CaMKII+ and GAD+ cell type–specific knockdown of M1-AChR in the mPFC. To determine whether glutamatergic pyramidal neurons and GABAergic interneurons in the mPFC express M1-AChR, double immunohistology was conducted with an M1-AChR antibody and Ca2+/calmodulin-dependent kinase II (CaMKII; pyramidal cell) or glutamate decarboxylase-67 (GAD67; GABA interneuron) (25). The results show that CaMKII+ pyramidal neurons display punctate M1-AChR labeling in the mPFC, and higher magnification shows the presence of M1-AChR+ neurons that lack CaMKII labeling (Figure 1, A and B). Double labeling with GAD67 shows that many of the CaMKII– neurons were GABA interneurons (Figure 1C).

Figure 1 M1-AChR colocalized with CaMKII+ and GAD67+ neurons in the mPFC. WT mice were perfused, and brains were processed for immunohistology. Representative confocal images of immunofluorescent labeling in the mPFC are shown. (A) Images of CaMKII (green) and M1-AChR (red) in the mPFC are shown. Original magnification, ×40. Scale bar: 100 μm. (B) Magnified images of the dashed portion are shown. Original magnification, ×40, zoom 3. Scale bar: 25 μm. (C) Subsequent sections labeled with GAD67 (green) and M1-AChR (red) are shown. Original magnification, ×40, zoom 3. Scale bar: 25 μm. Arrows show neurons colabeled for specific marker and M1-AChR, while arrowheads show neurons that only label with M1-AChR.

For cell-specific M1-AChR knockdown, we used a construct that expresses shRNA targeting M1-AChR (M1shRNA) in a Cre-recombinase–dependent manner (Supplemental Figure 1A). Both DsRed and EGFP are expressed unless recombination deletes EGFP and allows expression of only DsRed and M1shRNA. To test this construct, N2a cells were cotransfected with Cre-expressing (CAG-Cre) and M1shRNA plasmids (pM1shRNA). Transfection with pM1shRNA alone resulted in EGFP and DsRed expression (Supplemental Figure 1B), while cotransfection with CAG-Cre and pM1shRNA resulted in recombination, shown by reduced EGFP expression and retained DsRed expression (Supplemental Figure 1C). Cotransfection with CAG-Cre decreased EGFP mRNA levels in control and pM1shRNA cultures (main effect, F 1,14 = 38.86, P < 0.0001, Supplemental Figure 1D) and, importantly, resulted in robust knockdown of M1-AChR mRNA (interaction, F 1,22 = 16.07, P < 0.0006, Supplemental Figure 1E). There was no significant effect on mAChR subtypes (mAChR2–5), demonstrating specificity of the M1shRNA (Supplemental Figure 1F).

Knockdown of M1-AChR in GAD+, but not CaMKII+, neurons blocks the antidepressant effects of scopolamine. pM1shRNA was packaged into an adeno-associated virus-2 (AAV2M1shRNA) and infused bilaterally into the mPFC of Gad1-Cre and Camk2a-Cre mice or their WT littermate controls. The mPFC was targeted, as prior studies show direct infusion of scopolamine into this region caused antidepressant actions. Initial studies were conducted in Gad1-Cre mice, and 3 weeks after viral infusion, brains were collected for histology (Figure 2A). Figure 2B shows the spread of AAV2M1shRNA infusion in the mPFC, and higher magnification shows that there was no observed recombination in WT mice (Figure 2C). However, infusion with AAV2M1shRNA into Gad1-Cre mice resulted in a subset of cells displaying DsRed only (Figure 2C), demonstrating cell-specific expression of the M1shRNA construct.

