Inhibition of impulsive action by projection-defined prefrontal pyramidal neurons

Abstract

The prefrontal cortex (PFC) plays a critical role in curbing impulsive behavior, but the underlying circuit mechanism remains incompletely understood. Here we show that a subset of dorsomedial PFC (dmPFC) layer 5 pyramidal neurons, which project to the subthalamic nucleus (STN) of the basal ganglia, play a key role in inhibiting impulsive responses in a go/no-go task. Projection-specific labeling and calcium imaging showed that the great majority of STN-projecting neurons were preferentially active in no-go trials when the mouse successfully withheld licking responses, but lateral hypothalamus (LH)-projecting neurons were more active in go trials with licking; visual cortex (V1)-projecting neurons showed only weak task-related activity. Optogenetic activation and inactivation of STN-projecting neurons reduced and increased inappropriate licking, respectively, partly through their direct innervation of the STN, but manipulating LH-projecting neurons had the opposite effects. These results identify a projection-defined subtype of PFC pyramidal neurons as key mediators of impulse control.

Keywords: impulsive behavior; lateral hypothalamus; prefrontal cortex; subthalamic nucleus; two-photon calcium imaging.

Fig. 1.

Different dmPFC layer 5 neurons project to STN, V1, and LH. (A) Virus injection for tracing axonal projections from dmPFC layer 5 neurons. (B) Fluorescence images of the injection site (white box in coronal diagram) and several ipsilateral projection targets. Red, mCherry; blue, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). (Scale bar, 1 mm.) (C) Injection procedure for dual retrograde tracing. (D) Fluorescence images of retrogradely labeled PFC neurons. (Right) Enlarged view of the region in white box (Left). Red, mCherry; green, eGFP; blue, DAPI. (Scale bar, Left, 500 µm, and Right, 100 µm.) (E) Quantification of overlap between each pair of projection-defined populations (n = 3 mice each). Spatial distribution of (F) PFC^→STN (n = 6 mice), (G) PFC^→V1 (n = 6 mice), and (H) PFC^→LH (n = 6 mice) neurons. The three distributions are significantly different (PFC^→STN vs. PFC^→V1, P = 7.7 × 10⁻⁶; PFC^→LH vs. PFC^→V1, P = 0.03; PFC^→STN vs. PFC^→LH, P = 0.006; Kolmogorov–Smirnov test, Bonferroni–Holm corrected). All error bars indicate ±SEM.

Fig. 2.

Activity of PFC^→STN, PFC^→V1, and PFC^→LH neurons during go/no-go task. (A) Schematic of calcium imaging during task. (B) Schematic for the task structure (Top) and an example behavioral session (Bottom). Each tick indicates one lick. Yellow shading indicates delay period. Dashed line indicates end of response window. Numbers in brackets indicate the number of trials of each type. (C) (Left) Field of view in an example imaging session for PFC^→STN neurons. Red outlines indicate example cells shown on the right. (Scale bar, 100 µm.) (C) (Right) Raw fluorescence traces of the example ROIs. Yellow shading indicates delay period; blue/magenta stripes indicate cue periods with target/nontarget tones, respectively. Dashed line indicates end of response window. Black ticks on top are lick responses. Blue/cyan arrowheads indicate delivery of reward/punishment. (Vertical scale bar, 3 Z scores; horizontal scale bar, 5 s.) (D) Trial-averaged activity of the example ROIs. Shading indicates ±SEM. (Scale bar, 1 Z score.) (E and F) Similar to C and D but for PFC^→V1 neurons. (G and H) Similar to C and D but for PFC^→LH neurons. (I) Euclidean distance between the population activity in the correct go (hit) and no-go (CR) trials for PFC^→STN neurons. The distance during the cue and delay periods was significantly higher than baseline (P = 1.9 × 10⁻⁷, Wilcoxon rank sum test, n = 6 mice, ***P < 0.001). (J) Similar to I but for PFC^→V1 neurons (P = 0.69, n = 6 mice). (K) Similar to I but for PFC^→LH neurons (P = 1.2 × 10⁻⁵, n = 6 mice).

Fig. 3.

