Strength of Activation and Temporal Dynamics of BioLuminescent-Optogenetics in Response to Systemic Injections of the Luciferin

BioLuminescent OptoGenetics (“BL-OG”) is a chemogenetic method that can evoke optogenetic reactions in the brain non-invasively. In BL-OG, an enzyme that catalyzes a light producing reaction (i.e., a luciferase) is tethered to an optogenetic element that is activated in response to bioluminescent light. Bioluminescence is generated by injecting a chemical substrate (luciferin, e.g., h-Coelenterazine; h-CTZ) that is catalyzed by the luciferase. By directly injecting the luciferin into the brain, we showed that bioluminescent light is proportional to spiking activity, and this relationship scales as a function of luciferin dosage. Here, we build on these previous observations by characterizing the temporal dynamics and dose response curves of BL-OG effects to intravenous (IV) injections of the luciferin. We imaged bioluminescence through a thinned skull of mice running on a wheel, while delivering h-CTZ via the tail vein with different dosage concentrations and injection rates. The data reveal a systematic relationship between strength of bioluminescence and h-CTZ dosage, with higher concentration generating stronger bioluminescence. We also found that bioluminescent activity occurs rapidly (< 60 seconds after IV injection) regardless of concentration dosage. However, as expected, the onset time of bioluminescence is delayed as the injection rate decreases. Notably, the strength and time decay of bioluminescence is invariant to the injection rate of h-CTZ. Taken together, these data show that BL-OG effects are highly consistent across injection parameters of h-CTZ, highlighting the reliability of BL-OG as a non-invasive neuromodulation method.


HIGHLIGHTS
• Systematic injections of the luciferin, h-Coelenterazine (h-CTZ), parametrically drive bioluminescence activity in neocortical neurons.• The strength of bioluminescence activity is invariant to the injection rate of h-CTZ.
• Intravenous injections of h-CTZ lead to rapid bioluminescent activity in neocortex, regardless of the luciferin dosage • The BL-OG molecule produces strong and reliable bioluminescence activity that can be monitored noninvasively.

