Optogenetic stimulation of medial prefrontal cortex Drd1 neurons produces rapid and long-lasting antidepressant effects

Abstract

Impaired function in the medial prefrontal cortex (mPFC) contributes to depression, and the therapeutic response produced by novel rapid-acting antidepressants such as ketamine are mediated by mPFC activity. The mPFC contains multiple types of pyramidal cells, but it is unclear whether a particular subtype mediates the rapid antidepressant actions of ketamine. Here we tested two major subtypes, Drd1 and Drd2 dopamine receptor expressing pyramidal neurons and found that activating Drd1 expressing pyramidal cells in the mPFC produces rapid and long-lasting antidepressant and anxiolytic responses. In contrast, photostimulation of Drd2 expressing pyramidal cells was ineffective across anxiety-like and depression-like measures. Disruption of Drd1 activity also blocked the rapid antidepressant effects of ketamine. Finally, we demonstrate that stimulation of mPFC Drd1 terminals in the BLA recapitulates the antidepressant effects of somatic stimulation. These findings aid in understanding the cellular target neurons in the mPFC and the downstream circuitry involved in rapid antidepressant responses.

Fig. 1

Cre-dependent targeting of Chr2 to pyramidal cell subtypes in the mouse mPFC. a mPFC targeting strategy and representative images of Chr2 expression and cannula placement in Drd1-Cre and Drd2-Cre mice. b, c Action potential generation and inward current produced by light activation in Drd1-Cre and Drd2-Cre pyramidal cells, respectively. d Unilateral stimulation of Chr2 in Drd1-Cre and Drd2-Cre mice produces similar increases in cFos expression (normalized cells per mm²) in the stimulated hemisphere that are greater than those observed in the unstimulated hemisphere (Wilcoxon matched pairs signed rank, *p < 0.05 vs. respective stim−, n = 6 males/group). e Light-sensitive pyramidal cells in Drd1-Cre mice demonstrate lower voltage sag (open arrow) upon current injection than light-sensitive pyramidal cells in Drd2-Cre mice (closed arrow) (Factorial ANOVA, group ****p < 0.0001, Drd1-Cre 9 cells/2 male animals, Drd2-Cre 10 cells/3 male animals). Error bars represent mean ± SEM, scale bar 100 µM

Fig. 2

Antidepressant effect of prior stimulation of mPFC Drd1 expressing cells. a Drd1-Cre forced swim immobility 24 h after photostimulation (unpaired t-test, p < 0.05). b, c Drd1-Cre elevated plus maze open arm exploration time and arm entries 48 h after photostimulation (unpaired t-test, p < 0.05). d Fiber optic cannula placements for photostimulation of Drd1-Cre mice. e Drd1-Cre sucrose preference outcome 24 h after photostimulation. f Drd1-Cre novelty suppressed feeding times 72 h after photostimulation (Mantel–Cox (log rank), p < 0.05). g Drd1-Cre total immobility time in the forced swim test 7 days after photostimulation (unpaired t-test, p < 0.05). h Second cohort Drd1-Cre fiber optic cannula placements. i Drd2-Cre forced swim immobility time 24 h after photostimulation. j, k Drd2-Cre elevated plus maze open arm time and arm entries 48 h after photostimulation (unpaired t-test, p < 0.05). l Drd2-Cre time to feed in the novelty suppressed feeding test 72 h after photostimulation. m Fiber optic cannula locations for photostimulation of Drd2-Cre mice; n = a–d Drd1-Cre 5 males, 6 females; WT 4 males, 5 females, e–h Drd1-Cre 7 males, WT 7 males, i–m Drd2-Cre 7 males, 4 females; WT 7 males, 4 females; *p < 0.05. Error bars represent mean ± SEM. Female—closed circles, male—open circles

Fig. 3

Inhibition of mPFC Drd1 expressing cells blocks the antidepressant effect of ketamine. a Representative AAV-ARCH viral expression in ventral mPFC. b Action potentials induced by current injection in voltage clamp are potently inhibited by ARCH activation. c Current clamp recordings demonstrate hyperpolarization associated with light application. d Voltage clamp recordings demonstrate outward current during light application. e FST immobility time 24 h after photoinhibition and ketamine (Factorial ANOVA interaction p = 0.11). f Time to feed in the NSF test 72 h after photoinhibition and ketamine administration (Mantel–Cox (log rank) overall p < 0.01, WT-veh vs. WT-ket p < 0.05). g Representative AAV-hM4Di expression. h FST immobility time 24 h after hM4Di inhibition and ketamine (Factorial ANOVA interaction p < 0.01, post-hoc t-test p < 0.001). i Time to feed in the NSF 72 h after hM4Di inhibition and ketamine (Mantel–Cox (log rank) overall p < 0.05, WT-veh vs. WT-ket p < 0.01). e, f n = 5 males, 3 females per group; h, i n = 5 males, 5 females per WT group, n = 4 males, 5 females Cre ket, n = 5 males, 4 females Cre veh; *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent mean ± SEM, scale bar 100 µM. Male—closed data points, female—open data points

