C57Bl/6 × Sv129 hybrid mice were used in all experiments. All mice were housed according to guidelines from the Animal Care and Use Committee of Johns Hopkins University. Male and female mice age 2–8 months were housed in plastic translucent cages with steel-lined lids in an open room. Ambient room temperature and humidity were monitored daily and tightly controlled. Food and water were available ad libitum. All mice were maintained in a 12 h:12 h light–dark cycle with light intensity around 100 lux.

Clozapine-N-oxide (CNO, Sigma-Aldrich SKU:C0832-5MG) was dissolved in sterile 0.9% saline solution. CNO/saline solution was then diluted to achieve the dosage (mg/kg body weight) per mouse required for the experiment.

Clozapine-N-oxide was delivered either by eye-drop or intraperitoneal injection. For eye-drops, CNO was diluted based on mouse weight to achieve the correct dose within a 1–2 μl dose. 1–2 μl was then loaded into P10 micropipette followed by immobilizing the mouse via scruff. The 1–2 μl range was chosen because it is large enough to be accurately pipetted and small enough to not drip off of the eye after application. The solution in the pipette was then expelled slowly so that a stable droplet forms on the pipette tip. The droplet was then carefully touched to the cornea of the mouse eye and the mouse was released. The pipette tip never contacts the mouse’s eye.

All mice were dark-adapted for at least 30 min prior to CNO delivery and subsequent pupil measurements. For all experiments, mice were unanesthetized and restrained by hand. Videos of the eye were taken using a Sony Handycam (FDR-AX33) mounted on a tripod a fixed distance from the mouse. Manual focus was maintained on the camera to ensure that only one focal plane existed for each mouse and that therefore variable distance from the camera should not contribute to differences in relative pupil area throughout the video. Pupil size was first recorded under dim red light and an external infrared light source to capture the dark-adapted baseline pupil size. CNO was then delivered as an eye-drop or injection and the mouse was returned to their cage. Pupil size was monitored at intervals described in the results section. All pupil images presented in the paper were cropped to a fixed square area surrounding the eye using GNU Image Manipulation Program (GIMP). The images were made grayscale and then brightness and contrast were adjusted to enhance visibility of the pupil and exported as PNG files.

Videos were transferred from the camera to a computer as Audio Video Interleave (AVI) files and individual frames were taken using VLC media player¹ and saved in portable network graphics format (PNG). Pupil area was then quantified manually in ImageJ² software. The pupil area was measured in pixels using the oval tool in which the four cardinal points of the oval were touching their respective edges of the pupil. The relative pupil area was calculated using Microsoft Excel in which the area during the stimulus was divided by the area prior to CNO dosage.

The dose-response curve was fit using a variable slope sigmoidal dose-response curve in Graphpad Prism 6. The top and bottom of the fit were constrained to 1.0 and between 0 and 0.10, respectively.

For viral infection of the retina, Opn4^Cre (Ecker et al., 2010) and littermate control mice were anesthetized by IP injection of avertin (2, 2, 2-Tribromoethanol) and placed under a stereo microscope. 1 μl of adeno-associated virus (AAV)2-hSyn-DIO-hM3DG_q-mCherry (4.6 × 10¹² viral particles/ml, Roth lab, UNC Vector Core) was placed on a piece of Parafilm and drawn into a 10-μl microcapillary tube (Sigma P0674) that had been pulled to a needle (Sutter Instruments, Model P-2000). The loaded needle was then placed in the holster of a pico-injector (Harvard Apparatus PLI-90). The needle punctured the eye posterior to the ora serrata and air pressure was used to drive the viral solution into the vitreous chamber of the eye to ensure delivery specifically to the retina. Mice recovered from surgery on a heating pad until they woke from anesthesia. All experiments were performed 3–5 weeks following viral injection.

For viral infection of the brain, mice were anesthetized by IP injection of avertin, and rAAV5-hSyn-hM3D(G_q)-mCherry (3.4 × 10¹² viral particles/ml, Roth lab, UNC Vector Core) was stereotaxically delivered. All coordinates used follow the Paxinos and Franklin atlas (Franklin and Paxinos, 1997). A 10-μl microcapillary pipette was pulled and loaded with the AAV solution. A total volume of 30 nl of AAV was injected using a microinjector (Nanojector II, Drummond Scientific Company). A heating pad was used to maintain the body temperature at ∼35°C. Before and after the surgery, systemic analgesics (buprenorphine, 0.1 mg/kg) were administrated.

Four weeks after brain injections, c-Fos induction (in AAV-infected neurons) was immunohistochemically evaluated in mice that were perfused 90 min after application of an eye-drop of CNO 0.001, 0.01, 0.1, and 1.0 mg/kg doses); mice were kept in constant darkness during the experiment. After perfusion, brains were post-fixed overnight, cryoprotected in 30% sucrose and subsequently sectioned on a cryostat (coronal sections, 40 μm). Brain sections were blocked for 2 h in PBS containing 0.3% Triton X-100 and 3% heat-inactivated goat serum and then incubated with a mouse IgG1 α-c-Fos (EnCor MCA-2H2; 1:500) overnight, at 4°C. After rinse, sections were incubated with goat anti-mouse IgG1 Alexa 488 (1:500) secondary antibody. Finally, slides were mounted in AntiFade medium (Molecular Probes), and images were acquired using a LSM-700 confocal microscope (Zeiss). Zeiss Zen software and ImageJ were used for subsequent file export.

