Longitudinal assessment of DREADD expression and efficacy in the monkey brain
- Advanced Neuroimaging Center, National Institutes for Quantum Science and Technology, Japan
- System Neuroscience Section, Center for the Evolutionary Origins of Human Behavior, Kyoto University, Japan
- Department of Advanced Nuclear Medicine Sciences, National Institutes for Quantum Science and Technology, Japan
Figures
Figure 1
Time course of DREADD expression levels up to 150 days after injection.
(A, B) Parametric coronal images of the increase (relative to baseline) in specific binding of [11C]DCZ (ΔBPND) at 30 (A) and 64 (B) days after adeno-associated virus (AAV) injection, overlaid on MR images. Images were obtained from a monkey (#241) that received multiple injections of AAV with different constructs (see Table 2). (C) Time course of in vivo DREADD expression levels (ΔBPND) up to 150 days after the injections, summarized from 10 regions of interest (ROIs) obtained from seven monkeys. The value at day 0 indicates the baseline (before injection). The black curve is the best-fitted sigmoid curve, which provides a better fit (Bayesian information criterion, BIC = 61.1) than does the double logistic model (BIC = 62.9). Lowercase letters correspond to the DREADD-induced regions described in Table 2.
Figure 2
Longitudinal time-course of DREADD expression levels.
(A) Parametric coronal images of DREADD expression levels (ΔBPND) overlaid on MR images obtained from monkey #237, measured at 42, 496, 978, and 1,139 days after injection. Scale bar is 10 mm. (B, C) Time course of hM4Di (B) and hM3Dq (C) expression levels normalized to the peak ΔBPND observed between 40 and 80 days post-injection (time between the dotted red vertical lines). Each colored line represents one injection site, and the lowercase letters at the ends of each line correspond to the injection sites listed in Table 2. Each dot represents one [11C]DCZ PET measurement. The dashed black line indicates the group average at 6-month intervals. hM4Di expression levels remained stable for approximately 1.5 and those for hM3Dq about 1 year, after which variable patterns of decline were observed.
Figure 3
Duration of effective functional modulation induced by DREADDs.
(A, B) Duration of effective functional modulation induced by hM4Di (A) and hM3Dq (B). Blue and red horizontal bars indicate the duration of successful chemogenetic manipulation. Black and white ticks on the horizontal bars mark the timing of agonist administration, with CNO and Compound 21 (C21) explicitly stated; all other ticks correspond to DCZ. For example, repeated DCZ administration consistently induced behavioral change in monkey #229 and increased glucose metabolism in monkey #215. The corresponding functional assessments are summarized in Table 1. The dark blue bar indicates the duration of failed chemogenetic manipulation in monkey #234 (see also Figure 4). Total numbers of agonist administrations (DREADD activation) are shown at the end of each duration bar. Red ticks indicate the timing of DCZ-PET scans and red arrowheads indicate the timing of perfusion. (C) In vivo visualization of hM4Di expression in the dorsolateral prefrontal cortex 377 days after injection (monkey #229). The coronal PET image showing specific binding of [11C]DCZ is overlaid on MR images. (D, E) Nissl- (D) and DAB-stained (E) sections corresponding to (C), representing immunoreactivity against reporter protein. White and black arrowheads represent the dorsal and ventral borders of the target regions, respectively. Scale bars, 5 mm. (F) In vivo visualization of hM3Dq expression in the amygdala 980 days after the injection (monkey #215). The coronal PET image showing specific binding of [11C]DCZ is overlaid on MR images. (G, H) Nissl- (G) and DAB-stained (H) sections corresponding to (F), representing immunoreactivity against reporter protein.
Figure 4
Disappearance of behavioral effects in an hM4Di-extinguished monkey (#234).
