Differential attentional control mechanisms by two distinct noradrenergic coeruleo-frontal cortical pathways

Abstract

The attentional control of behavior is a higher-order cognitive function that operates through attention and response inhibition. The locus coeruleus (LC), the main source of norepinephrine in the brain, is considered to be involved in attentional control by modulating the neuronal activity of the prefrontal cortex (PFC). However, evidence for the causal role of LC activity in attentional control remains elusive. Here, by using behavioral and optogenetic techniques, we investigate the effect of LC neuron activation or inhibition in operant tests measuring attention and response inhibition (i.e., a measure of impulsive behavior). We show that LC neuron stimulation increases goal-directed attention and decreases impulsivity, while its suppression exacerbates distractibility and increases impulsive responding. Remarkably, we found that attention and response inhibition are under the control of two divergent projections emanating from the LC: one to the dorso-medial PFC and the other to the ventro-lateral orbitofrontal cortex, respectively. These findings are especially relevant for those pathological conditions characterized by attention deficits and elevated impulsivity.

Keywords: attentional control; locus coeruleus; norepinephrine; prefrontal cortex; response inhibition.

Fig. 1.

Double-transgenic strategy for the optogenetic targeting of LC neurons. (A) Expression of light-activated opsins in noradrenergic (NET+; red) LC neurons of Ai32⁺ (Top) or Ai35⁺ mice (Bottom) crossed with NET^Cre+ mice. (Scale bar, 100 µm.) (B) (Top) Strategy for optogenetic manipulation and simultaneous electrophysiological recording of LC neurons. (Bottom) Representative waveforms from a LC isolated unit. (Scale bar, 0.2 ms × 0.1 mV.) (C) Effect of blue laser (Top: six neurons, two mice) and green laser (Bottom: seven neurons, two mice) on LC firing rate in Ai32⁺ x NET^Cre+ (ChR2+) and Ai35⁺ x NET^Cre+ (Arch+) mice, respectively (7-s bins). (D) Histological sample showing the position of the optic fiber (solid line) and electrode track (dashed line). (Scale bar, 100 µm.) (E) Strategy for virally mediated expression of the fluorescent NE sensor GRAB_NE1m in mPFC and fiber photometry recordings during LC optogenetic stimulation in ChR2+ mice. (F) Representative traces (scale bar, 1% ΔF/F) and quantification (G) of NE release in the mPFC (two mice; three trials each) at different laser activation frequencies (7 s, 1.5 mW, 10-ms pulses). (H) Histological verification of GRAB_NE1m expression and optic fiber placement in the mPFC. (I) Color scale of heatmaps. (J) Heatmaps of locomotor activity for ChR2 mice in the OF test. (K) Effect of LC stimulation on locomotor activity in the OF test. (L) Effect of LC stimulation on the time spent in the center of the OF apparatus. (M) Heatmaps of ChR2 mice locomotor activity in the EPM test. (N) Effect of LC stimulation on the time spent in the open arms of the EPM apparatus. Data are presented as mean; error bars show ± SEM. Repeated measures (RM) one-way ANOVA followed by Sidak (C) or Dunnett (G; 5 Hz as reference variable) post hoc test. Unpaired t tests (K, L, N) are two-tailed. n.s., not significant; *P < 0.05; **P < 0.005; ***P < 0.0001.

Fig. 2.

LC neurons activity modulates sustained attention and response inhibition. (A) Schematic representation of the two-choice task. (B) Single-trial structure of the two-choice task (LH: limited hold). (C) Confocal tile scan of a histological sample of a bilateral optic fiber implant (dashed lines) placed just above the LC. (Scale bar, 500 µm.) (D) Strategy for the optogenetic stimulation of LC neurons in ChR2 mice. Effect of LC stimulation on correct responses (E), premature responses (F), and RTs (G) in ChR2 mice (ChR2+, blue bars, n = 11; ChR2–, gray bars, n = 9). (H) Strategy for the optogenetic inhibition of LC neurons in Arch mice. Effect of LC inhibition on correct responses (I), premature responses (J), and RTs (K) in Arch mice (Arch+, green bars; n = 9; Arch–, gray bars, n = 7). Data are presented as the mean of three sessions (150 trials/subject); Two-way RM ANOVA followed by Sidak post hoc test. Effect of laser: n.s., not significant; *P < 0.05; **P < 0.005; ***P < 0.0001.

Fig. 3.