All c-Fos staining was performed in parallel and images were taken with identical exposure. DREADD-mCherry expressing neurons co-labeled with c-Fos were quantified from six hippocampal slices at each CNO dose.

All statistical tests were performed in Graphpad Prism 6. Specific statistical comparisons are listed in the figure captions. Because the EC50 data appears to be a normal distribution on a log scale (log-normal distribution), all statistical tests and data analysis involving EC50 were performed on the log transformed data set.

We first sought to evaluate whether CNO eye-drops effectively activate DREADDs in the brain. To do so, we injected a G_q-DREADD-containing virus [AAV5-hSyn-hM3D(G_q)-mCherry] into a the hippocampus (Figure 1A). Four weeks following viral injection, we applied an eye-drop of saline or CNO (0.001, 0.01, 0.1, and 1.0 mg/kg doses) and evaluated neuronal activation 90 min afterward by assaying the expression of the immediate early-gene c-Fos. Using the DREADD-mCherry fusion protein to identify virally infected cells, we observed robust c-Fos staining following 1.0 mg/kg CNO in neurons expressing the DREADD-mCherry reporter, indicating that the CNO eye-drop successfully activated G_q-DREADD in those cells (Figure 1B). DREADD activation by 1.0 mg/kg CNO in the subpopulation of infected hippocampal neurons resulted in widespread activation and c-Fos induction of the entire hippocampus. Less intense c-Fos induction was observed following 0.1 mg/kg CNO and no obvious c-Fos induction followed 0.01 and 0.001 mg/kg CNO. A saline eye-drop or 1.0 mg/kg CNO dose applied to a non-infected mouse did not elicit hippocampal c-Fos expression (Figure 1B). Finally, we quantified the percentage of DREADD-mCherry+ cells clearly expressing c-Fos following saline and each dose of CNO [Saline: 8 of 272 cells (2.9%); 0.001 mg/kg CNO: 12 of 241 (4.98%); 0.01 mg/kg: 4 of 213 (1.88%); 0.1 mg/kg: 99 of 381 (25.99%); 1.0 mg/kg: 256 of 263 (97.34%)] (Figure 1C). 1.0 mg/kg CNO appears to be an effective dose for robust activation of DREADD-expressing neurons in the brain. This result confirms the ability of CNO delivered by eye-drop to enter the blood and activate DREADDs in the brain.

To visualize and quantify DREADD activity in vivo, we utilized a genetically defined subpopulation of melanopsin expressing retinal ganglion cells, intrinsically photosensitive retinal ganglion cells ipRGCs (ipRGCs), which we and others have shown to drive pupil constriction when activated by G_q-DREADD (Keenan et al., 2016; Milosavljevic et al., 2016b). This system gives us a readily observable and easily quantifiable output of DREADD activation in vivo, allowing us to quantitatively compare the effectiveness of eye-drops and IP injection in real-time The Opn4^Cre (Opn4 gene codes melanopsin) mouse (Ecker et al., 2010) provides us genetic access to ipRGCs, and a Cre-dependent G_q-DREADD AAV allows us to express G_q-DREADD specifically in these cells.

We first injected an AAV carrying a Cre-dependent G_q-DREADD construct (AAV2-hSyn-DIO-hM3DG_q-mCherry) into only the right eye of Opn4^Cre mice, leaving the left eye uninfected (Figure 2A). 3–5 weeks after infection, we applied a 1 μl CNO (0.1 mg/kg) eye-drop to the uninfected left eye. We observed robust pupil constriction (Figure 2B) as has been observed previously in response to IP CNO (Keenan et al., 2016; Milosavljevic et al., 2016b). This result further demonstrates that CNO delivered via eye-drop is absorbed into the blood and delivered to distant tissues at working concentrations in vivo.

After confirming the feasibility of eye-drops as a delivery method, we next compared the CNO doses required to elicit responses when using eye-drop or IP delivery (Figures 2C,D). To do so, we administered doses of 0.001, 0.01, 0.1, and 1.0 mg/kg CNO via eye-drop or IP injection and monitored pupil constriction (Figure 2C, n = 10). We observed similar dose responses for both methods, with eye-drops displaying a small but statistically significant decrease in the dose required to achieve half-maximal response (EC₅₀) (Figure 2D, P = 0.0195 by Wilcoxon matched-pairs signed rank test). This difference could be explained by changes in blood absorption efficiency or potentially by reduced stress responses in these mice after eye-drop as opposed to IP injection. However, the magnitude of the difference is minor and essentially irrelevant when considering the practical application of either technique.