Optogenetic stimulation of infralimbic PFC reproduces ketamine's rapid and sustained antidepressant actions

Abstract

Ketamine produces rapid and sustained antidepressant actions in depressed patients, but the precise cellular mechanisms underlying these effects have not been identified. Here we determined if modulation of neuronal activity in the infralimbic prefrontal cortex (IL-PFC) underlies the antidepressant and anxiolytic actions of ketamine. We found that neuronal inactivation of the IL-PFC completely blocked the antidepressant and anxiolytic effects of systemic ketamine in rodent models and that ketamine microinfusion into IL-PFC reproduced these behavioral actions of systemic ketamine. We also found that optogenetic stimulation of the IL-PFC produced rapid and long-lasting antidepressant and anxiolytic effects and that these effects are associated with increased number and function of spine synapses of layer V pyramidal neurons. The results demonstrate that ketamine infusions or optogenetic stimulation of IL-PFC are sufficient to produce long-lasting antidepressant behavioral and synaptic responses similar to the effects of systemic ketamine administration.

Keywords: antidepressant; glutamate; neural depolarization; prefrontal cortex; synapse.

Fig. 1.

IL-PFC stimulation is necessary and sufficient for the antidepressant behavioral actions of ketamine. (A–C) Neuronal silencing by muscimol infusion (1.25 µg per side) into the IL blocks the effects of ketamine (10 mg/kg, i.p.) in the FST (B) and NSFT (C). Immobility times in the FST or the latency to eat in NSFT are shown as the mean ± SEM (n = 4–10 per group). *P < 0.05, compared with PBS + Sal; analysis of variance (two-way or one-way ANOVA with LSD post hoc test). (D–F) Ketamine microinfusions into the IL produce an antidepressant response in the FST (E) and an anxiolytic effect in the NSFT (F). Doses of 10 and 30 ng per side produced a significant response in the FST, and the 10-ng dose was used for subsequent studies in the NSFT. Means are derived from 4–8 rats per group. *P < 0.05, compared with PBS; analysis of variance (one-way ANOVA with LSD post hoc test) or independent t test (C, E, and F). Ket, ketamine; Mus, muscimol; Sal, saline.

Fig. S1.

Muscimol infusions into the IL block the induction of Fos+ cell labeling by systemic ketamine administration. (A) Location of microinfusion. (B and C) Influence of muscimol preinfusion (30 min) on ketamine induction of Fos+ labeling in the (B) IL-PFC and (C) PrL-PFC, determined 1 h after ketamine administration. Right panels show representative Fos+ immunolabeling in the IL under the indicated conditions. *P < 0.05, compared with PBS + Sal; analysis of variance (two-way ANOVA with LSD post hoc test). Ket, ketamine; Mus, muscimol; Sal, saline.

Fig. S2.

Influence of muscimol microinfusions and systemic ketamine administration on locomotor activity. Muscimol microinfusions into IL in the absence or presence of systemic ketamine administration had no effect on locomotor activity. Activity measures were determined 24 h after muscimol and ketamine infusions, the same time point used for analysis of FST and NSFT. Results are the mean ± SEM of controls.

Fig. S3.

Influence of ketamine and muscimol infusions into the Prl on immobility in the FST. (A) Location of muscimol microinfusion into PrL. (B) Muscimol infusion into the PrL had no effect on the antidepressant actions of ketamine in the FST. (C) Microinfusions of ketamine (10 ng per side) into the PrL had no effect on immobility in the FST (D) or latency to feed in the NSFT (E). Immobility times in the FST or latency to feed in NSFT are shown as the mean ± SEM (n = 4–10 per group). *P < 0.05, compared with PBS; analysis of variance (one-way ANOVA with LSD post hoc test, B) or independent t test (D and E). Ket, ketamine; Mus, muscimol; Sal, saline.

Fig. S4.