Figure 2 Infusion of AAV2M1shRNA in mPFC of Gad1-Cre mice blocked the antidepressant effects of scopolamine. WT or Gad1-Cre mice received bilateral infusion of AAV2M1shRNA in the mPFC. After 3 weeks, mice were tested for baseline behavioral changes, then received saline or scopolamine, followed by additional behavioral tests. (A) Schematic showing experimental approach and time line. A subset of mice were perfused, and brains were processed for histology. Representative confocal images of fluorescence in the mPFC are shown. OF, open field. (B) Low magnification image in WT/AAV2M1shRNA mouse to show representative infusion in mPFC. Original magnification, ×4. Scale bar: 1 mm. (C) Representative image of AAV2M1shRNA in prelimbic mPFC of WT mouse. Original magnification, ×20. Inset shows all neurons have colocalization of eGFP (green) and DsRed (red) fluorescence. Scale bars: 100 μm; 10 μm (inset). (D) Representative image of AAV2M1shRNA in prelimbic mPFC of Gad1-Cre mouse. Original magnification, ×20. Scale bar: 100 μm. Inset shows some neurons have colocalization of eGFP (green) and DsRed (red) fluorescence, while others only have DsRed fluorescence. Scale bar: 10 μm. Prior to scopolamine treatment, mice were tested in open-field activity and FST (preswim). Total distance in the open field (E), time spent in center of open field (F), and time spent immobile in the preswim are shown (G) for each set of experiments. Following repeated doses of scopolamine, WT or Gad1-Cre mice were tested in FST and NSFT. Time spent immobile in the FST (H) and latency to feed in NSFT are shown (I). Scop, scopolamine; Sal, saline. Bars represent the mean ± SEM, n = 7–9/group. Numbers in the bars represent the total sample size for each group. *P < 0.05, means significantly different from the respective saline group based on ANOVA.

Behavioral studies using the same design demonstrate that infusions of AAV2M1shRNA into Gad1-Cre mice had no significant effects on baseline behaviors in the open field test (total distance and center time, anxiety measure) or forced swim test (FST) (immobility, behavioral despair measure) compared with those of WT controls receiving the same viral infusions (Figure 2, E–G). Mice then received 3 doses of scopolamine (25 μg/kg, i.p.) every other day, a dosing regimen based on the schedule used in depressed human subjects and shown to produce robust signaling and behavioral responses in rodents (23). In the FST, scopolamine significantly decreased immobility time in WT mice infused with AAV2M1shRNA, but these effects were completely blocked in Gad1-Cre mice receiving AAV2M1shRNA infusions into the mPFC (interaction, F 1,28 = 8.17, P < 0.0008; Figure 2H). In the novelty suppressed feeding test (NSFT), scopolamine decreased latency to feed in WT mice infused with AAV2M1shRNA, but again had no effect in Gad1-Cre mice receiving the same viral infusions (main effect of scopolamine, F 1,28 = 6.44, P < 0.02; main effect of genotype, F 1,28 = 3.88, P < 0.06; interaction, F 1,28 = 2.71, P < 0.11; Figure 2I).

Similar studies were conducted in Camk2a-Cre mice with infusions of AAV2M1shRNA in the mPFC (Figure 3, A and B). There was no recombination observed in WT mice infused with AAV2M1shRNA (Figure 3, B and C), but in Camk2a-Cre mice, the majority of neurons showed recombination (DsRed+ only) due to the high density of glutamatergic pyramidal neurons in mPFC (Figure 3D). Behavioral testing showed that AAV2M1shRNA had no effect on baseline behavior in the open field test (distance or center time) or immobility in the FST (Figure 3, E–G). Scopolamine administration (3-dose regimen) significantly decreased immobility in the FST in both WT and Camk2a-Cre mice infused with AAV2M1shRNA (main effect, F 1,32 = 18.58, P < 0.0001, Figure 3H). Similar results were observed in the NSFT, where scopolamine administration reduced the latency to feed in both WT and Camk2a-Cre mice receiving AAV2M1shRNA infusion (main effect, F 1,35 = 27.28, P < 0.0001, Figure 3I).

