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Published April 23, 2021 | Accepted Version + Supplemental Material
Journal Article Open

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


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.

Additional Information

© 2021 American Association for the Advancement of Science. This is an article distributed under the terms of the Science Journals Default License. Received 14 August 2020; resubmitted 22 December 2020. Accepted 12 March 2021. We thank K. Brose, H. Marie, B. Philpot, and J. McNamara for input on the manuscript; A. Florwick, R. Settelen, K. Tsukayama, T. Greber, C. R. Means, and R. M. Rodriguiz for technical assistance; and the researchers and staff who made this work possible, as a substantial portion of this study occurred under the hardships of the COVID-19 pandemic. Research was supported by funding from the following sources: NIH BRAIN initiative (NS110059, N.C.; MH123017, B.D.T.), NIH (NS076708-06, M.C.-M.; NS051156 and NS095653, J.E.R.), NSF (DGE-1644868, V.L.H.), Dystonia Medical Research Foundation (N.C., A.R.H.), Tyler's Hope for a Dystonia Cure (N.C.), and a Holland-Trice Scholar award (N.C.). Author contributions: A.R.H. performed most of the SPOTlight imaging, DREADD manipulation, immunohistochemical experiments, and data analysis; designed behavioral experiments; and analyzed results from core facility for MWM, OFT, rotarod, and fear conditioning. V.L.H. designed, supervised, and analyzed lever press CReP studies. R.H.-M. initiated study of ISR in CINs and designed, performed, and analyzed all CIN recording experiments. M.L.O. contributed to SPOTlight validation and imaging analyses and designed and analyzed p-PERK immunohistochemical measures. Z.F.C. and J.E.R. conceived of and designed the SPOTlight reporter. B.D.T. designed, performed, and analyzed the dLight experiments. M.K.S. performed all viral surgeries and a subset of immunohistochemical staining and documented viral spread for DREADD and CReP experiments. C.S.K. performed single-cell RNA-sequencing analyses. V.G. advised on the project and contributed unpublished reagents. C.G. participated in early SPOTlight image analyses and interpretations. M.C.-M. contributed conceptually and supervised R.H.-M. during the Eif2s1 phosphomutant studies. A.R.H. and N.C. wrote the manuscript with substantial input from all authors. N.C. participated in conceptual development, research oversight, and interpretation. The authors declare no competing interests. Data and materials availability: All data are available in the main text or the supplemental materials. Plasmids will be made available through Addgene or upon request.

Attached Files

Accepted Version - nihms-1717314.pdf

Supplemental Material - abe1931-Helseth-SM-Reproducibility-Checklist.pdf

Supplemental Material - abe1931-Helseth-SM.pdf


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Additional details

August 20, 2023
December 22, 2023