INTRODUCTION
Methods of neuromodulation can provide fundamental insight into the role that neural ensembles play in mediating perceptual functions and motor actions.In a seminal study, Salzman and colleagues (Salzman et al., 1990) solidified the role of medio-temporal (MT) cortex in driving perception of visual motion by showing that intracortical microstimulation (ICMS) in MT systematically biases visual motion judgements.ICMS was also used to show that perception of flutter (i.e., low frequency vibrations) is mediated, at least in part, by neurons in primary somatosensory cortex (SI) (Romo et al., 1998).ICMS has become one of the leading methods used in brain computer interface (BCI) applications, especially in clinical applications for human patients that rely on neuroprosthetic devices (Ajiboye et al., 2017;Chaudhary et al., 2022;Collinger et al., 2014Collinger et al., , 2013;;Downey et al., 2016).Yet, although ICMS has led to fundamental discoveries, the technique can only provide unidirectional neuromodulation effects by upregulating activity of neural populations (i.e., it cannot be used to cause inhibitory effects).Further, ICMS can generate non-specific effects by modulating widespread networks through stimulation of passing fibers (Histed et al., 2009;Kumaravelu et al., 2022).ICMS also fails to provide selective modulation of cell-type specific populations (e.g., inhibitory vs. excitatory cells).Indeed, understanding how individual cell types contribute to a particular neural or perceptual function can lead to fundamental insight into the circuit mechanisms that mediate such function.
Neuromodulation methods that rely on genetic modification strategies (e.g., optogenetics and chemogenetics) can provide precise excitatory and/or inhibitory modulation of cell-type specific circuits (Aston-Jones and Deisseroth, 2013a;Bernstein et al., 2012;Bernstein and Boyden, 2011).In particular, optogenetics affords high spatio-temporal precision, even at the single cell level (Marshel et al., 2019).However, similar to ICMS, optogenetics requires an invasive surgical procedure to generate neuromodulation effects in the brain (e.g. a cranial surgery to install a chronic implant to support the external light source and provide optical access to the brain).In contrast, chemogenetic methods are minimally invasive, largely because they do not require a cranial implant to generate neuromodulation (Berglund et al., 2016;Dimidschstein et al., 2016;Gomez-Ramirez et al., 2020;Hori et al., 2023;Song et al., 2022;Urban and Roth, 2015;Vlasov et al., 2018;Zhang et al., 2022).Further, chemogenetics can modulate activity across broad spatial scales by systemically injecting the chemical that drives the chemogenetic molecule, a useful feature for large-brain animals such as non-human primates (Cushnie et al., 2023;Hori et al., 2023;Raper and Galvan, 2022).However, chemogenetics has less temporal precision, in comparison to optogenetics and ICMS, with some methods producing effects that last hours as in the case of designer receptors exclusively activated by designer drugs (DREADDs) (Alexander et al., 2009;Claes et al., 2022).Thus, all neuromodulation methods come with inherent advantages and disadvantages, and it is incumbent on the researcher to use the approach (or approaches) best suited to answer the question of the study.
Indeed, a recent study showed lower bioluminescence but stronger BL-OG effects on spiking activity in cells that use FRET to drive opsins (i.e., LMO7 vs. LMO3) (Björefeldt et al., 2024).(Björefeldt et al., 2024) study also show higher photonic activity in cells that express a light emitter without an opsin vs. cells that express an LMO (e.g., LMO3 or LMO7), which highlights the coupling efficiency between light-emitting molecule and the optogenetic element.We previously showed that the bioluminescence and BL-OG effects of the LMO3 molecule is highly proportional to injections of the substrate (Gomez-Ramirez et al., 2020).Here, we study whether the bioluminescence response of the LMO7 molecule is also commensurate to the injection parameters of its substrate, h-CTZ.
The rapid catalytic reaction that produces bioluminescence can generate relatively short-term effects (e.g., minutes to tens of minutes).BL-OG can also provide moment-tomoment neuromodulation by conventional activation of opsins using a fiber optic cable.
A critical feature of BL-OG is that the bioluminescent light created from the BL-OG mechanism provides an optical readout of the neuromodulation effect itself (Gomez-Ramirez et al., 2020).We recently showed that bioluminescent light is directly proportional to multi-unit spiking activity, and this relationship scales as a function of CTZ amount (Gomez-Ramirez et al., 2020).Taken together, BL-OG represents a highly feasible and handy method for generating and tracking neuromodulation effects in celltype specific circuits via minimally invasive approaches.
To further establish and facilitate the use of BL-OG as a minimally invasive modulation method, it is key to establish the temporal properties and strength of the neuromodulation effects of BL-OG in response to different injection parameters of the luciferin.Here, using bioluminescence as a proxy for spiking activity (Gomez-Ramirez et al., 2020), we quantified the temporal dynamics and dose response functions of the LMO7 molecule to different concentrations and rate of injections of the luciferin, h-Coelenterazine (h-CTZ).
Activity was imaged through the thinned-skull of a mouse to further highlight the minimally invasive properties of BL-OG.We found that bioluminescence increases linearly as a function of h-CTZ dosage, and, importantly, the overall strength of bioluminescence is consistent across the rate of injection.We also observed that the onset of bioluminescence in neocortex occurs rapidly (~10 seconds after h-CTZ injection), regardless of h-CTZ concentration.However, as expected, the onset time of bioluminescence is systematically delayed as a function of injection rate.Taken together, our data show that bioluminescence response, and, by extension, BL-OG effects (Gomez-Ramirez et al., 2020), are highly predictable across injection parameters of the luciferin, highlighting the consistency and feasibility of BL-OG as a chemogenetic neuromodulation method.

