Article Figures & Data
Figures
- Figure 1.
Selective expression of hM3Dq DREADD in forebrain excitatory neurons. A, Shown is a schematic of experimental strategy. hM3Dq DREADD was selectively expressed in the CamKIIα-positive excitatory neurons in the forebrain. tTA: tetracycline transactivator. B, Shown is a schematic of the extent of expression. Representative confocal images showing expression of hM3Dq identified by fluorescent immune-staining of HA-tag in the CA1 (C), DG (D), and cortex (E). No HA-tag immunoreactivity was observed in the genotype-control animals (F–H). Blue: DAPI; red: HA-Tag. Scale bar = 50 μm. I, Shown is a schematic showing whole-cell patch-clamp recording from the somata of CA1 pyramidal cells. J, Bath application of CNO (1 μM) resulted in robust spiking activity of CA1 pyramidal neurons. Refer to Extended Data Fig. 1-1 for spiking activity following bath application of 20 μM CNO. R: recording electrode.
- Figure 2.
Acute chemogenetic activation of hippocampal excitatory neurons differentially regulates field transmission in Schaffer collaterals in a dose-dependent manner. A, Shown is a schematic of placement of stimulating and recording electrode in the Schaffer collaterals. B, Shown are representative fEPSP traces before (1) and 30 min (2) following 1 μM CNO treatment and representative fEPSP traces before (3) and 30 min (4) following 20 μM CNO treatment. Refer to Extended Data Fig 2-1 for genotype-controls and bath application of 1 μM, 5 μM, 10 μM, and 20 μM CNO. Refer to Extended Data Fig 2-2 for fEPSP time-course following CNO-washout. Bath application of CNO did not affect fEPSP time course at 1 μM, whereas it led to LTD of fEPSP at 20 μM. Stimulus intensity: 50–80 μA; 20–30 μs. The FV peak was not altered with increasing stimulus intensity either at 1 μM (C) or 20 μM (E). The fEPSP slope was not different at 1 μM (D), but significantly lower in response to increasing stimulus intensity at 20 μM (F). G, Shown are representative paired-pulse-evoked fEPSP traces before (1) and 30 min (2) following 1 μM CNO treatment. A significant potentiation of PPR was observed (G) following 30 min of 1 μM CNO bath application. H, Shown are representative paired-pulse-evoked fEPSP traces before (3) and 30 min (4) following 20 μM CNO treatment. No significant alteration in the PPR was noted following 30 min of 20 μM CNO bath application (H). Results are expressed as the mean ± SEM; *p < 0.05 as compared between 1 and 20 μM CNO treatment (linear regression followed by ANCOVA). S: stimulating electrode; R: recording electrode.
- Figure 3.
Dose-dependent effects of acute chemogenetic hM3Dq activation on TBS-induced LTP in hippocampal slices. A, Shown are representative fESPS traces before, 30 min after CNO treatment and 20 min after LTP induction. B, Slices with 30 min of 1 μM CNO bath application or sham-administered control, show robust induction in LTP, with failure to induce LTP at 20 μM CNO treatment. Bath application of 1 μM CNO results in a significant increase in early-phase LTP as compared to sham-treated controls. C, fEPSP slope normalized to pre-LTP baseline shows robust potentiation with bath application of both sham and 1 μM CNO, but not at 20 μM CNO; *p < 0.05 as compared between 1 and 20 μM CNO treatment (linear regression followed by ANCOVA) for time-course analysis. The average fEPSP slope in 5-min time bins is significantly increased in 5- to 10-, 25- to 30-, and 55- to 60-min bins as compared to pre-LTP in aCSF bath (sham; D) as well as with 1 μM CNO bath application (E). The average fEPSP slope in 5-min time bins was significantly increased 5- to 10- and 25- to 30-min bins as compared to pre-LTP with 20 μM CNO bath application (F). The average fEPSP slope was not significantly different in 55- to 60-min bin as compared to pre-LTP with 20 μM CNO bath application (F). Results are expressed as the mean ± SEM; *p < 0.05 as compared between pre-LTP and post-LTP (5–10 min), post-LTP (25–30 min), or post-LTP (55–60 min; one-way ANOVA followed by post hoc Bonferroni multiple comparison). G, The average fEPSP slope in 5-min time bins is significantly increased in 5- to 10 and 25- to 30-min bins as compared to pre-LTP with 1 μM CNO bath application. The average fEPSP slope with 20 μM CNO bath application in 5- to 10- and 25- to 30-min bins was significantly lower compared to both sham-treated control and 1 μM CNO treatment. The average fEPSP slope in the 55- to 60-min bin was significantly lower with both 1 and 20 μM CNO bath application compared to sham-treated control. Results are expressed as the mean ± SEM; *p < 0.05 as compared between sham, 1 μM, and 20 μM CNO treatment for each time bin (one-way ANOVA followed by post hoc Bonferroni multiple comparison).
- Figure 4.