(A) Top: coronal PET image showing ΔBPND of [11C]DCZ data overlaid on an MR image, obtained 44 days after viral vector injection into the right SID2. Filled and open arrowheads represent the central and ipsilateral sulci, respectively. Middle: performance of fine grasping using a modified Brinkman board task, assessing the monkey’s ability to pick up small food pellets with its thumb and index fingers. Values indicate the change in total duration to complete the task between pre- and post-DCZ administration sessions. Ipsi (Contra) refers to performance using the hand ipsilateral (contralateral) to the hM4Di-expressing SID2. Gray lines connect performances from the same sessions using different hands. Error bars, s.e.m. *P<0.002, paired t-test. +P<0.004, paired t-test, adjusted for multiple comparisons. n=5 sessions. Bottom: DCZ-induced change in the foot withdrawal latency in response to cold (magenta) or control (cyan) stimulation. Negative values indicate faster withdrawal latency following DCZ administration. Gray lines connect performances from the same sessions. Error bars, s.e.m. *P<0.001, paired t-test. +P<0.001, paired t-test comparing between pre- and post-DCZ administration. n=7 sessions. (B) Same types of measurements as in (A), but following the extinction of hM4Di expression. The PET image was obtained about 3 years (1051 days) post-vector injection. n=5 and n=4 sessions for Brinkman board task and foot withdrawal test, respectively. (C) A Nissl-stained section demonstrating the absence of neuronal loss at the vector injection sites (left panel) and the contralateral side (right panel). The locations of the filled and open arrowheads correspond to those shown in (A) and (B). Scale bars are 2.5 mm and 250 μm.
Figure 5 with 1 supplement
Effect of protein tags on peak DREADD expression levels.
Peak expression levels (ΔBPND) for different constructs showing the presence and type of protein tag. Box plots represent the median (central line), interquartile range (box), and the range (whiskers) of the data. Individual data points are labeled with lowercase letters and correspond to the injection sites listed in Table 2. Note that two cases (right putamen of #223 and left putamen of #255) were identified as outliner and excluded from the analysis (see ‘Materials and methods’).
Figure 5—figure supplement 1
Effect of promoter and DREADD type on peak expression levels.
Peak expression levels (ΔBPND) are shown for different constructs combining two promoter types and two DREADD types. Box plots represent the median (central line), interquartile range (box), and the range (whiskers) of the data. Individual data points labeled with lowercase letters correspond to the injection sites listed in Table 2.
Author response image 1
Tables
Table 1
Summary of subjects, DREADD used, and functional assessments used in this study.
| ID | Species | Sex | weight (kg) | Age (years) | DREADD | [11C]DCZ | Behavior | FDG | Electrophysiology |
|---|---|---|---|---|---|---|---|---|---|
| 153 | R | M | 6.8 | 10 | hM4Di | ✓* | |||
| 163 | R | M | 6.1 | 13 | hM4Di | ✓† | |||
| 193 | R | M | 4.4 | 9 | hM4Di | ✓ | |||
| 201 | R | M | 6.3 | 8 | hM4Di | ✓ ‡ | ✓ | ||
| 207 | R | M | 6.8 | 6 | hM4Di | ✓ ‡ | ✓ | ||
| 212 | R | F | 3.4 | 8 | hM4Di | ✓ | |||
| 218 | J | M | 7.2 | 4 | hM4Di | ✓ § | |||
| 221 | J | M | 6.4 | 4 | hM4Di | ✓ | ✓* | ||
| 225 | J | F | 6 | 7 | hM4Di | ✓ | ✓ ¶ | ||
| 229 | R | M | 4.6 | 4 | hM4Di | ✓ | ✓** | ||
| 234 | J | M | 5.8 | 5 | hM4Di | ✓ | ✓ ¶ | ||
| 237 | J | M | 6.9 | 5 | hM4Di | ✓ | |||
| 238 | J | M | 6.2 | 4 | hM4Di | ✓ | ✓ § | ||
| 245 | J | F | 5.9 | 6 | hM4Di | ✓ | ✓** | ||
| 215 | R | F | 3.4 | 8 | hM3Dq | ✓ | ✓ | ||
| 223 | C | M | 4.2 | 4 | hM3Dq | ✓ | ✓ | ||
| 224 | J | M | 9.9 | 9 | hM3Dq | ✓ | ✓ | ||
| 236 | J | M | 7.1 | 6 | hM3Dq | ✓ | ✓ | ||
| 241 | J | M | 5.1 | 3 | hM3Dq | ✓ | ✓ | ||
| 255 | C | M | 5.2 | 4 | hM3Dq | ✓ | ✓ |
-
Weight and age are the values recorded at the beginning of the experiments.