Animals
Experiments were conducted using C57/BL6 mice (N = 17 across all experiments; The Jackson Laboratory stock #000664) bred in the vivarium at the University of Rochester.All experimental methods are consistent with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at the University of Rochester (UCAR).
All surgical procedures were conducted under general and local anesthesia.Mice were anesthetized with isoflurane (3% for induction and 1-2% for the remainder of the surgical procedure) and stereotaxically fixed.The fur was removed from the top of the head.The area was treated with 4 or 5% lidocaine cream, and then aseptically prepared.
A custom-made head post was affixed to the skull using C&B Metabond® Quick Adhesive Cement (Parkell).The skull was shaved down until the cranium was translucent throughout a 3mm radius in the area of left primary somatosensory cortex (SI; -1.25 AP and +3.5mm ML from the Bregma).Skull thinning was performed using a modified approach established in mice (Steinzeig et al., 2017).A NeoBurr sterile carbide bur drill bit (Microcopy, FG-4) was used to shave the skull until the cranium was translucent.The drill bit was lightly brushed across the skull in a circular and side-to-side motion every two seconds to achieve a smooth surface and avoid overheating of the skull.The skull was regularly flushed with cooled saline to increase visibility and reduce heating from the drilling.The virus with the LMO7 was injected through three burr holes in the thinned skull, 400nl in each burr hole at an injection rate of 10nl/minute and a depth of 400m through custom-pulled micropipettes using a Stoelting™ Quintessential Stereotaxic Injector (QSI).
The thinned skull and burr holes were then covered with clear nail polish (Electron Microscopy Sciences, Manufacturer Part Number: 72180).Mice were imaged at a minimum of two weeks after the surgery to allow time for the virus to express in the brain (Figure 1B).

Luciferin injections
Bioluminescence was produced using the substrate h-Coelenterazine-SOL (h-CTZ) for in vivo applications (NanoLight Technology, CAT#3011).Vials were stored at -80C until shortly before use.Once removed from the -80C freezer, the h-CTZ vial was wrapped in aluminum foil to protect it from light, and left on the lab counter for a few minutes to reach room temperature.One hundred microliters of sterile water was added to each vial, with vials placed in a 55C water bath for the powder to dissolve (Crespo et al., 2021).An additional two hundred microliters of sterile saline for injection was then  .74, 0.98, 1.72, 2.46, 3.19, and 3.93 mM).A volume of 250L of diluted h-CTZ was injected for each dose concentration at a rate of 1ml/min.For the Injection Rate experiment, we injected two hundred and fifty microliters of h-CTZ at a concentration of 3.93 mM and rates of 0.1, 0.2, 0.3, or 1 ml/min.

Bioluminescence and fluorescence imaging
Imaging was done through the thinned skull using an EMCCD camera (Falcon III; Raptor Photonics) fitted with a 2X objective from Thorlabs while the mouse was awake, head-fixed, and running on a 3D-printed wheel in a light-proofed chamber that was built in-house.The timeline of events is illustrated in and was maintained with isoflurane through a nose cone (~1%) for the duration of catheter placement.After placing the catheter in the tail vein, the animal was allowed to recover before the start of the imaging experiment (Figure 1D).We injected the fluorescein dye (Ak-Fluor 10%) to confirm that the tail vein catheter was placed correctly.After observing fluorescence emitted by Ak-Fluor, the catheter was taped firmly in place, and anesthesia was removed.Bioluminescence imaging was performed approximately 20 minutes after the brief anesthesia protocol to let the animal recover and be fully awake.Unless noted, bioluminescence was imaged with 60 seconds exposure time, and with the EM-gain of the EMCCD camera set at 3000.Thus, based on our imaging acquisition rate, time = '0' represents the aggregated bioluminescence activity between 0 and 60 seconds post h-CTZ injection.In follow-up studies, we imaged bioluminescence activity with a faster exposure time < 10 seconds to study the temporal dynamics of the bioluminescence response at a finer detail.We also imaged fluorescence in response to injections of Ak-Fluor 10% via the tail vein to estimate the time that it takes a chemical substance to reach the brain (exposure time = 0.5 Hz).The camera was connected to a chiller (Solid State Cooling Systems, UC160) with thermoelectric cooling temperature set to -70C to minimize noise emitted from heat mechanisms.Micromanager was used to control the EMCCD camera (Edelstein et al., 2010).We collected ten minutes of images (i.e., 10 frames) before injecting the h-CTZ.On frame eleven, we injected h-CTZ via the infusion pump, and imaging was continued for fifty additional minutes (i.e., 50 extra frames).
Figure 1D shows a representative example of the averaged bioluminescence map across one imaging session.
For Vector Laboratories, H-1800) and imaged using a widefield Zeiss Microscope.