Fig. 4

mPFC Drd2-expressing cells are not necessary for the ketamine response. a Representative AAV-hM4Di viral expression in ventral mPFC. b Forced swim immobility time 24 h after hM4Di inhibition and ketamine (post-hoc t-test, p < 0.001). c Time to feed in novelty suppressed feeding test 72 h after photoinhibition and ketamine administration (Mantel–Cox (log rank) overall p < 0.0001, WT-veh vs. WT-ket p < 0.0001, Cre-veh vs. Cre-ket p < 0.05). n = 5 males, 2 females per WT group, n = 4 males, 3 females per Cre group; *p < 0.05, ***p < 0.001. Error bars represent mean ± SEM, scale bar 100 µM. Male—closed data points, female—open data points

Fig. 5

Antidepressant effect of prior stimulation of mPFC Drd1 terminals in the BLA. a Experimental strategy (infusions and cannulations were bilateral) and representative cannula placements over mPFC Drd1-Cre or WT BLA terminals (green—EYFP, magenta—DAPI); scale bar 100 µM. b Photostimulation of mPFC Drd1 cell terminal field in the BLA increases cFos expression (normalized cells per mm², Mann–Whitney, p < 0.01, Drd1-Cre (n = 5) vs. Drd1-WT (n = 4), green—EYFP, red—cFos, blue—NeuN). c FST immobility time 24 h after photostimulation of mPFC Drd1 terminals in the BLA (Mann–Whitney p < 0.05) Drd1-WT, 11 males, 5 females; Drd1-Cre, 7 males, 7 females. d NSF time 24 h after photostimulation of mPFC Drd1 terminals in the BLA (Mantel–Cox (log rank) p < 0.01). Drd1-WT, 11 males, 5 females; Drd1-Cre, 6 males, 6 females; *p < 0.05, **p < 0.01. Error bars represent mean ± SEM. Male—closed data points, female—open data points

Fig. 6

mPFC projections to the BLA play a role in the ketamine response. a Viral strategy and representative mCherry and hM4Di expression (red = mCherry, hM4D1, cyan = DAPI). b FST immobility time 24 h after CNO and ketamine administration (factoral ANOVA interaction p < 0.05, post-hoc t-test *p < 0.05). c Time to feed in the NSF test 24 h after CNO and ketamine administration (Mantel–Cox (log rank) overall p = 0.06); n = 9 mCherry Veh, hM4Di Veh, hM4Di Ket, n = 10 mCherry Ket. All male. Error bars represent mean ± SEM, scale bar 100 µM

Fig. 7

mPFC D1r pharmacological manipulations impact the effects of ketamine. a FST immobility time 24 h after D1r agonist administration (ANOVA p < 0.05, post-hoc t-test p < 0.05 vs. Veh). b Locomotor activity 24 h after D1r agonist infusion. c Time to feed in the NSF test 48 h after D1r agonist infusion (Mantel–Cox (log rank) overall p < 0.05, Veh vs. 1.0 µg p < 0.05). d FST immobility time 24 h after ketamine treatment with, or without, D1r antagonist (500 ng/side) pretreatment (Factorial ANOVA interaction p < 0.05, post-hoc t-test p < 0.01). e Locomotor activity 24 h after ketamine treatment with, or without, D1r antagonist pretreatment. f NSF time 72 h after treatment with combinations of ketamine and D1r antagonist (Mantel–Cox (log rank) overall p < 0.001, Veh–Veh vs. Veh–Ket p < 0.01); *p < 0.05, **p < 0.01. Error bars represent mean ± SEM. All data points—male

Fig. 8

Proposed model of ketamine associated activity at Drd1 cells in the mPFC. Antagonism of NMDA receptors on GABAergic interneurons produces a non-specific glutamate burst. Activation of Drd1 expressing pyramidal cells modulates activity in output structures. mPFC Drd1 cell projections to the BLA play a role in the antidepressant response to photostimulation and ketamine, but are likely not the only downstream targets of mPFC activation. Drd1 cell activity concurrent with D1r activation results in rapid and long-lasting antidepressant effects. SST somatostatin, PV parvalbumin