Figure 3 Infusion of AAV2M1shRNA in mPFC of Camk2a-Cre mice did not influence the antidepressant effects of scopolamine. WT or Camk2a-Cre mice received bilateral infusion of AAV2M1shRNA in the mPFC. After 3 weeks, mice were tested for baseline behavioral changes, then received saline or scopolamine, followed by additional behavioral tests. (A) Schematic showing experimental approach and time line. A subset of mice were perfused, and brains were processed for histology. Representative confocal images of fluorescence in the mPFC are shown. (B) Low-magnification image in WT/AAV2M1shRNA mouse to show representative infusion in mPFC. Original magnification, ×4. Scale bar: 1 mm. (C) Representative image of AAV2M1shRNA in prelimbic mPFC of WT mouse. Original magnification, ×20. Inset shows all neurons have colocalization of EGFP (green) and DsRed (red) fluorescence. Scale bars: 100 μm; 10 μm (inset). (D) Representative image of AAV2M1shRNA in prelimbic mPFC of Camk2a-Cre mouse. Original magnification, ×20. Inset shows that many neurons have only DsRed fluorescence, while few have colocalization of EGFP (green) and DsRed (red) fluorescence. Scale bars: 100 μm; 10 μm (inset). Prior to scopolamine treatment, mice were tested in open-field activity and FST (preswim). Total distance in the open field (E), time spent in center of open field (F), and time spent immobile in the preswim are shown (G) for each set of experiments. Following repeated doses of scopolamine, WT or Gad1-Cre mice were tested in FST and NSFT. Time spent immobile in the FST (H) and latency to feed in NSFT are shown (I). Bars represent mean ± SEM, n = 8–11 / group. *P < 0.05, means significantly different from respective saline group based on ANOVA. Numbers in the bars represent the total sample size for each group.

M1-AChR knockdown in GAD+ interneurons blocks scopolamine stimulation of FosB. Prior studies indicate that scopolamine increases Fos labeling in the mPFC, further evidence of increased glutamate neurotransmission and neuronal activation (23). To determine whether M1-AChR knockdown blocks scopolamine induction of neuronal activation in the mPFC, we examined FosB immunohistology (27). WT and Gad1-Cre mice infused with AAV2M1shRNA received a final injection of scopolamine (25 μg/kg), and then 1 hour later, brains were collected and processed for FosB immunohistology. Scopolamine increased the number of FosB+ neurons in the mPFC of WT, but not Gad1-Cre, mice following AAV2M1shRNA infusions (Figure 4A). Scopolamine induction of FosB+ neurons was observed in both the infralimbic and prelimbic cortices of WT/AAV2M1shRNA, but not in Gad1-Cre/AAV2M1shRNA mice (infralimbic, interaction, F 1,14 = 17.51, P < 0.0009; prelimbic, interaction, F 1,14 = 4.22, P < 0.06; Figure 4, B and C). Studies of Camk2a-Cre mice showed that scopolamine induction of FosB+ neurons was observed in both WT/AAV2M1shRNA and Camk2a-Cre/AAV2M1shRNA mice (infralimbic, main effect, F 1,12 = 83.09, P < 0.0001; prelimbic, main effect, F 1,12 = 67.52, P < 0.0001; Supplemental Figure 3). Knockdown of M1-AChR was confirmed by immunolabeling studies that showed that Camk2a-Cre/AAV2M1shRNA significantly reduced M1-AChR immunolabeling in pyramidal neurons compared with WT/AAV2M1shRNA mice (P < 0.02, Supplemental Figure 2). These findings provide further evidence for functional knockdown of M1-AChR in Gad1-Cre mice and blockade of scopolamine-induced neuronal activity.

Figure 4 M1-AChR knockdown in Gad1-Cre mice decreased FosB activation following scopolamine treatment. WT or Gad1-Cre mice received bilateral infusion of AAV2M1shRNA in the mPFC. Following behavioral tests, mice received an acute scopolamine injection (25 μg/kg) and were perfused 1 hour later. Brains were collected and processed for immunohistology. (A) Representative images of FosB labeling in the prelimbic mPFC of WT/AAV2M1shRNA and Gad1-Cre/AAV2M1shRNA mice treated with saline or scopolamine. Scale bar: 100 μm. (B) Quantification of FosB+ neurons in the (B) prelimbic or (C) infralimbic mPFC. Bars represent the mean ± SEM, n = 4–5/group. *P < 0.05, means significantly different from respective saline group based on ANOVA.