Imaging analyses
Images were taken at 16-bit resolution, and analyzed using custom-based scripts in MATLAB.For each mouse, a bioluminescence region of interest (ROI) was computed by drawing an area that contained the most amount of bioluminescence (Gomez-Ramirez et al., 2020).Note that the same ROI was used for all experimental conditions.Baseline activity was derived by averaging the signal in the ROI across the first ten frames of each experimental condition (i.e., 600 seconds).Baseline activity was then subtracted from each frame collected during the experiment.
Peak bioluminescence was calculated by averaging the image frame with maximum activity together with its two nearest neighboring frames.We performed randomization tests to statistically determine the onset and offset of bioluminescence activity in each mouse.Surrogate distributions (N = 5000) for estimating the onset of bioluminescence were generated by averaging across one hundred randomly sampled pixels from the first ten frames in the experiment (i.e., prior to h-CTZ injection).The initial frame that showed bioluminescence was estimated by determining the first two consecutive frames from the observed data that showed higher bioluminescence relative to the surrogate baseline distribution (probability value < 0.05).To estimate the offset of bioluminescence, we built surrogate distributions using image pixels that were randomly sampled from the last eight frames in the imaging session.Offset bioluminescence was estimated as the first frame of the observed data (after h-CTZ injection) that failed to show greater bioluminescence relative to the surrogate distribution built from the last eight frames in the session.Time of decay of the bioluminescence response was calculated as the time difference between the peak bioluminescence response and the bioluminescence offset.Area under the curve (AUC, i.e., total bioluminescence) was calculated by averaging activity across the start and end frame of the observed data.For the highest concentration in the Dose Response experiment, the response and time dynamics of bioluminescence was estimated by averaging across the values in each of the three sessions.Statistical testing at the group level was performed using Friedman repeated measures tests.
A Friedman test was used, instead of a parametric ANOVA, because the bioluminescence responses were not normally distributed, and our sample size was relatively small (N = 8).Effect sizes were estimated using Kendall's W statistic described by Equation 1:

Equation 1
Where X 2 = the Friedman test statistic value, k = the number of groups, and n = the total number of observations.Follow-up tests were done using Mann-Whitney U tests or curve fitting using a linear, quadratic, or exponential function.The model that provided 25% higher adjusted-R 2 values, relative to the adjusted-R 2 of the linear function, was determined to be the model that best explains the data.However, if none of the models provided an adjusted-R 2 greater than 0.1, then we deemed that none of the models were reliable predictors of the data.

The bioluminescence response is proportional to luciferin dosage
Bioluminescence activity systematically increases as a function of h-CTZ dosage concentration.Figure 2A shows the average bioluminescence time course for each h-CTZ concentration injected at 1ml/min.The traces are time-locked to the onset of the injection.A Friedman test revealed significant differences in the bioluminescence peak response across h-CTZ conditions (X 2 (5) = 33.71,p = 2.72 x 10 -6 ; W = 0.84; Figure 2B), with bioluminescence having a linear relationship to the dosage of the luciferin (adjusted-R 2 = 0.46; Figure 2B dashed lines).Note that a W greater than 0.5 is considered a large effect size.We also observed significant differences between h-CTZ dosage and area under the curve (AUC, defined as the mean activity between the onset and offset bioluminescence response; X 2 (5) = 33.43,p = 3.91 x 10 -6 ; W = 0.84; Figure 2C).Similar to the bioluminescence peak response, we observed a linear relationship between the AUC and h-CTZ (adjusted-R 2 = 0.57; Figure 2C dashed line).Taken together, these data highlight the consistency and predictability of the bioluminescence response and, by proxy, BL-OG effects to a particular h-CTZ concentration.