-
R, Rhesus; J, Japanese; C, Cynomolgus; M, male; F, female.
-
*
Multi-reward task, #153 and #221, Oyama et al., 2022a.
-
†
Delayed-reward task, #163, Hori et al., 2021.
-
‡
Delayed matching-to-sample task, #201 and #207, Hirabayashi et al., 2024.
-
§
Reversal learning task, #218 and #238, Oyama et al., 2024.
-
¶
Brinkman board/foot sensation, #225 and #234, Hirabayashi et al., 2021.
-
**
Delayed response task, #229 and #245, Nagai et al., 2020.
Table 2
Summary of injection location, type of virus vector, titer, and injected volume used in this study.
| ID | Region | Vector | Titer(×1013 gc/ml) | Volume (µl) | Symbols in figures | |||
|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 5 | S1 | |||||
| 153 | R-OFC | AAV2-CMV-hM4Di | 1.0 | 54 | ||||
| L-rmCD | AAV2-CMV-hM4Di | 2.0 | 3 | |||||
| 163 | L-dCDh | AAV2.1-hSyn-hM4Di-IRES2-AcGFP | 4.7 | 6 | ||||
| R-dCDh | AAV2.1-hSyn-hM4Di-IRES2-AcGFP | 4.7 | 6 | |||||
| 193 | R-Amygdala | AAV2-CMV-hM4Di | 2.0 | 6 | a | a | a | |
| 201 | R-OFC (microinjected) | AAV2-CMV-hM4Di | 2.2 | 2 | ||||
| L-OFC (microinjected) | AAV2-CMV-hM4Di | 2.2 | 2 | |||||
| 207 | R-OFC (microinjected) | AAV2-CMV-hM4Di | 1.3 | 3 | ||||
| L-OFC (microinjected) | AAV2-CMV-hM4Di | 1.3 | 3 | |||||
| 212 | R-Put | AAV2-CMV-hM4Di | 2.6 | 6 | b | b | b | b |
| 215 | L-Amygdala | AAV2-CMV-hM3Dq | 1.2 | 6 | c | c | c | c |
| 218 | R-OFC | AAV2-CMV-hM4Di | 2.3 | 50 | ||||
| L-OFC | AAV2-CMV-hM4Di | 2.3 | 54 | |||||
| 221 | L-OFC | AAV2-CMV-hM4Di | 2.0 | 50 | ||||
| R-rmCD | AAV2-CMV-hM4Di | 2.0 | 3 | k | k | k | ||
| 223 | R-Cd | AAV2.1-hSyn-hM3Dq-IRES2-AcGFP | 1.0 | 3 | d | d | d | d |
| R-Put | AAV2.1-hSyn-hM3Dq-IRES2-AcGFP | 5.0 | 3 | l | ||||
| L-Cd | AAV2-CMV-hM3Dq-IRES-AcGFP | 1.0 | 3 | |||||
| L-Put | AAV2-hSyn-hM3Dq-IRES2-AcGFP | 1.0 | 3 | r | r | |||
| 224 | R-Amygdala | AAV2.1-hSyn-hM3Dq-IRES-AcGFP | 2.0 | 5 | m | m | m | |
| L-Amygdala | AAV2.1-hSyn-hM3Dq-IRES-AcGFP | 2.0 | 4 | s | s | |||
| 225 | L-SID2 | AAV2-CMV-hM4Di | 1.5 | 4 | t | t | ||
| 229 | R-dlPFC | AAV2.1-hSyn-hM4D-IRES2-AcGFP | 4.7 | 35 | n | |||
| L-dlPFC | AAV2.1-hSyn-hM4D-IRES2-AcGFP | 4.7 | 37 | |||||
| 234 | R-SID2 | AAV2.1-hSyn-hM4D-IRES2-AcGFP | 3.8 | 4 | o | o | o | |
| 236 | R-Amygdala | AAV2-CMV-hM3Dq | 1.2 | 6 | ||||
| 237 | L-Amygdala | AAV2.1-hSyn-hM4Di-IRES2-AcGFP | 2.0 | 4 | e | e | e | e |
| 238 | R-OFC | AAV2.1-CaMKII-hM4Di-IRES-AcGFP | 1.0 | 49 | q | |||