PV and SST interneurons show varied distribution and expression of M1-AChR in the mPFC. GABAergic interneurons in the PFC have diverse physiological properties and can be categorized into subtypes based on expression of calcium-binding proteins, such as PV, and neuropeptides, such as SST. Each interneuron subtype has a dynamic role in modulating the activity of glutamatergic pyramidal cells based on firing rates and synaptic connections (28, 29). To further characterize the role of GABA interneurons in the mPFC, we used double immunohistology to determine distribution and colocalize expression of M1-AChR on PV and SST interneuron subtypes (Figure 5, A and B). PV interneurons were concentrated in deeper cortical layers (III–V), while SST interneurons were dispersed evenly across cortical layers (Supplemental Figure 4 and Figure 5D). Further analyses showed that M1-AChR is more frequently colocalized with SST compared with PV interneurons, with approximately 62% of SST and 24% of PV interneurons colocalized with M1-AChR across both mPFC regions (Figure 5, C and D).

Figure 5 Parvalbumin and SST interneurons have varied expression of M1-AChR in the mPFC. WT mice were perfused, and brains were processed for immunohistology. Representative confocal images of immunofluorescent labeling in the mPFC are shown. (A) Images of PV (green) and M1-AChR (red) in the mPFC are shown. Original magnification, ×40, zoom 2. (B) Images of SST (green) and M1-AChR (red) in the mPFC are shown. Original magnification, ×40, zoom 2. Scale bars: 50 μm. Arrows show interneurons colabeled for specific marker and M1-AChR, while arrowheads show interneurons that do not colabel with M1-AChR. (C and D) Number and proportion of PV and SST interneurons that colocalized with M1-AChR in layers I, II/III, and V of the (C) prelimbic or (D) infralimbic mPFC. Proportions represent the percentage of M1-AChR+ interneurons in each subpopulation and cortical layer indicated.

M1-AChR mediates cholinergic stimulation of SST interneurons in the mPFC. To determine the role of M1-AChR in SST interneuron responses to cholinergic stimulation, brain slice electrophysiology was performed with SST-tdTomato mice. ACh or muscarine was applied to mPFC SST interneurons in mPFC slices under whole-cell voltage clamp (layer II/III and V). The SST-tdTomato interneurons had a mean resting membrane potential equal to –75.4 ± 1.4 mV, and ACh induced an inward current (mean = 85.7 ± 17.8 pA). Muscarine application also produced an inward current (mean = 43.3 ± 9.9 pA), while nicotine application caused no change in SST-tdTomato interneuron currents (Figure 6A). In all, 92% of SST-tdTomato interneurons showed inward current responses. We also found that SST-tdTomato interneurons displayed increased excitatory postsynaptic currents (EPSCs) after muscarine application, as a result of excitatory inputs from pyramidal neurons (Figure 6B). Prior application of the mACh receptor antagonist telenzepine blocked both muscarine-induced inward current (musc: mean = 40 ± 9.6 pA; telenzepine + musc: mean = 0 ± 0 pA, Figure 6B) and EPSCs in SST-tdTomato interneurons (Figure 6C).

Figure 6 M1-AChR mediates cholinergic stimulation of SST interneurons in the mPFC. SST-tdTomato and Sst-Cre/AAV2M1shRNA mice were used for brain slice electrophysiology. (A) Representative electrophysiology traces from SST-tdTomato interneurons in the mPFC following application of ACh, muscarine, or nicotine. Consecutive traces shown in the lower panel depict the spontaneously occurring excitatory, inward synaptic currents recorded at –70 mV in the absence and presence of agonists. Representative image of SST-tdTomato neuron with whole cell patch clamp. Scale bar: 10 μm. (B) Representative electrophysiology traces from SST-tdTomato interneurons in the mPFC after application of ACh alone or telenzepine followed by ACh. Lower traces show SST-tdTomato interneurons in the mPFC after application of muscarine alone or telenzepine followed by muscarine. (C) Proportion of SST-tdTomato interneurons that exhibited inward current and EPSCs following stimulation with muscarine (n = 14) or telenzepine followed by muscarine (n = 6). (D) Schematic and representative electrophysiology traces from DsRed+ interneurons in the mPFC of Sst-Cre/AAV2M1shRNA mice following application of muscarine alone or telenzepine followed by muscarine. (E) Proportion of DsRed+ interneurons that exhibited inward current and EPSCs after stimulation with muscarine (n = 6) or telenzepine followed by muscarine (n = 3).