Temporal dynamics of bioluminescence are consistent across luciferin dosage
Systemic h-CTZ injection generates bioluminescent activity within the first 60 seconds for all dosage concentration.Figure 3A shows the median onset time of bioluminescence for each h-CTZ concentration.A Friedman test did not reveal differences in bioluminescence onset time across h-CTZ conditions (X 2 (5) = 0.56, p = 0.98).A Friedman test also showed no significant differences in peak activation timing across all h-CTZ dosages (X 2 (5) = 1.32, p = 0.93; Figure 3B), with the peak activity occurring between 60 and 120 seconds following h-CTZ injection.However, we found that the decay time of the bioluminescence response systematically increases as a function of dosage concentration (Figure 3C).A Friedman test revealed significant differences in time decay between h-CTZ dosage (X 2 (5) = 33.21,p = 3.42 x 10 -6 ; W = 0.83), with bioluminescence having a linear relationship to h-CTZ dosage (adjusted-R 2 = 0.48; Figure 3C dashed lined).Bioluminescence was imaged using a 60 seconds exposure time to obtain a strong and reliable measure of the signal (see e.g., Gomez-Ramirez et al 2020).However, this image acquisition rate provides a coarse estimate of the time onset properties of bioluminescence.As such, we performed additional imaging experiments where bioluminescence was sampled at a faster rate (i.e., a short exposure time of 6 seconds) to have a better estimate of the temporal dynamics of the bioluminescence response across h-CTZ dosage conditions.We increased the EM gain of the camera to its maximum to capture images with larger SNRs.These additional experiments were conducted on a separate cohort of mice (N = 3) who expressed the LMO7 molecule.Imaging was done via a thinned skull, and injecting a smaller set of the h-CTZ dosage conditions (0.98, 1.72, and 3.93 mM). Figure 3D shows the time course of the bioluminescence in response to the three h-CTZ dosage conditions imaged with a short exposure time, with higher h-CTZ dosage generating greater bioluminescence activity.The left inset in Figure 3D shows the onset time of bioluminescence for the three h-CTZ conditions imaged with a short exposure time.
Although the largest condition seemed to have the shortest onset time, a Friedman test failed to show a significant difference in bioluminescence onset time between the dosage conditions (X 2 (2) = 2.17, p = 0.34).Although we imaged activity using the camera's highest EM gain, the delayed onset time for the lower dosage conditions may be caused by lower SNR due to the faster sampling rate of imaging.In support, we observed that the peak onset time of bioluminescence was similar across the three dosage conditions (Peak Time = 66, 54, and 72 seconds for the 0.98, 1.72, and 3.93 mM dosages, respectively; X 2 (2) = 1.5, p = 0.47).We also observed that the time of decay increased as a function of h-CTZ dosage (X 2 (2) = 6.26, p = 0.04; adjusted-R 2 = 0.63; right inset Figure 3D).These findings from the short exposure time experiments are highly consistent with those results from experiments using a longer exposure time.Taken together, these data show that although the onset and peak responses of bioluminescence are consistent across dosage, the duration of the bioluminescence response systematically increases as a function of the luciferin concentration.
We performed an additional control experiment where we injected fluorescein via the tail vein in 3 mice to determine the time it takes for the injected substrate to reach the brain.Figure 3E shows the time course of the normalized fluorescence response (left axis, purple trace), and normalized bioluminescence response to the highest h-CTZ dosage (3.93nM; right axis, burgundy color).Three mice were imaged with an exposure time of 6 seconds, and two mice were imaged with a 10 seconds exposure time.The median onset time of bioluminescence is a bit delayed relative to the fluorescence onset from the fluorescein injection (18 vs. 9.6 seconds; see Figure 3E inset), but bioluminescence still occurred relatively quickly after the h-CTZ injection.Taken together, these data indicate that systemic injections of luciferin generate rapid bioluminescence responses in the brain.

Bioluminescence dynamics are largely stable across luciferin injections with different rates
Bioluminescence activity is similar regardless of the injection rate of h-CTZ of high concentration (i.e., 3.93 mM). Figure 4A shows the average bioluminescence time course for each h-CTZ injection rate.The traces are time-locked to the onset of the injection.A Friedman test did not reveal significant differences in peak responses (X 2 (3) = 3.19, p = 0.36; Figure 4B) or AUC activity (X 2 (3) = 2.36, p = 0.50; Figure 4C) across h-CTZ injection rate conditions.Thus, the strength of BL-OG effects appears to be insensitive to the injection rate of the luciferin.

