Figures and data
Summary of subjects, DREADD used, and functional assessments used in this study
Time course of the DREADD expression levels up to 150 days after the 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 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) until 150 days after the injections, summarized from 10 ROIs obtained from 7 monkeys. The value at day 0 indicates the baseline (before injection). The red dots and numbers correspond to the value at the ROIs depicted in A and B. The black line is the best-fitted sigmoid curve.
Summary of injection location, type of virus vectors, titer, and injected volume used in this study
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. (B-C) Time course of hM4Di (B) and hM3Dq (C) expression as a ratio of peak expression level (ΔBPND). The dotted red vertical lines indicate the range of peak times across 11 monkeys (40 to 80 days). Expression levels of remained stable for about 1 (hM3Dq) or 1.5 years (hM4Di), after which they gradually decreased.
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. For example, agonist administration (black tics) 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 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 and 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 and H) Nissl-(G) and DAB-stained (H) sections corresponding to F, representing immunoreactivity against reporter protein.
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 withdrawal latency of the foot sole 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 measurements as in A, but following the extinction of hM4Di expression. The PET image was obtained about three years (1,051 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. 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.
Peak DREADD expression levels among different viral vector constructs.
(A) Relationship between peak expression levels (ΔBPND) and virus titer. Regression lines are plotted for each DREADD (colored) and overall (black). (B) Peak expression levels (ΔBPND) for different constructs with the presence and type of protein tag. Box plots represent the median (central line), interquartile range (box), and the range (whiskers) of the data. Points outside the whiskers indicate outliers.