To confirm neuron subtype-specific knockdown of M1-AChR, SST interneurons from Sst-Cre/AAV2M1shRNA mice were recorded (Figure 6D). DsRed+ SST interneurons in these mice had resting membrane potentials equal to –77.3 ± 2.8 mV, comparable to SST-tdTomato interneurons shown above. In Sst-Cre/AAV2M1shRNA mice, a minimal muscarine-induced inward current was observed in 1 out of 6 (17%) interneurons tested. Muscarine-induced EPSCs were observed in 5 out of 6 (83%) interneurons tested (Figure 6E), indicating that synaptic inputs onto SST interneurons were not affected in Sst-Cre/AAV2M1shRNA mice. These EPSCs were blocked when telenzepine was applied prior to muscarine (Figure 6E). Collectively, these data show that cholinergic stimulation of SST interneurons in the mPFC is mediated by M1-AChR and that AAV2M1shRNA effectively blocked muscarinic stimulation of SST interneurons. Knockdown of M1-AChR was confirmed by immunolabeling studies, which showed that infusion of AAV2M1shRNA into Sst-Cre mice significantly reduced M1-AChR immunolabeling compared with that of Sst-Cre mice infused with a scrambled shRNA control (AAV2SCR) (P < 0.02, Supplemental Figure 2).

Knockdown of M1-AChR in SST, but not PV, interneurons prevents the antidepressant-like effects of scopolamine. To determine the GABAergic interneuron subtype that mediates the behavioral responses to scopolamine, WT, Sst-Cre, and PV-Cre mice received bilateral infusion of AAV2M1shRNA into the mPFC and were analyzed 3 weeks later (Figure 7A). Infusion of AAV2M1shRNA into the mPFC of Sst-Cre mice resulted in recombination in a subset of neurons, shown by expression of only DsRed (Figure 7, B and C). AAV2M1shRNA infusion in Sst-Cre mice produced no significant changes in baseline behavior other than a small increase in preswim immobility compared with that of WT mice (P < 0.003; Supplemental Figure 5, B–D). In littermate WT/AAV2M1shRNA mice, scopolamine reduced immobility in the FST as expected, but this effect was completely absent in Sst-Cre/AAV2M1shRNA mice (interaction, F 1,27 = 7.95, P < 0.009; Figure 7E). In the NSFT, scopolamine decreased latency to feed in WT/AAV2M1shRNA mice, but had no effect in Sst-Cre/AAV2M1shRNA mice (interaction, F 1,26 = 3.75, P = 0.06, Figure 7F). In PV-Cre mice, infusion of AAV2M1shRNA resulted in recombination in a subset of neurons, shown by expression of DsRed only (Figure 7D). M1-AChR knockdown in PV-Cre mice caused no baseline behavioral effects in either the open field test or FST (Supplemental Figure 5, E–G) and had no effect on the antidepressant-like actions of scopolamine in the FST (main effect, F 1,23 = 22.35, P < 0.0001, Figure 7G) or NSFT (main effect, F 1,24 = 13.12, P < 0.001, Figure 7H). Following behavioral testing, studies were conducted to determine whether M1-AChR knockdown in SST interneurons blocks scopolamine induction of FosB. In WT/AAV2M1shRNA mice, scopolamine increased FosB+ staining in the mPFC, but there were no significant differences in Sst-Cre/AAV2M1shRNA mice (Figure 8A). Scopolamine increased the number of FosB+ neurons in both the prelimbic (interaction, F 1,14 = 21.64, P < 0.004; Figure 8B) and infralimbic (interaction, F 1,14 = 7.68, P < 0.01; Figure 8C) cortices of WT/AAV2M1shRNA mice, but there were no significant effects in Sst-Cre/AAV2M1shRNA mice (Figure 8, B and C).