Bidirectional dose-dependent modulation of excitability following 30 min of hM3Dq chemogenetic activation of hippocampal excitatory pyramidal neurons. A, Shown is a schematic depicting whole-cell patch-clamp recording from somata of CA1 pyramidal cells. B, The number of spikes generated with increasing amount of current injection is enhanced with bath application of 1 μM CNO but reduced with 20 μM CNO. C, Shown is a representative trace of action potentials from CA1 neurons before CNO treatment, after a 30-min bath application of 1 μM CNO, and 20 μM CNO. Results are expressed as the mean ± SEM; *p < 0.05 as compared between 1 and 20 μM CNO treatment (linear regression followed by ANCOVA). Shown are representative sPSC traces before (D) and 30 min after 1 μM (E) or 20 μM (F) CNO treatment. Bath application of 1 μM CNO for 30 min resulted in significantly altered sPSC amplitude with decreased cumulative probability at lower amplitude and increased cumulative probability at higher amplitude (>35 pA; G), whereas no significant effect was observed on interevent interval (H). Bath application of 20 μM CNO for 30 min significantly decreased sPSC amplitude (I) and sPSC interevent interval (J). Results are expressed as cumulative probabilities; #p < 0.001 as compared to pre-CNO-treated group (Kolmogorov–Smirnov two-sample comparison). R: recording electrode.
- Figure 5.
Dose-dependent effects of acute chemogenetic hM3Dq activation on AMPAR- and NMDAR-mediated currents. A, Shown is a schematic depicting whole-cell patch-clamp recording from somata of CA1 pyramidal cells with stimulating electrodes places on the Schaffer collateral input pathway. B, Shown is a schematic demonstrating the measurement of AMPAR- and NMDAR-mediated currents, respectively. C, Shown are representative AMPAR-mediated-evoked PSC traces before and after 10 and 30 min of CNO bath application. Voltage clamped at –70 mV. D, Shown are representative NMDAR-mediated-evoked PSC traces before and after 10 and 30 min of CNO bath application. Voltage clamped at +40 mV. Bath application of both 1 and 20 μM CNO results in reduced AMPAR-mediated currents, with significantly lower currents at 20 μM (E, G). Bath application of both 1 and 20 μM CNO results in reduced NMDAR-mediated currents, with significantly lower currents at 20 μM (F, H). AMPAR/NMDAR ratio is significantly reduced with bath application of 20 μM CNO (I). The time constant of NMDAR-mediated current decay (τ) is significantly decreased following 30 min of both 1 and 20 μM CNO administration (J). Results are expressed as the mean ± SEM; *p < 0.05 as compared between 1 and 20 μM CNO treatment (linear regression followed by ANCOVA) for time-course analysis; *p < 0.05 as compared between pre-CNO, 1 and 20 μM CNO treatment (one-way ANOVA followed by post hoc Bonferroni multiple comparison) for others. S: stimulating electrode; R: recording electrode.
- Figure 6.
Acute administration of CNO results in a dose-dependent increase in intracellular calcium levels in primary hippocampal neurons. A, Shown are representative images of primary hippocampal neurons before CNO-treatment and 4, 8, and 12 min following 1 μM (top) or 20 μM (bottom) CNO treatment. B, Application of both 1 and 20 μM CNO significantly increased intracellular calcium levels as observed by increased fluorescence intensity. Treatment with 20 μM CNO resulted in a significantly faster increase in intracellular calcium levels as compared to 1 μM CNO. Results are expressed as the mean ± SEM; *p < 0.05 as compared between 1 and 20 μM CNO treatment (linear regression followed by ANCOVA).
Tables
- Table 1.
Acute CNO-mediated hM3Dq DREADD activation of hippocampal excitatory neurons does not alter intrinsic membrane properties
RMP (mV) Input resistance (MΩ) AP threshold (mV) τ (ms) Sag (mV) Sag (%) Accomodation index Pre-CNO -61.31 ± 0.62 194.9 ± 10.95 -53.61 ± 0.59 26.15 ± 1.57 -5.67 ± 0.61 6.39 ± 0.61 0.45 ± 0.04 30 min Post-CNO (1 μM) -59.24 ± 1.52 173.7 ± 17.47 -56.04 ± 2.11 23.11 ± 1.66 -4.84 ± 0.85 5.82 ± 0.84 0.36 ± 0.09 30 min Post-CNO (20 μM) -62.30 ± 1.31 201.8 ± 13.21 -53.39 ± 1.22 27.05 ± 3.03 -4.71 ± 0.72 5.30 ± 0.74 0.47 ± 0.08 AP threshold: action potential threshold; τ: membrane time constant.
Extended Data Figure 1-1
A, Shown is a schematic showing whole-cell patch-clamp recording from the somata of CA1 pyramidal cells. B, Bath application of CNO (20 μM) resulted in spiking activity of CA1 pyramidal neurons. R: recording electrode. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Bath application of hM3Dq DREADD agonist CNO had no effect on fEPSP at low dose (1 μM; A). CNO treatment induced LTD at a subsequently high dosage of 5 μM (B), 10 μM (C), and 20 μM (D). No effect on fEPSP was observed at 1 μM (E) and 20 μM (F) CNO in slices taken from background C57Bl/6J animals. Results are expressed as the mean ± SEM. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
A, Bath application of 20 μM CNO for 30 min resulted in decline in fEPSP slope. The fEPSP slope continued to decrease for ∼30 min after washout with aCSF before reaching a steady-state. Recovery to baseline was not observed for at least 60 min following wash with aCSF. Download Figure 2-2, TIF file.
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