Muscimol infusion into the PrL blocks the induction of Fos+ cell labeling by systemic ketamine administration. (A) Location of microinfusion. (B and C) Influence of muscimol preinfusion (30 min) on ketamine induction of Fos+ labeling in the (B) PrL-PFC and (C) IL-PFC, determined 1 h after ketamine administration. Right panels show representative Fos+ immunolabeling in the PrL and IL under the indicated conditions. *P < 0.05, compared with PBS + Sal; analysis of variance (one-way ANOVA with LSD post hoc test). Ket, ketamine; Mus, muscimol; Sal, saline.

Fig. 2.

Electrophysiological validation of ChR2 activity in IL layer V pyramidal cells. (A) Two-photon image of a recorded ChR2-eYFP–expressing cell in layer V of IL (green fluorescence, Left), colabeling with neurobiotin/strepavidin-Alexa594 (red fluorescence, Middle), and merged image showing double labeling (Right). (B) Spikes induced in this cell by depolarizing pulses. (C) Whole-cell recordings showing spikes or (D) ChR2 slow-wave currents induced in this cell by laser pulses (15 ms, 10 Hz, marked by blue dashes). (E) A low-magnification (5×) fluorescence image in a PFC slice expressing ChR2-eYFP in the IL; a cloud of green fluorescence can be seen both in in the layer I (apical tuft) and in the layer V (cell body) region. Recorded cells are indicated by the presence of Alexa594-labeled cells; arrow shows track of injection needle. (F) Confocal image (20×) of the apical shaft and apical tuft of double-labeled recorded cells (expanded from area within white box in E). (G) High-magnification (100×) merged image shows punctate green ChR2-eYFP fluorescence surrounding an apical branch of a double-labeled recorded cell; arrows show green punctate labeling in close proximity to neurobiotin/Alexa594-labeled spines; these may represent collateral synaptic connections with nearby unlabeled cells. (H and I) Traces showing laser-induced EPSCs that are evoked in cells with discernable ChR2 slow-wave currents. (J) Example of a cell that does not have detectable ChR2 currents but in which laser stimulation evokes EPSCs; note the variable frequency and amplitude of EPSCs by light pulses (blue dashes). The EPSCs may be generated through axon collaterals of neighboring cells that do express ChR2-eYFP.

Fig. 3.

Optogenetic stimulation of IL and induction of Fos+ labeling. Control (rAAV2-GFP) or active (rAAV2-ChR2-eYFP) viral vectors were infused into the IL-PFC, and 2 wk were allowed for viral infection. (A) Location of viral infusions and conditions for in vivo optogenetic stimulation of IL. Both rAAV2-GFP and rAAV2-ChR2-eYFP animals received laser stimulation. (B) Influence of laser stimulation on Fos+ cell labeling. Representative images of Fos+ expression after photostimulation of IL in rAAV2-ChR2-eYFP and rAAV2-GFP control animals (×20). (Scale bar, 50 μm.) Optical stimulation in IL significantly increased the number of Fos+ cells in IL (t₁₆ = 9.760, P < 0.01).

Fig. S5.

Influence of optogenetic stimulation of IL-PFC on Fos+ cell labeling in the subregions of medial PFC. (A) Map of IL and PrL in the medial PFC of rat. (B and C) Expression of active virus (rAAV2-ChR2-eYFP) in deep layers of IL and location of optic fiber. (C) Stimulation of IL in rats infused with either control virus (rAAV-GFP) or active virus (rAAV2-ChR2-eYFP) (20 min, 10 Hz, 15 ms pulse width; 1 min on and 1 min off) increases the density of Fos+ cells in the IL as well as the PrL. (Inset) High-magnification (20×) view of Fos immunolabeling in IL. **P < 0.05 compared with control virus by two-tailed t test. ACC, anterior cingulate; CC, corpus callosum; IL, infralimbic; PrL, prelimbic.

Fig. 4.