Figure 7 Infusion of AAV2M1shRNA in mPFC of Sst-Cre mice blocked the antidepressant effects of scopolamine. WT or Sst-Cre mice received bilateral infusion of AAV2M1shRNA in the mPFC. After 3 weeks, mice were tested for baseline behavioral changes, then received saline or scopolamine, followed by additional behavioral tests. (A) Schematic showing experimental approach and time line. A subset of mice were perfused, and brains were processed for histology. (B) Representative image of AAV2M1shRNA in prelimbic mPFC of WT mouse. Inset shows all neurons have colocalization of EGFP (green) and DsRed (red) fluorescence. Scale bars: 100 μm; 10 μm (inset). (C and D) Representative image of AAV2M1shRNA in prelimbic mPFC of (C) Sst-Cre or (D) PV-Cre mouse. Insets show that many neurons have colocalization of EGFP (green) and DsRed (red) fluorescence, while some have only DsRed (red) fluorescence. Scale bar: 100 μm; 10 μm (insets). Following repeated doses of scopolamine, Sst-Cre or PV-Cre mice with corresponding WT littermates were tested in FST and NSFT. Time spent immobile in the FST (E and G) and latency to feed in NSFT are shown (F and H). Bars represent the mean ± SEM, n = 6–8/group. *P < 0.05; #P = 0.06, means significantly different from respective saline group. Numbers in the bars represent the total sample size for each group.

Figure 8 M1-AChR knockdown in Sst-Cre mice decreased FosB activation following scopolamine treatment. WT or Sst-Cre mice received bilateral infusion of AAV2M1shRNA in the mPFC. Following behavioral tests, mice received an acute scopolamine injection (25 μg/kg) and were perfused 1 hour later. Brains were collected and processed for immunohistology. (A) Representative images of FosB labeling in the prelimbic mPFC of WT/AAV2M1shRNA and Sst-Cre/AAV2M1shRNA mice treated with saline or scopolamine. Scale bar: 100 μm. Quantification of FosB+ neurons in the (B) prelimbic or (C) infralimbic mPFC. Bars represent the mean ± SEM, n = 4–5/group. *P < 0.05, means significantly different from respective saline group based on ANOVA.

To test the possibility that the effects of AAV2M1shRNA knockdown might occur via nonspecific actions of shRNA expression, we examined the effects of a scrambled shRNA control (AAV2SCR) in the mPFC of Sst-Cre mice. Immunohistology showed that M1-AChR knockdown occurred in Sst-Cre/AAV2M1shRNA mice (P < 0.003, Supplemental Figure 2). Similarly to what occurred in prior studies (Figure 7, E and F), Sst-Cre/AAV2M1shRNA mice showed no antidepressant-like response in the FST following scopolamine treatment (Figure 9, A–C). In contrast, Sst-Cre/AAV2SCR mice showed antidepressant-like responses comparable to those of WT/AAV2M1shRNA mice, with significant reductions in FST immobility (interaction, F 2,27 = 5.15, P < 0.01, Figure 9B). In the NSFT, no significant interaction was observed; however, there was a trend for an effect of scopolamine across groups (main effect, F 1,27 = 3.29, P = 0.08, Supplemental Figure 6). Ad hoc analyses showed that WT/AAV2M1shRNA and Sst-Cre/AAV2SCR mice treated with scopolamine exhibited a decrease in latency to feed (P < 0.02 for both, Supplemental Figure 6).

Figure 9 Infusion of AAV2M1shRNA in mPFC of Sst-Cre mice blocked the antidepressant-like effects of scopolamine compared with scrambled control. Sst-Cre mice received bilateral infusion of AAV2M1shRNA or AAV2SCR in the mPFC. After 3 weeks, mice were tested for baseline behavioral changes, then received saline or scopolamine, followed by additional behavioral tests. (A) Schematic showing experimental approach and time line. Following repeated doses of scopolamine, Sst-Cre mice were tested in FST, FUST, and NSFT. Time spent immobile in the FST (B) and interaction time in FUST (C) are shown. Bars represent the mean ± SEM, n = 5–6/group. *P < 0.05, means significantly different from respective saline group. Numbers in the bars represent the total sample size for each group.