Noradrenergic terminal short-term potentiation enables modality-selective integration of sensory input and vigilance state

Abstract

Recent years have seen compelling demonstrations of the importance of behavioral state on sensory processing and attention. Arousal plays a dominant role in controlling brain-wide neural activity patterns, particularly through modulation by norepinephrine. Noradrenergic brainstem nuclei, including locus coeruleus, can be activated by stimuli of multiple sensory modalities and broadcast modulatory signals via axonal projections throughout the brain. This organization might suggest proportional brain-wide norepinephrine release during states of heightened vigilance. Here, however, we have found that low-intensity, nonarousing visual stimuli enhanced vigilance-dependent noradrenergic signaling locally in visual cortex, revealed using dual-site fiber photometry to monitor noradrenergic Ca²⁺ responses of astroglia simultaneously in cerebellum and visual cortex and two-photon microscopy to monitor noradrenergic axonal terminal Ca²⁺ dynamics. Nitric oxide, following N-methyl-d-aspartate receptor activation in neuronal nitric oxide synthase-positive interneurons, mediated transient acceleration of norepinephrine-dependent astroglia Ca²⁺ activation. These findings reveal a candidate cortical microcircuit for sensory modality-selective modulation of attention.

Fig. 1.. Nonarousing visual stimulation region specifically potentiates vigilance-dependent V1 astrocyte Ca²⁺ activation.

(A) In vivo dual-site fiber photometry schematic for recording astroglia Ca²⁺ dynamics in cerebellum (Cb) and primary visual cortex (V1) from awake, head-fixed mice. (B) Ca²⁺ dynamics during enforced locomotion (green bars) or during simultaneous enforced locomotion and high-intensity (2.2 cd/m²) visual stimulation (gray bars) in V1 and Cb of Aldh1l1-CreER^T2;Ai95 mice. Black traces represent mean Ca²⁺ responses within the regions of interest (ROIs) defined in (A). Gray traces represent voluntary locomotion contaminated trials; see Materials and Methods. (C) Left/middle: Population data of mean Ca²⁺ elevations from onset of locomotion to peak of locomotion or costimulation (Costim; high intensity) in V1 (left) and Cb (middle). Right: Ca²⁺ elevations during costimulation normalized to respective locomotion response in both regions (n = 9 mice). Gray lines, same mouse. Red symbols, means ± SEM. Repeated-measures analysis of variance (ANOVA) followed by Tukey-Kramer correction. Individual P values represent comparisons to 1 (blue dashed line), respectively. (D) Same as (B) but for low-intensity (0.3 cd/m²) visual stimulation. (E) Left/middle: Same as (C) left/middle but low intensity. Right: Same as (C) right but low intensity (n = 6 mice). Source data are provided as a source data file.

Fig. 2.. Visual cortex–specific potentiation of vigilance-dependent noradrenergic terminal Ca²⁺ elevations by nonarousing visual stimulation.

(A) Schematic of two-photon volume Ca²⁺ imaging. (B) Top: V1 section from 8-week-old Dbh-Cre;Ai14 mouse with tdTomato fluorescence (magenta) and immunostained for tyrosine hydroxylase (green). Bottom: Same for Cb molecular layer from same mouse. (C) V1 noradrenergic terminal Ca²⁺ responses in Dbh-Cre;Lck-GCaMP6f^flox mice to enforced locomotion (green bars) or costimulation [low intensity, blue filtered (0.1 cd/m²); gray bars]. Pseudo-colored plot represents Ca²⁺ changes in ROIs schematized in (A). Black traces represent mean Ca²⁺ change in all ROIs. (D) Same for Cb molecular layer noradrenergic terminals. Gray traces represent voluntary locomotion contaminated trials. (E) Normalized means ± SEM (gray shading) of population Ca²⁺ dynamics in (C) and (D) (normalized to respective average locomotion trials). (F) Overlay of stimulus-aligned Ca²⁺ fluorescence traces in response to locomotion (left), costimulation (right), in V1 (top) or Cb (bottom). Magenta or cyan traces represent mean in V1 or Cb, respectively. (G) Chained-dot plots compare mean Ca²⁺ change during 10 s from onset of locomotion between locomotion and costimulation trials (V1, n = 7 mice; Cb, n = 8 mice). Gray lines, same mouse. Red symbols, means ± SEM or median. (H) Mean time to peak of the same data analyzed in (G). Wilcoxon signed-rank tests followed by Bonferroni correction. Source data are provided as a source data file.

Fig. 3.. Sensory stimulus-evoked potentiation of noradrenergic terminals is short-lived.

