostasis and is important in the brain for learning and memory. Typically, the pathway is thought to be activated transiently to fulfill these roles but may become persistent in disease states. Helseth et al. developed a reporter to study ISR activation brainwide and found a class of cells that engage ISR for steady-state cell function (see the Perspective by Ingebretson and Lemos). In mice, cholinergic interneurons required the ISR to maintain properties of tonic firing and response to dopamine. Inhibiting the ISR in these cells changed dopamine neuromodulation and enhanced performance in learned tasks. Thus, ISR engagement can play a steady-state role that is independent of cell stress and important in learning. Science, this issue p. eabe1931; see also p. 345 A fluorescent reporter reveals high steady-state integrated stress response activation in a subset of neurons. INTRODUCTION The integrated stress response (ISR) is a highly conserved biochemical pathway that, upon activation, markedly shifts which proteins are synthesized. Its roles in proteostasis, synaptic plasticity, learning, and memory have made the pathway an attractive therapeutic target for systemic and brain diseases. Preclinical studies showing the capacity of small-molecule ISR inhibitors to enhance some forms of learning and memory have further highlighted its translational potential. Despite strong and accumulating evidence for the ISR as a potent modifier of plasticity, learning, and memory in diverse behavioral paradigms, the cellular sites of action and time course of ISR engagement are less well understood. RATIONALE To better understand ISR roles in the brain, we developed a two-color reporter of ISR activation state, SPOTlight, and delivered it through a viral vector for brainwide imaging with cellular resolution. SPOTlight was designed to differentially translate green or red fluorescent proteins from a single transcript based on ISR activation state. We first established that reporter readouts corresponded to known manipulations of the ISR using immunohistochemical analyses. SPOTlight signal indicating high ISR activation at steady state in striatal cholinergic interneurons (CINs) was validated using immunohistochemical analyses. To understand the factors driving ISR activation in CINs, we inhibited CIN activity chemogenetically and assessed the ISR state. Selective pharmacological and genetic manipulations were combined with electrophysiological CIN recordings to establish the mechanisms by which ISR state influences CIN physiology. Fluorescent dopamine reporter imaging was used to examine the circuit-level effects of CIN ISR state on evoked striatal dopamine transients in acute brain slices. Finally, task training assays were used to measure the behavioral effects of CIN ISR activity. RESULTS Pharmacological ISR activators and inhibitors delivered in vivo differentially modified levels of SPOTlight-encoded fluorescent proteins and corresponded to immunohistochemical markers of ISR activation in the mouse brain. As expected, in the normal mature mouse brain, SPOTlight revealed only sparse cells with evidence of ISR activation. Unexpectedly, we also found a class of neurons that showed population-wide ISR activation: tonically firing striatal CINs. Chemogenetic inhibition of CIN firing reduced ISR activation, indicating an activity-dependent component. CINs also appeared to be distinctive in ISR engagement; a survey of SPOTlight in other cell types with tonic or high-firing properties did not show similarly high and population-wide ISR engagement. In CINs, manipulations inhibiting the ISR inverted the normal type 2 dopamine receptor (D2R) response from slowing to increasing CIN firing through a mechanism that reduced small-conductance calcium-activated potassium channel currents. Cell-autonomous ISR inhibition in CINs also inverted D2R modulation of evoked striatal dopamine and altered skill learning by increasing the velocity of movement in two learned tasks. CONCLUSION Our study defines a distinct role for the ISR in brain, neuromodulation, which expands our understanding of how the ISR influences learning and memory. We show that steady-state ISR activation in striatal cholinergic interneurons determines their response to dopaminergic modulation, shapes circuit-level dopamine release, and regulates learned skill performance. In this context, ISR activation is activity dependent and influences CIN cellular excitability. As ISR-inhibiting drugs move toward the clinical setting, our results highlight an unappreciated mechanism for their effects on learned behaviors. Our results also prompt further examination of the sites of ISR action in various forms of synaptic plasticity given the importance of cholinergic and dopaminergic neuromodulation in this process. Finally, SPOTlight provides a tool with which to explore when and where the ISR is activated across diverse contexts, including developmental, learning-related, and diseased states. The ISR in striatal CINs. Using a two-color viral reporter of ISR activation state, striatal CINs were distinguished from other cell types by showing steady-state ISR activation. ISR inhibition altered CIN excitability, inverted the sign of dopamine modulation of CIN firing rate and evoked dopamine release, and increased movement vigor in learned tasks. mAHP, medium afterhyperpolarization; AP, action potential; DA, dopamine. The integrated stress response (ISR) maintains proteostasis by modulating protein synthesis and is important in synaptic plasticity, learning, and memory. We developed a reporter, SPOTlight, for brainwide imaging of ISR state with cellular resolution. Unexpectedly, we found a class of neurons in mouse brain, striatal cholinergic interneurons (CINs), in which the ISR was activated at steady state. Genetic and pharmacological manipulations revealed that ISR signaling was necessary in CINs for normal type 2 dopamine receptor (D2R) modulation. Inhibiting the ISR inverted the sign of D2R modulation of CIN firing and evoked dopamine release and altered skill learning. Thus, a noncanonical, steady-state mode of ISR activation is found in CINs, revealing a neuromodulatory role for the ISR in learning.