R26-DIO-SPOTlight design and expression in Nestin-Cre line

(A) SPOTlight reporter design. (B) R26-DIO-SPOTlight transgene insertion strategy and Nestin-Cre::DIO-SPOTlight mouse line generation. (C & D) Example images of expression in P30 mice. (C) Cortex (CTX), scale bar 100 µm. Cerebellum (CBM), scale bar 100 µm. Cervical spinal cord (SC), scale bar 200 µm. (D) Full sagittal brain slice, scale bar 1000 µm.

Nestin-Cre::DIO-SPOTlight fluorescence functionally differentiates rare subsets of cells.

(A-C) Examples of “red hot” (strongly red, minimal green) neurons. Additional images available in Figure 2 – Supplement 1. (A) Image from striatum in live acute brain slice tissue using multi-photon microscopy, Scale bars 100 µm, 20 µm, 5 µm. Remainder of images are of fixed tissue with immunohistochemistry (B, C, E). (B) Hippocampus, scale bar 100 µm. (C) Cerebellum, scale bar 100 µm. (D) Sub-regional SPOTlight red:green ratio analysis shown as log2 fold change of ChAT over non-ChAT neurons. N = 6 mice per region, DLS-Tail: ChAT(+) n = 41 cells, non-ChAT n = 128 cells; DLS: ChAT(+) n = 143 cells, non-ChAT n = 507 cells; DMS: ChAT(+) n = 154 cells, non-ChAT n = 519 cells; NAc core: ChAT(+) n = 93 cells, non-ChAT n = 434 cells; NAc shell: ChAT(+) n = 112 cells, non-ChAT n = 342 cells. Statistics summary tables and relative intensities of fluorescent proteins for panel D available in Figure 2 –Supplement 2. (E) Representative images of dorsolateral striatum and nucleus accumbens shell, scale bars 50 µm.

Acute tunicamycin exposure modifies SPOTlight readouts.

(A) Schematic of ISR manipulation strategy. (B-D) Quantification of SPOTlight readouts: TM n = 5 mice, n = 771 cells; Vehicle n = 5 mice, n = 798 cells. (B) Red/green ratio: t = 1.614, F(1,8) = 2.604, P = 0.1453. (C) Anti-RFP quantification t = 3.639, F(1,8) = 13.24, P = 0.0066. (D) Anti-GFP quantification: t = 1.73, F(1,8) = 2.994, P = 0.1218. (E) Representative images of red and green fluorescence in hippocampal pyramidal cell layer from transgene-negative control mice and Nestin Cre::DIO-SPOTlight hemizygous mice under vehicle and tunicamycin conditions. (F) Time course model for kinetics of ISR and SPOTlight reporter components after acute stressor/ISR activator. Following stressor, serial waves of eIF2α kinase activation, eIF2α phosphorylation, and ATF4 synthesis ensue. (Not shown, increased synthesis of other ISR effectors, phosphatases and downregulation of general protein synthesis.) Time course of SPOTlight EGFP translation is predicted to follow the period of general translation inhibition. Time course of SPOTlight tdTomato translation is predicted to follow ATF4 translation. Relative to the kinetics of deactivation and/or degradation of each ISR component, the half-lives of the SPOTlight reporters (tdTomato and EGFP) are relatively long (t1/2 approx. 24 h). In the particular case of tunicamycin as a stressor, there are predicted to be additional transient effects of inhibiting proteasomal degradation (Menéndez-Benito et al., 2005; Shenkman et al., 2007). The light shaded areas on each reporter are intended to indicate this effect which would cause additional accumulation of existing fluorophores relative to steady-state.

CNS-wide Atlas of SPOTlight Activity in ChAT-positive neurons.

(A) Brain and spinal cord regional analysis of R:G ratio values in ChAT vs non-ChAT neighboring neurons. (B) Regional analysis of normalized Log2 (fold change) values of R:G ratios in ChAT(+)/non-ChAT neighboring neurons. (A-B) Statistical summary tables and relative intensities of fluorescent proteins for A-B available in Figure 4 – Supplement 1A-C. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. X: n = 4 mice, ChAT(+) n = 84 cells, Non-ChAT n = 66 cells; III/IV: n = 5 mice, ChAT(+) n = 144 cells, Non-ChAT n = 145 cells; Lumbar spinal cord n = 3 mice, ChAT(+) n = 250 cells, non-ChAT n = 165 cells; Cervical spinal cord n = 3 mice, ChAT(+) n = 257 cells, non-ChAT n = 182 cells; PBG: n = 3 mice, ChAT(+) n = 76 cells, Non-ChAT n = 69 cells; V: n = 6 mice, ChAT(+) n = 151 cells, Non-ChAT n = 136 cells; MH: n = 3 mice, ChAT(+) n = 241 cells, Non-ChAT n = 173 cells; Thoracic spinal cord n= 3 mice, ChAT(+) n = 342 cells, non-ChAT n = 210 cells; DS: n = 6 mice, ChAT(+) n = 338 cells, Non-ChAT n = 1154 cells; PPn: n = 5 mice, ChAT(+) n = 123 cells, Non-ChAT n = 200 cells; NDB: n = 5 mice, ChAT(+) n = 105 cells, Non-ChAT n = 144 cells; LDT: n = 5 mice, ChAT(+) n = 194 cells, Non-ChAT n = 241 cells; GPi/GPe: n = 4 mice, ChAT(+) n = 34 cells, Non-ChAT n = 77 cells; VII: n = 3 mice, ChAT(+) n = 91 cells, Non-ChAT n = 56 cells; XII: n = 4 mice, ChAT(+) n = 146 cells, Non-ChAT n = 101 cells; VI: n = 5 mice, ChAT(+) n = 52 cells, Non-ChAT n = 98 cells; NAc: n = 6 mice, ChAT(+) n = 205 cells, Non-ChAT n = 776 cells; MS: n = 4 mice, ChAT(+) n = 61 cells, Non-ChAT n = 111 cells; OT: n = 4 mice, ChAT(+) n = 39 cells, Non-ChAT n = 94 cells. (C) Representative images from select regions: Ventral horn of cervical spinal cord (VH), scale bar 50 µm; Oculomotor nucleus/trochlear nucleus (III/IV), scale bar 50 µm; Laterodorsal tegmental nucleus (LDT), scale bar 50 µm; Dorsal motor nucleus of the vagus nerve (X) and Hypoglossal nucleus (XII), scale bar 100 µm; Pedunculopontine nucleus (PPn), scale bar 50 µm. Example images for other regions are available in Figure 4 – Supplement 2.