Photostimulation of IL-PFC produces antidepressant and anxiolytic behavioral responses. (A) Timeline for behavioral testing, starting 1 d after laser stimulation. Both rAAV2-GFP and rAAV2-ChR2-eYFP animals received laser stimulation, with the rAAV-GFP serving as controls. Experiments included animals receiving either unilateral (Uni-stim) or bilateral (Bistim) optical stimulation. The results demonstrate that both uni- and bistim of rats infused with rAAV2-ChR2-eYFP produced an antidepressant effect in the FST (B) (F_2,20 = 21.229, P < 0.01) and an anxiolytic effect in the NSFT (C) (F_2,17 = 14.619, P < 0.01) compared with animals receiving rAAV2-GFP control virus and blue light stimulation. Optical stimulation of IL also produced a significant effect in the SPT (D), although only in animals receiving bilateral stimulation (F_2,21 = 3.935, P = 0.037). The antidepressant effect of IL optical stimulation in the FST was still present 17 d after the stimulation (E); at this time point, the effect was more robust in the animals receiving bilateral stimulation (F_2,20 = 35.313, P < 0.01). (F–H) In contrast to IL, bilateral stimulation of PrL had no significant effect in the FST (F) (t₆ = 0.594, P = 0.574), NSFT (G) (t₆ = 0.226, P = 0.829), or SPT (H) (t₆ = 1.302, P = 0.241). Data are the mean ± SEM (n = 4–11 per group). *P < 0.05, **P < 0.01 compared with control animals; analysis of variance (one-way ANOVA with LSD post hoc test) or independent t test.

Fig. S6.

Location of rAAV2-ChR2-eYFP infusion into PrL. (A) Map of PrL and IL in the medial PFC of rat. (B) rAAV2-ChR2-eYFP was infused into the deep layers of the PrL-PFC, and levels of eYFP expression were determined after 3 wk to allow for virus expression. Infusions were made into the dorsal PrL to avoid infection of the underlying IL-PFC.

Fig. S7.

Influence of optogenetic stimulation of IL or PrL on locomotor activity. Optogenetic stimulation of pyramidal neurons in IL or PrL had no significant effect on locomotion, which was measured on day 6 after laser stimulation. The effects of optogenetic stimulation in IL (p, t₂₀ = 0.331, P = 0.744; q, F_2,21 = 0.054, P = 0.948) and in PrL (r, t₆ = 0.339, P = 0.746) on the number of total beam breaks in10 min are shown.

Fig. 5.

Optogenetic stimulation in vivo increases the number and function of spine synapses in IL-PFC pyramidal neurons. (A) Two-photon confocal z-stack projections of apical tuft dendritic segments of layer V pyramidal cells taken from rAAV2-GFP control virus or rAAV2-ChR2-eYFP–infused animals, both receiving in vivo light stimulation. (Scale bar, 10 μm.) (B) Spine density analysis; the results are the mean ± SEM (45 images from 11 cells from 5 rats for control; 66 images from 15 cells from 5 rats for stimulated; **P < 0.01; Student t test). (C) Examples of layer V pyramidal cell recording traces taken from control or stimulated animals; note the marked 5-HT–induced EPSCs in the cell taken from the stimulated animal. (D) Summary data showing both increased frequency (12.2 ± 1.8 and 18.8 ± 2.5 Hz, control and stimulated, respectively; P < 0.05) and amplitude (31.4 ± 1 and 42.3 ± 3.5 pA, respectively; P < 0.05) of 5-HT–induced EPSCs (*P < 0.05; Student t test; n = 15 neurons). (E) Cumulative probability distributions showing significant increases in amplitude (Kolmogorov–Smirnov two-sample test; P < 0.0000; z value = 12.9) and frequency (Kolmogorov–Smirnov two-sample test; P < 0.0000; z value = 10.9) of 5-HT–induced EPSCs (n = 15 neurons per group).

Fig. S8.

Characteristics of dendrite segments used for analysis of spine morphology. Analysis of spine diameter was conducted using Neurolucida 10.21 and Neurolucida Explorer. Shown are the base and average diameters, as well as length, of dendrite segments, none of which were not significantly different between control virus (rAAV2-GFP) or active virus (rAAV2-ChR2-eYFP), both of which received laser stimulation. The number of segments analyzed was 45 and 66 for control and stimulated, respectively.