Temporal dynamics of bioluminescence vary by rate of luciferin injection
Injection rate of h-CTZ systematically modulates the onset of bioluminescent activity.Figure 5A shows the median onset time of bioluminescence for each injection rate condition.A Friedman revealed significant differences in bioluminescence onset time across injection rate (X 2 (3) = 12.97, p = 0.043; W = 0.54), with the onset time exponentially decreasing as a function of injection rate (adjusted-R 2 = 0.51; Figure 5A dashed lines).The data further show that the peak time of activation also decreases as a function of injection rate (X 2 (3) = 13.05,p = 0.0045; W = 0.54; Figure 5B), with the bioluminescence peak time linearly decreasing with injection rate speed (adjusted-R 2 = 0.24; Figure 5B dashed lines).However, the data did not show an effect of injection rate on the time decay of bioluminescence (X 2 (3) = 3.79, p = 0.285, Figure 5C).These data reveal that the early dynamics of the bioluminescence response are systematically modulated by the rate of injection of the luciferin.

Bioluminescence activity is specifically generated by the LMO7 molecule
We performed an additional control experiment to determine how much of the bioluminescence response is driven by 'auto-bioluminescence' of the h-CTZ itself (i.e., uncaging of photons not created by the luciferase/luciferin interaction).In these control experiments, we imaged activity in three mice that did not express LMO7.Three different concentrations of h-CTZ were injected via the tail vein (1.72, 2.46, and 3.93 mM).We also imaged activity without injecting h-CTZ (i.e., 0 mM dosage) to estimate the 'photonic noise' of the EMCCD camera.Figure 6A shows very weak photonic activity for all h-CTZ dosage conditions.However, we observed similar values for the 0 mM dosage condition (X 2 (3) = 1, p = 0.8013), indicating that this weak bioluminescence activity is driven by 'photonic noise' from the EMCCD camera.Figure 6B shows that total bioluminescence (area under the curve) across h-CTZ dosages for the control group vs. the mouse cohort expressing the LMO7 molecule.These data show a ~15 fold increase in the bioluminescence response observed in the LMO7 vs. the control group (averaged across h-CTZ dosages).These data indicate that the bioluminescence activity observed in animals expressing the LMO7 molecule is not produced by 'auto-bioluminescence' from h-CTZ.

DISCUSSION
We characterized dose response functions and temporal dynamics of bioluminescence in response to different h-CTZ injection parameters in mice expressing the BL-OG molecule LMO7, a fluorescent protein-luciferase fusion tethered to VChR1.Bioluminescence was imaged through a thinned-skull, while delivering injections of h-CTZ via the tail vein.The data show that the strength of bioluminescence, measured during the peak activity and AUC (i.e., the total bioluminescence across time), linearly increases as a function h-CTZ concentration (see Figures 2B and 2C).
Importantly, the peak and AUC of the bioluminescence response is unaffected by the rate of injection of h-CTZ (see Figure 4B and 4C).Future projects using singleunit electrophysiology or 2-photon fluorescence imaging will determine whether these concentration-dependent increases in BL-OG bioluminescence lead to more neurons being recruited and/or amplifications of individuals' neural firing themselves.
The findings in this study build on our previous work that show robust and systematic bioluminescence activity in response to direct injection of the luciferin in the brain (Gomez-Ramirez et al., 2020).Here, we further demonstrate this robust and parametric relationship between the strength of the bioluminescence response and luciferin dosage using systemic injections of the luciferin.Our data also reveal that bioluminescence activity produced by the BL-OG molecule can be reliably imaged through a thinned skull of a mouse, highlighting BL-OG's feasibility to track neuromodulation effects through minimally invasive procedures.In sum, our study provides strong evidence that IV injections of h-CTZ generate concentrationdependent BL-OG effects in the brain, and that these effects are independent of the rate at which the luciferin is delivered.