LC neurons control goal-directed attention. (A) Schematic representation of the four trial types in the cued version of the two-choice task (Movie S1). (B) Timeline of events within a cued trial (LH: limited hold). (C) Effects of trial type on the RT of ChR2 mice. (D) Effect of laser stimulation on the RT Validity effect (Invalid RTs – Valid RTs). (E) Effect of trial type on the RT of Arch mice. (F) Effect of laser stimulation on the RT Validity effect in Arch mice. (G) Effect of trial type on the proportion of correct responses in ChR2 mice. (H) Effect of laser stimulation on the Validity effect of response accuracy (Valid % correct – Invalid % correct) in ChR2 mice. (I) Effect of trial type on correct responses in Arch mice. (J) Effect of laser activation on the Validity effect of response accuracy in Arch mice. Data are expressed as averages of six sessions (300 trials/subject) ± SEM. ChR2–, ChR2+, and Arch–: n = 9, Arch+: n = 8. RM ANOVA followed by Dunnett post hoc test (or the equivalent nonparametric Friedman test followed by Dunn post hoc test) with Neutral trials (empty bars) as the reference variable (C, E, G, I). Paired t tests (D, F, H, J) are two-tailed. Lasers were activated on every trial. Effect of trial type (C, E, G, I) or genotype (D, F, H, J): *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig. 4.

LC calcium activity reflects a trade-off between top-down and bottom-up attention. (A) Approach used for FP recordings of LC calcium activity. (B) Confocal tile scan showing optic fiber placement (Left; scale bar, 500 µm) and GCaMP6f expression in LC neurons (Right; Scale bar, 100 µm). (C) Representative FP traces of LC recordings during white-noise stimulation (Left; scale bar, 10 s × 5% ΔF/F) and FP traces aligned to 1 s white noise stimuli (Right; 144 trial, four mice; scale bar, 1% ΔF/F). (D) Strategy for the recording of calcium transients in Ai95 x NET^Cre mice during the cued version of the two-choice task. (E) Trial types used in the cued version of the two-choice task during FP recordings. (F) Peri-event histogram showing LC calcium activity during correct trials segregated by trial type and aligned to target stimuli for the analysis of spontaneous (gray, 4 s) or evoked (orange and yellow, 0.5 s) LC activity. (G) Comparison of LC spontaneous activity between correct and incorrect trials. (H) Comparison of LC spontaneous activity between correct and premature trials. (I) Comparison of LC activity evoked by cues during correct and premature trials. LC spontaneous activity divided by Fast and Slow trials during Neutral (J), Valid (K), or Invalid (L) trial types. LC activity evoked by target stimuli divided by Fast and Slow trials during Neutral (M), Valid (N), or Invalid (O) trial types. (n = 4 mice; 150 trials/subject). Correct trials do not include premature trials (– prem) except in G. Data are expressed as mean ± SEM of n = 4 mice. Box and whiskers plots represent the five-number summary. TW: time window analyzed (in seconds). Paired t tests are two-tailed. n.s., not significant; *P < 0.05; **P < 0.005.

Fig. 5.

Dissociable roles of divergent coeruleo-cortical projections in attentional control. (A) Schematic representation of the modified version of the of the two-choice task with distractors on a subset of trials. (B) Single-trial structure of the task. (C) Approach used for the expression of ChR2 in coeruleo-cortical terminals and their stimulation (5 Hz). (D) Confocal tile scan showing ChR2-eYFP (green) expression in LC neurons. (Scale bar, 500 µm.) (E) Magnified view of LC neurons expressing ChR2. (Scale bar, 50 µm.) (F) Histological samples showing LC terminals (NET; red) expressing ChR2 (eYFP; green) in the dmPFC (Left) and the vlOFC (Right). (G) Histological sample showing optic fiber placement (dashed lines) in the dmPFC (solid line). (Scale bar, 400 µm.) Effect of LC terminal stimulation in the dmPFC (n = 7) on correct responses (H), premature responses (I), and RTs (J). (K) Histological sample showing optic fiber placement (dashed lines) in vlOFC (solid line). (Scale bar, 400 µm.) Effect of LC terminal stimulation in vlOFC (n = 8) on correct responses (L), premature responses (M), and RTs (N). Data are presented as the mean of three sessions (150 trials/subject). Two-way RM ANOVA followed by Sidak post hoc test. AP: anterior-posterior coordinates. Effects of laser: n.s., not significant; *P < 0.05; **P < 0.005; ***P < 0.0005.