| L-OFC | AAV2.1-CaMKII-hM4Di-IRES-AcGFP | 1.0 | 53 | p | ||||
| 241 | R-Cd | AAV2-hSyn-hM3Dq-IRES-AcGFP | 2.0 | 3 | u | u | ||
| L-Cd | AAV2-hSyn-hM3Dq | 2.0 | 3 | g | g | g | g | |
| R-Put | AAV2.1-hSyn-hM3Dq-IRES2-AcGFP | 2.0 | 3 | h | h | h | h | |
| L-Put | AAV1-hSyn-hM3Dq-IRES2-AcGFP | 2.0 | 3 | f | f | f | f | |
| 245 | R-dlPFC | AAV2.1-hSyn-hM4D-IRES2-AcGFP | 4.7 | 44 | ||||
| L-dlPFC | AAV2.1-hSyn-hM4D-IRES2-AcGFP | 4.7 | 40 | |||||
| 255 | L-VPL | AAV5-hSyn-HA-hM3Dq | 2.7 | 3 | j | j | j | j |
| R-VPL | AAV2-hSyn-hM3Dq | 1.8 | 3 | i | i | i | i | |
| L-Cd | AAV2-hSyn-hM4Di | 1.2 | 3 | x | x | |||
| R-Cd | AAV2-hSyn-hM4Di | 1.7 | 3 | v | v | |||
| L-Put | AAV5-hSyn-hM4Di | 4.6 | 3 | |||||
| R-Put | AAV2-hSyn-hM4Di | 1.7 | 3 | w | w | |||
-
R, right; L, left; OFC, orbitofrontal cortex; rmCD, rostromedial caudate nucleus; dCDh, dorsal part of caudate nucleus head; CD, caudate nucleus; Put, putamen; S1D2, hand index finger region of primary somatosensory cortex; dlPFC, dorsolateral prefrontal cortex; VPL, ventral posterolateral nucleus of thalamus.
Table 3
Results of the linear model analysis.
| Effect | DFn | DFd | F-value | P value | Effect size (η²G) |
|---|---|---|---|---|---|
| Titer | 1 | 4 | 2.093 | 0.222 | 0.343 |
| DREADD | 1 | 4 | 2.565 | 0.184 | 0.391 |
| Promoter | 1 | 4 | 5.280 | 0.083 | 0.569 |
| Tag | 2 | 4 | 19.84 | 0.008* | 0.908 |
| Serotype | 3 | 4 | 2.105 | 0.242 | 0.612 |
| Volume | 1 | 4 | 0.090 | 0.779 | 0.022 |
| Titer:promoter | 1 | 4 | 2.346 | 0.200 | 0.370 |
| DREADD:promoter | 1 | 4 | 10.74 | 0.031* | 0.729 |
| Titer:tag | 2 | 4 | 1.494 | 0.328 | 0.428 |
| Titer:volume | 1 | 4 | 3.199 | 0.148 | 0.444 |
| DREADD:volume | 1 | 4 | 1.497 | 0.288 | 0.272 |
-
The ANOVA table describes the factors contributing to the level of expression. The optimal model, selected based on Akaike’s information criterion, was the following: ΔBPND = viral titer + DREADD type + promoter + reporter tag + serotype + injection volume + viral titer:promoter + DREADD type:promoter + viral titer:reporter-tag + viral titer:injection volume + DREADD type:injection volume.
-
DFn, degrees of freedom numerator; DFd, degrees of freedom denominator; η²G, generalized eta-squared.
-
*
P<0.05.
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Longitudinal assessment of DREADD expression and efficacy in the monkey brain
eLife 14:RP105815.
https://doi.org/10.7554/eLife.105815.3