Go vs. no-go preference of PFC^→STN and PFC^→LH neurons. (A) Color-coded difference between averaged Z-scored activity in CR and hit trials (CR − hit) for each PFC^→STN neuron. Neurons are sorted based on the averaged difference during the late delay period (3 to 4 s). Black rectangle indicates the late delay period quantified in B. Red arrows indicate the neurons shown in Fig. 2D. (B) CR – FA vs. CR – hit activity difference during the late delay period for PFC^→STN neurons. Magenta/blue circles indicate neurons with higher/lower activity in CR than hit trials (P < 0.05, two-sided t test). Red arrows indicate the neurons shown in Fig. 2D. (C and D) Similar to A and B but for PFC^→LH neurons. Red arrows indicate the neurons shown in Fig. 2H. (E and F) Similar to A and B but for the difference between CR and FA trials. (G and H) Similar to E and F but for PFC^→LH neurons.

Fig. 4.

Inputs to PFC^→STN, PFC^→V1, and PFC^→LH neurons. (A) Schematic for RV-mediated transsynaptic retrograde tracing. (B) Fluorescence images of starter cells in dmPFC (white box in coronal diagram). Red, RV-tdTomato; green, TVA-eGFP; blue, DAPI. White arrowheads indicate starter cells expressing both RV-tdTomato and TVA-eGFP. (Scale bar, 100 µm.) (C) Fluorescence images of presynaptic cells in several brain regions (black boxes in coronal diagrams). Red, RV-tdTomato; blue, DAPI. MO, somatomotor areas; ACA, anterior cingulate area; NDB, diagonal band nucleus; VM, ventral medial nucleus of the thalamus; VIS, visual cortical area. (Scale bar, 500 µm.) (D) Percentages of total presynaptic inputs (PFC^→STN, n = 4 mice; PFC^→V1, n = 3 mice; PFC^→LH, n = 4 mice; *P < 0.05; **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey’s post hoc test). Only regions with >2% inputs to at least one of the three populations are shown. Dashed boxes indicate regions shown in C. ORB, orbital area; PL, prelimbic area; ILA, infralimbic area; SS, somatosensory areas; RSP, retrosplenial area; AM, anteromedial nucleus of the thalamus; CL, central lateral nucleus of the thalamus; MD, mediodorsal nucleus of the thalamus; PO, posterior complex of the thalamus; VAL, ventral anterior-lateral complex of the thalamus; LP, lateral posterior nucleus of the thalamus; LD, lateral dorsal nucleus of the thalamus. Error bars indicate ±SEM.

Fig. 5.

Causal functions of PFC^→STN and PFC^→LH neurons. (A) Schematic of optogenetic activation/inactivation of PFC^→STN neurons (Left) and fluorescence image of dmPFC expressing ChR2-eYFP (Right). Green, ChR2-eYFP; blue, DAPI. (Scale bar, 400 µm.) (B) Similar to A but for PFC^{→ LH} neurons. (C) An example session with PFC^→STN neuron activation. (Left) Laser on trials and (Right) laser off trials. Blue shading indicates laser stimulation. Each tick indicates one lick. (D) Activation of PFC^→STN neurons caused a marked improvement in task performance by reducing the FA rate (correct rate, P < 0.001; hit rate, P = 0.77; FA rate, P < 0.001; bootstrap). Each circle represents one mouse (n = 6 mice). (E and F) Similar to C and D but for behavioral impairment caused by PFC^→LH neuron activation (correct rate, P = 0.04; hit rate, P = 0.44; FA rate, P = 0.03; n = 9 mice). (G and H) Similar to C and D but for PFC^→STN neuron inactivation (correct rate, P < 0.001; hit rate, P = 0.45; FA rate, P < 0.001; n = 6 mice). (I and J) Similar to G and H but for PFC^→LH neuron inactivation (correct rate, P = 0.04; hit rate, P = 0.46; FA rate, P = 0.04; n = 7 mice).

Fig. 6.

Effects of activating PFC^→STN axons or STN neurons. (A) Schematic of optogenetic activation of dmPFC axon terminals in STN. (B) Fluorescence image showing ChETA-eYFP expressing dmPFC axons in STN. Green, ChETA-eYFP; blue, DAPI. (Scale bar, 1 mm.) (C) Activation of dmPFC axon terminals in STN (50 Hz) caused a significant improvement in task performance by reducing the FA rate (correct rate, P = 0.03; hit rate, P = 0.53; FA rate, P < 0.001; bootstrap, n = 6 mice). (D) Schematic of optogenetic activation of STN cells. (E) Fluorescence image showing ChR2-eYFP expression in STN neurons. Green, ChR2-eYFP; blue, DAPI. (Scale bar, 1 mm.) (F) Activation of STN cell (10 Hz) caused a significant improvement in task performance (correct, hit, and FA rates are all P < 0.001; bootstrap, n = 7 mice).