Temporal dynamics of the bioluminescence response
The onset time of bioluminescence in neocortex is rapid and invariant to h-CTZ concentration (see Figure 3A).In particular, bioluminescence emerges within the first 60 seconds after h-CTZ injection in all concentration conditions.However, using shorter exposure times (i.e., higher sampling rates), we found that the onset time of bioluminescence is ~18 seconds after injection.This rapid onset time highlights the robust bioavailability of h-CTZ to the brain, and the fast-acting mechanisms of BL-OG to elicit neuromodulation effects.We also found that the onset peak response is unaffected by the concentration of h-CTZ.Further, as expected, the decay time of bioluminescence linearly increases as a function of h-CTZ concentration, likely due to the large amount of the substrate that is catalyzed by the luciferase.
As expected, the onset time of bioluminescence modulates as a function of injection rate.Specifically, we observed a nonlinear relationship (exponential) between bioluminescence onset and injection rate, with faster rates generating earlier onset times (see Figure 5A).We note that, although bioluminescence onset times are fit well by an exponential function, the pattern may be driven (at least in part) by the sparse sampling in the imaging and injection rate conditions.As such, future experiments should sample bioluminescence activity much faster to determine whether the onset of bioluminescence activity in the brain scales linearly vs. non-linearly (e.g., exponentially) with the rate of injections of the luciferin.The data also show that the peak time of bioluminescence decreases as the rate of injection is increased (see Figure 5B).However, the decay time of bioluminescence is unaffected by injection rate.Thus, our data indicate that the rate of injection of h-CTZ modulates the bioluminescence response by shifting the onset and peak activation time, without modifying the decay dynamics of the bioluminescence response.These effects of injection rate are notable because they generate bioluminescence activations (e.g., AUC responses) that are largely homogenous regardless of the injection rate (see e.g., Figure 4C).
The onset time of BL-OG effects appear to be substantially faster than the neuromodulation onset times of other chemogenetic methods such as DREADDs (Deffains et al., 2021;Nagai et al., 2020).For instance, it was shown that deschloroclozapine (DCZ) administered IV at a rate of 0.2 ml/sec leads to neuromodulation effects that commence around 5 minutes after injection (Nagai et al., 2020).In contrast, using the same injection rate of h-CTZ, we observed bioluminescence occurring as early as 18 seconds after injection (see Figure 3D).
The difference in neuromodulation onset times between the two methods is unclear, but DREADDs operate on secondary messenger systems (i.e., G protein-coupled receptors; GPCRs) which have significantly slower kinetics as compared to ionotropic channels in which optogenetic elements operate on (Armbruster et al., 2007;Whissell et al., 2016).Our data also show that BL-OG effects can be short-lasting (~3 to ~12 minutes), providing repeated BL-OG-mediated neuromodulation effects within a single experimental day.In contrast, the duration of the neuromodulation effects of DREADDs are thought to last hours (Alexander et al., 2009;Whissell et al., 2016).
Taken together, our data demonstrate that the duration of BL-OG effects can be regulated by the concentration amount, volume (see (Gomez-Ramirez et al., 2020)), or injection rate of the luciferin, indicating that the dynamics of neuromodulation effects of BL-OG can be systemically controlled by experimenters.
Our study provides a generalized framework of the experimental parameters that control the strength and dynamics of BL-OG effects generated by the LMO7 molecule in the brain.In particular, our data provide evidence that BL-OG effects driven by LMO7 can be fast, with the strength and duration of the neuromodulation effects modulated by the injection parameters of the luciferin.Our data further show that the BL-OG method is unique in providing online tracking of the neuromodulation effects via the bioluminescent activity, and this tracking can be achieved in lieu of chronically-implanted cranial windows.
In sum, this study highlights the reliability of BL-OG as a major neuromodulation method and demonstrates its feasibility to monitor neuromodulation effects through minimally invasive approaches.

FIGURES FIGURE 1
added to the vial to form a stock solution of 300 L volume.For the Dose Response experiment, different levels of h-CTZ concentration (of the same volume) were derived by further diluting the stock solution with different amounts of sterile saline (e.g., 10L and 244L of sterile saline to achieve 3.93mM and 0.1 mM dosage concentrations of 250L, respectively).For each concentration condition, 250L of prepared solution was preloaded into a microbore tubing extension set (Smith's Medical, 536040C), with one end connected directly to the tail vein catheter and the other end connected to another extension tubing filled with sterile water for injection.The end tube was connected to an infusion pump (Harvard Apparatus, Model 11) that controlled the rate of injection of the luciferin.For the Dose Response experiment, we injected h-CTZ with six different concentrations (0

Figure 1C .
Prior to bioluminescence imaging, the mouse was put under light anesthesia to insert a tail vein catheter to deliver h-CTZ (Instech, Manufacturer Part Number: C10SS-MTV1417P).The lightly anesthetized plane was induced by placing the animal in an isoflurane chamber (~1.5 to 2% induction)