(A) Astroglia Ca²⁺ dynamics recorded with dual-site fiber photometry in Aldh1l1-CreER^T2;Ai95 mice during a pseudo-randomized (five each) order of locomotion-alone (green bars) or simultaneous locomotion and low-intensity (0.3 cd/m²) visual stimulation trials (gray bars) in both regions. Black traces represent mean Ca²⁺ responses in V1 (top) or Cb (bottom). Traces in shaded gray represent voluntary locomotion contaminated trials. (B) Top: If the first “uncontaminated” locomotion-alone trial happened before the first costimulation trial, then its mean Ca²⁺ change in V1 (left) or Cb (right) was compared to the last locomotion-alone trial. Bottom: Ca²⁺ elevations of the last locomotion-alone trial normalized to respective first uncontaminated locomotion-alone trial in both regions (n = 7 mice). Gray lines connect values from the same mouse. Red symbols indicate means ± SEM. Statistical analysis used repeated-measures ANOVA followed by Tukey-Kramer correction. Individual P values for normalized Ca²⁺ changes in V1 or Cb represent comparisons to 1 (blue dashed line), respectively. Source data are provided as a source data file.

Fig. 4.. NMDA receptors and NO mediate local, sensory stimulus-induced enhancement and acceleration of noradrenergic terminal Ca²⁺ elevations in V1.

(A) Experimental design as for Fig. 2 and pseudo-randomized stimulation as for Fig. 3. Incubation with drugs in artificial cerebrospinal fluid (aCSF) started 20 min before imaging (solution replacement every 10 min). (B) Ca²⁺ responses to enforced locomotion (green bars) or simultaneous enforced locomotion and blue stimulation (gray bars) in aCSF. Pseudo-colored plot represents Ca²⁺ changes in individual ROIs, and black traces represent mean Ca²⁺ change in all ROIs. (C) Same as (B) with NMDA receptor antagonist D-AP5 (1 mM). (D) Same as (B) with NOS inhibitor l-NMMA acetate (600 μM). (E) Chained-dot plot to compare mean Ca²⁺ change during 10 s from onset of locomotion in all noradrenergic terminal ROIs between locomotion and costimulation trials from all mice locally exposed to aCSF (left, n = 7 mice), D-AP5 (middle, n = 8 mice), and l-NMMA acetate (right, n = 6 mice). Gray lines, same mouse. Red symbols, means ± SEM. Repeated-measures ANOVA for three groups, followed by Tukey-Kramer correction. (F) Same as (E) but comparing mean time to peak. Red symbols, median. Wilcoxon signed-rank tests followed by Bonferroni correction. Source data are provided as a source data file.

Fig. 5.. Sensory stimulus-evoked potentiation of astrocyte Ca²⁺ elevations requires NMDA receptor activation and NO.

(A) Schematic illustrating same experimental design as described for Fig. 4 using Aldh1l1-CreER^T2;Ai95 mice. (B) Ca²⁺ responses to enforced locomotion (green bars) or simultaneous enforced locomotion and blue stimulation (gray bars). The surface of V1 was incubated in aCSF from 20 min before until the end of the experiment (aCSF was replaced every 10 min). Pseudo-colored plot represents Ca²⁺ changes in individual ROIs, and black traces represent mean Ca²⁺ change in all ROIs. (C) Same design as (B) but incubation with NMDA receptor antagonist D-AP5 (1 mM). (D) Same design as (B) but incubation with NOS inhibitor l-NMMA acetate (600 μM). (E) Chained-dot plot to compare mean Ca²⁺ change during 10 s from onset of locomotion in all astrocytes between locomotion-alone and costimulation trials from all mice locally exposed to aCSF (left, n = 7 mice), D-AP5 (middle, n = 8 mice), and l-NMMA acetate (right, n = 8 mice). Gray lines connect values from the same mouse. Red symbols indicate means ± SEM. Statistical analysis used repeated-measures ANOVA for three groups, followed by Tukey-Kramer correction. (F) Same as (E) but comparing mean time to peak. Gray lines connect values from the same mouse. Red symbols indicate means ± SEM. Statistical analysis used repeated-measures ANOVA for three groups, followed by Tukey-Kramer correction. Source data are provided as a source data file.

Fig. 6.. Induced knockout of GluN1 receptors on nNOS⁺ interneurons impairs the acceleration of noradrenergic signaling by visual input.

(A) Schematic diagram of generating nNOS-CreER^T2;cGrin1^flox/flox mice and AAV-PHP.B-gfaABC1D-GCaMP3. (B) Schematic of viral vector delivery by retro-orbital injection for in vivo astroglia Ca²⁺ imaging. (C) Population data with two-dimensional plots visualizing the mean Ca²⁺ change (y axis) and mean time to peak (x axis) for nNOS-CreER^T2;cGrin1^wt/wt (red) and nNOS-CreER^T2;cGrin1^flox/flox (green) mice during locomotion-alone trials (Motor) and costimulation trials (Costim). (D) Chained-dot plot to compare mean Ca²⁺ change between locomotion-alone and costimulation trials in nNOS-CreER^T2;cGrin1^wt/wt and nNOS-CreER^T2;cGrin1^flox/flox mice [n = 9 fields of view (FOVs) from three mice and n = 11 FOVs from four mice, respectively]. Gray lines connect values from the same mouse. Red and green symbols indicate means ± SEM. Statistical analysis used repeated-measures ANOVA for two groups, followed by Tukey-Kramer correction. (E) Same as (D) but comparing mean time to peak. Gray lines connect values from the same mouse. Red and green symbols indicate means ± SEM. Statistical analysis used repeated-measures ANOVA for two groups, followed by Tukey-Kramer correction. Source data are provided as a source data file.